Nutraceuticals and Human Blood Platelet Function: Applications in Cardiovascular Health 1119376009, 9781119376002

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Nutraceuticals and Human Blood Platelet Function

Nutraceuticals and Human Blood Platelet Function Applications in Cardiovascular Health

Asim K. Duttaroy Department of Nutrition Faculty of Medicine University of Oslo Norway

This edition first published 2018 © 2018 John Wiley & Sons Ltd All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. The right of Asim K. Duttaroy to be identified as the author of this work has been asserted in accordance with law. Registered Office(s) John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial Office The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Wiley also publishes its books in a variety of electronic formats and by print-on-demand. Some content that appears in standard print versions of this book may not be available in other formats. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Library of Congress Cataloging-in-Publication Data Names: Duttaroy, Asim K., author. Title: Nutraceuticals and human blood platelet function : applications in cardiovascular health / by Asim K. Duttaroy. Description: First edition. | Hoboken, NJ : Wiley, 2018. | Includes bibliographical references and index. | Identifiers: LCCN 2018000528 (print) | LCCN 2018000730 (ebook) | ISBN 9781119376002 (pdf ) | ISBN 9781119376057 (epub) | ISBN 9781119376019 (hardback) Subjects: | MESH: Cardiovascular Diseases–prevention & control | Cardiovascular Diseases–etiology | Dietary Supplements | Functional Food | Platelet Activation–physiology Classification: LCC RC672 (ebook) | LCC RC672 (print) | NLM WG 120 | DDC 616.1/05–dc23 LC record available at https://lccn.loc.gov/2018000528 Cover Design: Wiley Cover Images: © Monika Wisniewska/Shutterstock; © Lotus_studio/Shutterstock; © Amarita/iStockphoto; © STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY/ Getty Images; © SCIEPRO/Getty Images Set in 10/12pt WarnockPro by SPi Global, Chennai, India Printed in Great Britain by TJ International Ltd, Padstow, Cornwall. 10 9 8 7 6 5 4 3 2 1

Nor weep nor doubt that still the spirit is whole, And life and death but shadows of the soul. – Bhagavad Gita This book is dedicated to my elder brother, the late Subir K.  Duttaroy (01.05.1947–10.05.2004), who encouraged and supported me through the entire postgraduate process, and never got to read this book. I am forever indebted to my other elder brother, Mr. Asish K. Duttaroy, who gave me unrelenting support during my studies in India. Both of them routinely went beyond their duties to help me and to instil great confidence in my studies. I would also like to thank my wife Sujata and children, Shayantan and Sanghita, and my brothers and sisters for their unremitting encouragement. Without their help, best wishes, and willingness to share, the book would not have even been possible. In the same vein, I would like to thank numerous people who offered their time, support, and commitment to this book project. Lastly, I thank Wiley UK for publishing this book.

vii

Contents 1

Human Blood Platelets and Their Role in the Development of Cardiovascular Disease  1

Abbreviations Used in This Chapter  1 1.1 Introduction  1 1.2 Human Blood Platelets: Structure and Function  4 1.3 Platelet Activation Pathways  10 1.4 Platelets and Vessel Wall Interactions  12 1.5 Roles of Platelets in Atherosclerosis and Inflammatory Processes  13 1.6 Platelets and Their Role in the Development of Cardiovascular Disease  17 1.7 Conclusions  22 References  22 Epidemiology of Cardiovascular Disease  29 Abbreviations Used in This Chapter  29 2.1 Introduction  29 2.2 Dietary Lipids and Cardiovascular Disease  32 2.3 Environmental Factors and Cardiovascular Disease  34 2.4 Genetic Basis of Cardiovascular Disease Incidence  35 2.5 Fruits and Vegetables Consumption and Cardiovascular Disease Risk Reduction  37 2.6 Conclusions  40 References  40

2

n‐3 Fatty Acids and Human Platelets  47 Abbreviations Used in This Chapter  47 3.1 Introduction  47 3.2 Epidemiology of n‐3 Fatty Acids Intake and Cardiovascular Disease  51

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Contents

3.3 3.4 3.5 3.6

n‐3 Fatty Acids and Platelet Function  52 Platelet Function and Eicosanoids  56 Clinical Trials with n‐3 Fatty Acids  59 Dietary Recommendation and Sources of n‐3 Fatty Acids  61 3.7 Conclusions  62 References  62 4

Effects of Garlic, Onion, Ginger, and Turmeric on Platelet Function  69

Abbreviations  69 4.1 Introduction  69 4.2 Effects of Garlic (Allium Sativum) on Platelet Function  71 4.3 Effects of Onion (Allium Cepa L.) on Platelet Function  74 4.4 Effects of Ginger (Zingiber Officinale) on Platelet Function  75 4.5 Effects of Turmeric (Curcuma Longa) on Platelets  76 4.6 Conclusions  78 References  79 Herbs and Platelet Function  83 Abbreviations Used in This Chapter  83 5.1 Introduction  83 5.2 In Vitro Platelet Aggregation Studies: Effects of Different Herb Extracts  87 5.2.1 Andrographis (Andrographis Paniculata)  89 5.2.2 Cranberry (Vaccinium Macrocarpon)  90 5.2.3 Feverfew (Tanacetum Parthenium)  90 5.2.4 Green Tea (Camellia Sinensis)  91 5.2.5 Hawthorn (Crataegus Oxyacantha)  92 5.2.6 Horse Chestnut (Aesculus Hippocastanum)  92 5.2.7 Motherwort (Leonurus Japonicus)  93 5.2.8 St John’s Wort (Hypericum Perforatum)  93 5.2.9 Willow Bark (Salix Alba)  94 5.3 Effects of Herbs on Signaling Molecules in Human Platelets  95 5.4 Conclusions  97 References  98

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Contents

6 Tomato Extract and Human Platelet Functions  101 Abbreviations Used in This Chapter  101 6.1 Introduction  101 6.2 Epidemiology of Tomato Consumption and Cardiovascular Disease Risk Reduction  104 6.3 In Vitro Studies with Water‐Soluble Tomato Extract on Human Blood Platelet Aggregation  105 6.4 Fruitflow®: Compositional and Structural Aspects  111 6.5 Human Trials  112 6.6 Comparing the Dietary Anti‐Platelet Fruitflow® with the Anti‐Platelet Drug Aspirin  115 6.8 EFSA Approval of Fruitflow®  117 6.8 Conclusions  117 References  118 Dietary Nitrates and Their Anti‐Platelet Effects  125 Abbreviations Used in This Chapter  125 7.1 Introduction  125 7.2 Nitrate and Cardiovascular Health  129 7.3 Effects of Nitrates on Human Blood Platelet Function In Vitro  131 7.4 Clinical Studies with Dietary Nitrate: Effects on Ex Vivo Platelet Function  133 7.5 Conclusions  134 References  135

7

8 Kiwifruit and Human Platelet Function  139 Abbreviations Used in This Chapter  139 8.1 Introduction  139 8.2 Kiwifruit and Its Bioactive Phytochemicals  140 8.3 Kiwifruits and Human Blood Platelet Function  141 8.4 Human Trials Using Kiwifruit and Kiwifruit Extract  147 8.5 Conclusions  150 References  151 Polyphenols and Human Platelets  155 Abbreviations Used in This Chapter  155 9.1 Introduction  155

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Contents

9.2 9.3 9.4

Polyphenols: Structure and Activity  157 Sources of Polyphenols  159 Dietary Intakes and Bioavailability of Polyphenols  160 9.5 Roles of Polyphenols in Platelet Function  161 9.6 Conclusions  167 References  168 10

Effects of Ginkgo Biloba, Ginseng, Green Tea, and Dark Chocolate on Human Blood Platelet Function  171

Abbreviations Used in This Chapter  171 10.1 Introduction  171 10.2 Ginkgo Biloba Extract and Platelet Function  172 10.3 Clinical Trial with EGB761  175 10.4 Ginseng and Platelet Function  177 10.5 Green Tea (Camellia Sinensis) and its Effects on Platelet Function  181 10.6 Dark Chocolate and Platelet Function  183 10.7 Conclusions  185 References  187 Plant Alkaloids and Platelet Function  191 Abbreviations Used in This Chapter  191 11.1 Introduction  191 11.2 Alkaloids as Anti‐Platelet Agents  193 11.3 Mechanism of Actions of Alkaloids  197 11.4 Conclusions  198 References  199

11

Strawberries and Human Platelet Function  203 Abbreviations Used in This Chapter  203 12.1 Introduction  203 12.2 Polyphenols in Strawberries  204 12.3 Strawberry and its Cardio‐Protective Effects  206 12.4 Anti‐Platelet Factors in Strawberry  207 12.5 Discussion  209 References  211

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Contents

13 Effects of Metal Ions on Platelet Function  215 Abbreviations Used in This Chapter  215 13.1 Introduction  215 13.2 Zinc and Human Blood Platelet Function  216 13.3 Calcium and its Regulation of Platelet Function  218 13.4 Chromium and Platelet Function  221 13.5 Iron (Fe) and Platelet Function  221 13.6 Magnesium and Platelet Function  222 13.7 Platelet Function and Selenium  223 13.8 Conclusions  225 References  226 14

Individual Compounds with Anti‐Platelet Activity Isolated from Plant Sources  231

Abbreviations Used in This Chapter  231 14.1 Introduction  231 14.2 Effects of Taurine and Glycine on Human Platelets  233 14.3 Anthocyanins and Human Platelets  234 14.4 Coumarins and Their Anti‐Platelet Effects  235 14.5 Atractylenolides and Their Anti‐Platelet Effects  236 14.6 Flavonolignans and Blood Platelet Function  238 14.7 Protocatechuic Acid on Human Platelet Aggregation  238 14.8 KOK and Platelet Function  240 14.9 Inhibitors of Platelet Granules Secretion  241 14.10 Hydroxychavicol and Platelet Function  243 14.11 Compounds Isolated from Guttiferae Species with Anti‐Platelet Activity  243 14.12 Conclusions 244 References  244

Index  247

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1 Human Blood Platelets and Their Role in the Development of Cardiovascular Disease ­Abbreviations Used in This Chapter Cardiovascular disease, CVD; Glycoprotein IIb/IIIa, GPIIb/IIIa; von Willebrand factor, vWF; Tissue factor, TF; plasminogen activator inhibitor‐1, PAI‐1; cyclooxygenase, COX; TREM‐like transcript‐1, TLT‐1; P‐Selectin, CD62P; arachidonic acid,20: 4n‐6, ARA; CD40 ligand, CD40L; phospholipase C; PLC, Phosphatidylinositol‐4,5‐bisphosphate, PIP2; Inositol‐1,4,5‐­ trisphosphate, IP3; Purinergic receptor P2Y12, P2Y12

1.1 ­Introduction Human blood platelets are non‐nucleated cells, produced in bone marrow from megakaryocytes [1]. Although very dynamic, blood platelets (around 2μm in diameter) usually prefer to remain in inactive state in circulation, and get activated only when a blood vessel is damaged [1, 2]. The human body pro­ duces and removes 1011 platelets daily to maintain a normal steady state platelet count. Platelet production must be regu­ lated to avoid spontaneous bleeding or arterial occlusion and organ damage. The primary physiological role of platelets is to sense the damaged vessel endothelium and rapidly accumulate at the damaged site of the vessel, where they initiate blood coagulation process to stop the bleeding (Figure 1.1).

Nutraceuticals and Human Blood Platelet Function: Applications in Cardiovascular Health, First Edition. Asim K. Duttaroy © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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Nutraceuticals and Human Blood Platelet Function

Vessel before injury

Injured vessel

Hemostatic plug

Figure 1.1  The role of platelets in the blood vessel.

Circulating inactive platelets are biconvex but, upon activa­ tion, they become irregular and sticky, extending pseudopods and adhering to neighboring structures or aggregating with one another. The rapid interactions between activated platelets, their secreted components, or thrombin and endothelium at sites of damaged vessels, ensure the intravascular growth of the stable haemostatic plug [2]. Two different pathways mediate vascular homeostasis and thrombosis depending on vascular damage or vessel structure [1]. One is the intrinsic pathway mediated by collagen, while the other is the extrinsic pathway mediated by tissue factor(TF)‐fac­ tor‐VII complex. During normal hemostasis, damage to the endothelium may occur, and collagen from the sub‐endothelial space is exposed. Platelets, through their glycoproteins (GP) GPVI and GPIb/V/ IX, interact with collagen and von Willebrand factor (vWF). Collagen exposure leads to platelet adhesion and formation of a  platelet monolayer on the damaged surface of the vessel. Platelets form a three‐dimensional structure by aggregating through their activated GPIIb/IIIa complexes, the fibrinogen receptors. Activated platelets aggregate with other circulating platelets by secreting platelet aggregatory/activating agents,

Human Blood Platelets and Their Roles Collagen

Thrombin ADP

Adrenaline Arachidonic acid

Aspirin TxA2

GP IIb/IIIa

(Fibrinogen receptors)

Figure 1.2  Expression of fibrinogen receptors. Fibrinogen receptors (GPIIb/IIIa complex) on the platelet surface represents the final common pathway, whereby platelet stimulation by various agonists leads to fibrinogen binding, platelet aggregation, and thrombus formation. Aspirin can inhibit collagen, adrenaline and, to some extent, ADP‐induced expression of fibrinogen expression, but not the thrombin‐induced expression of GPIIb/IIIa complex on platelet surface.

such as thrombin, ADP, collagen, TxA2, and adrenaline. All these lead to the expression of fibrinogen receptors (GPIIb/IIIa ­complexes) [2]. Figure 1.2 shows the expression of platelet membrane surface GPIIb/IIIa complexes induced by different aggregating agents through TxA2 formation from ARA, liberated from membrane phospholipids. The tissue damage or plaque rupture leads to the release of TF from smooth muscle, adventitial cells, and peri­ cytes. TF, with the help of activated factor VII(VIIa), mediates the conversion of pro‐thrombin to thrombin, fibrin generation, and thus initiates the clotting cascade [1]. Activated platelets also accelerate the action of prothrombinase complex to pro­ duce thrombin from prothrombin. Apart from hemostasis, platelets are involved in several processes in the cardiovascular system, such as atherosclerosis process, immune system, inflammation, and cardiac events [3–5]. Thus, human blood platelets play many pivotal roles in the pathophysiology of different diseases, from CVDs to tumor metastasis [6].

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Nutraceuticals and Human Blood Platelet Function

Upon activation, platelets secrete more than 300 components from their intracellular stores. Platelet dense granule compo­ nents, such as ADP and polyphosphates, and Ca2+, contribute to blood coagulation and platelet aggregation. α‐Granules secrete multiple cytokines, mitogens, and other components that con­ tribute to the CVD processes. Anucleated platelets have stable mRNA transcripts with a long life, and use a variety of mecha­ nisms to translate these mRNAs into proteins [7]. There are two important key regulators of translation pro­ cesses such as ELF‐4e and ELF‐2a that are used by platelets. Platelets synthesize several proteins, such as integrins, αIIbβ3, TF, plasminogen activator inhibitor‐1 (PAI‐1), cyclooxygenase (COX), Factor XI, protein C inhibitor CCL5/ RANTES, and IL‐1B [7]. mRNA translation is also regulated by miRNA in human platelets. In fact, the possibility of using miRNAs as bio­ markers of atherosclerosis and cardiac episodes has been sug­ gested. In platelets constitutively are synthesized proteins such as actin, PDGF, glycoproteins GPIIb/IIIa, and P‐selectin [7]. Platelet hyperactivity, as occurs in obesity, smoking, sedentary life styles, and diabetes, insulin resistance is associated with secretion of different components, along with the shedding of membrane particles that play important roles in the develop­ ment of CVD risk, especially in the development of atheroscle­ rosis, blood flow, inflammation, and hypertension (Figure 1.3). Continued research has revealed that platelet micro‐particles have numerous functions. In addition to atherosclerosis, they are involved in thrombus and foam cells formation, and inflam­ mation. Platelet membrane proteins GPIbα, GPV, GPVI, amy­ loid βA4, TLT‐1 (TREM‐like transcript‐1), P‐selectin (CD40L), amyloid‐like protein 2 and semaphorin 4D are the most abun­ dantly shed platelet proteins. In this chapter, current understanding of human blood plate­ lets and their roles in the development of CVD is discussed.

1.2 ­Human Blood Platelets: Structure and Function Platelets have granular cytoplasm with no nucleus, and their diameter averages 2.5 µm, with a subpopulation of larger diameter 4–5 µm. Individual platelets, however, vary in terms of

Hemostatic and thrombosis Adhesion Activation Secretion Vasoconstriction Aggregation Tissue repair Clot dissolve

Hypertension Blood flow NO production Cytokine production Membrane particle shedding

Platelet function

Killing of bacteria Superoxide proudction Release of platelet compounds Complement system interaction Platelet-leukocytes interaction

Inflammation

Vascular tuning Uptake of serotonin Release of serotonin, Synthesis and release of TxA2, PGE1 Lipoxygenase products NO production Membrane particle shedding

Host defence

Tumor biology Tumor metastasis Tumor growth Tumor killing

Figure 1.3  The multiple roles of platelets in different diseases.

Atherosclerosis Membrane particle shedding Cytokine production Platelet-leukocytes interaction Conversion of monocytes to macrophages Foam cells generation

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Nutraceuticals and Human Blood Platelet Function

volume, density and reactivity towards agonists. The normal blood platelet count is in the range of 150–400 × 109/L. Under normal conditions, platelets circulate in the bloodstream for 8–10 days. Under conditions of hemostatic requirements, plate­ lets move from the spleen to the peripheral blood circulation (70% of total platelets). The normal peripheral blood platelet count is 150–400,000/μL [8]. This count represents only two‐thirds of available plate­ lets,  because the spleen retains the rest of the platelets. Megakaryocytes develop in the bone marrow from hematopoi­ etic stem cells [9]. The megakaryocyte undergoes endomito­ sis  as chromosomes duplicate, and a polyploid nucleus is synthesized. Within a five‐day maturation period, DNA amplification occurs while mitochondria, endoplasmic reticulum, Golgi appa­ ratus, membranous systems, and secretory granules develop [9]. Megakaryocytes mature into pro‐platelets, and subsequently fragment into platelets [10]. Organelles and granules are synthe­ sized in the megakaryocytes and then move towards the outer periphery using pseudopodia‐like extensions. As the megakary­ ocytes enter the circulation, the pseudopodia‐like extensions fragment, as a result of shear flow changes [10]. Fragmentation of megakaryocyte produces pseudopodia‐like extensions, with around 10–20 anuclear, dumbbell‐shaped pro‐platelets (diame­ ter of ≈ 2–3 µm). The pro‐platelet buds fragment again to form platelets. More than 120 proteins have been detected in platelets, including high concentrations of vWF, platelet factor 4, Factor V, Factor XIII, and plasminogen activator inhibitor‐1 (PAI‐1) [11]. These proteins are synthesized in the megakaryocytes, but are released when platelets become activated. In contrast, coagula­ tion proteins, such as fibrinogen, high molecular weight kinino­ gen, α2‐antiplasmin, and α2‐macroglobulin, are taken up from plasma by the blood platelets. Platelets contain two unique membrane systems – the dense tubular system and the open canalicular systems [10]. The dense tubular system is a closed membrane system that is the primary storage reservoir of intracellular Ca2+. The open canalicular sys­ tem is a continuous channel, connected to the outer membrane surface, which allows the secretory granules contents to exit

Human Blood Platelets and Their Roles

from the platelet during activation [12]. As the megakaryocyte reaches maturity, it enters the sinusoidal blood vessels of the bone marrow and migrates towards circulatory vessels [10]. A circumferential bundle of microtubules maintains the microtu­ bules, actin microfilaments, and intermediate microfilaments. These components regulate platelet shape change, extension of pseudopods, and secretion of granule contents. Platelets contain two types of secretory organelles – alpha and dense granules – which, together, store over 300 different pro­ teins and other small molecules [13] (Table 1.1). The alpha granules are the largest in size, at ≈ 200–500 nm, and are the most abundant at 50–80 per platelet cell. The gran­ ules contain proteins that participate in coagulation, aggrega­ tion and inhibition of coagulation. As the platelet becomes activated, alpha granule membranes fuse, and their contents exit to the nearby microenvironment, where they participate in platelet adhesion and aggregation and coagulation. The dense tubular system sequesters Ca2+, and contains proteins that acti­ vate platelets. These are phospholipase A2, COX‐1, and TxA2 synthetase responsible for liberation and metabolism of mem­ brane phospholipid arachidonic acid, 20:4n‐6 (ARA) to produce TxA2. A platelet has more than 50 different types of surface mem­ brane receptors. The predominant platelet membrane receptors are purinergic receptor (P2Y12), fibrinogen receptor (GPIIb/ IIIa), vWF receptor (GPIb/Factor V‐Factor IX), and collagen Table 1.1  Platelet granules contents released on activation. Alpha granules

Dense granules

Beta thromboglobulin

ADP

Platelet factor 4

ATP

Fibrinogen

Calcium ions

Adhesive glycoproteins

Serotonin (5‐HT)

P‐selectin Factor V Growth factors

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Nutraceuticals and Human Blood Platelet Function

receptors, GPIa/IIa, and GPVI. The dense granules are formed later than α‐granules during megakaryocyte differentiation. Small molecules are endocytosed, and are stored in the dense granules. Compared with alpha granules, the dense granules are less abundant, numbering 3–8 per platelet. Dense granules contain ions and signaling molecules such as ADP, ATP, serotonin, histamine, Ca2+, Mg2+, pyrophosphate, and polyphosphate. The dense granules are referred to as being electron‐rich, because they store ≈ 70% of the total platelet content of ions such as Ca2+, Mg2+, and polyphosphates [14]. In addition, transmembrane proteins, such as CD63, GP1b, and α2β3, are stored in the dense granules, and translocate to the membrane during platelet acti­ vation. Platelets contain lysosomes that participate in macro­ molecule degradation, mediated by acid hydrolases that can degrade proteins, carbohydrates and lipids. The platelet membrane phospholipids are asymmetrically arranged. The neutral phosphatidylcholine and sphingomyelin predominate in the plasma membrane layer, while the anionic phospholipids. phosphatidylinositol (PI), phosphatidylethanola­ mine (PE), and phosphatidylserine (PS) predominate in the inner cytoplasmic layer. These phospholipids, especially PI, sup­ port platelet activation by supplying ARA that is converted to different eicosanoids such as prostaglandins, lipoxins, and TxA2 during platelet activation [15, 16]. PS flips to the outer surface upon platelet activation, and on which coagulation factor complexes such as factors VIII‐IX and factors X‐V can assemble. The plasma membrane phospholip­ ids support platelet activation internally, and plasma coagula­ tion externally. Platelet plasma membrane lipid rafts are involved in signaling and intracellular trafficking. These membrane proteins include CD36, CD63, CD9, G protein‐coupled receptor (GPCR), GPIIb/IIIa, and GLUT‐3. Human platelets also express thrombin receptors, protease‐activated receptors‐1 and 4 (PAR1 and PAR4), and activation of either is sufficient enough to trig­ ger platelet activation secretion, and aggregation. Thrombin, generated by blood coagulation pathways (extrin­ sic and intrinsic pathways), is the most potent platelet aggregat­ ing agent, and also activates endothelial cells and other important responses in vascular biology. Thrombin and platelets play a

Human Blood Platelets and Their Roles

central role in CVD and other pathological processes. The thrombin surface receptors of platelets also trigger the release of  granules which play a role in multiple functions  –  namely, coagulation, inflammation, atherosclerosis, anti‐microbial host defense, angiogenesis, wound repair, and tumorigenesis. Among these surface receptors, GPCR has been reported to play a cru­ cial role in ADP secretion from dense granules. Asymmetrically arranged phospholipids (phosphatidylserine and phosphati­ dylinositol) present in the inner layer of the plasma membrane maintain the stability of platelet membrane surface during the inactive state. Platelet aggregation response can be analyzed using various assays and measures of platelet activation. Platelet aggregation remains the gold standard, but other testing methods offer advantages for specific applications, such as detecting overall platelet hyper‐reactivity in the presence of antiplatelet therapy, or detecting inhibition of the ADP receptor P2Y. Platelet behav­ ior is dependent on metabolic potential and, therefore, the secretory granules, mitochondria, and glycoprotein receptors. Light transmittance aggregometry, initially described by Born, can be used to measure platelet activity. This method was developed on the basis that plasma enriched with platelets (as a result of agonist‐induced platelet aggregation) has altered light transmittance. Light transmittance aggregometry is poten­ tially a good method for comparing the in vitro effects of differ­ ent pharmacological agents (such as aspirin and clopidogrel). However, this method is limited in the ability to predict in vivo platelet activity in the pathological state, probably because a sin­ gle agonist used in the test cannot reflect the complexity of pathophysiological signaling. The PFA‐100 (Platelet Function Assay https://en.wikipedia. org/wiki/PFA‐100 ‐ cite_note‐PFA‐1) is a platelet function ana­ lyzer. The membrane of the cartridges are coated with collagen and ADP, or collagen and epinephrine, inducing a platelet plug to form, which closes the aperture. The PFA test result is dependent on platelet function, plasma vWF level, platelet number. The normal value range is 84–160 seconds. The CT above 160 seconds suggests possible PLT hemostatic dysfunc­ tion in vivo. Standard testing protocols for platelet aggregation are needed to achieve consistency among studies.

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Nutraceuticals and Human Blood Platelet Function

1.3 ­Platelet Activation Pathways The hemostatic roles of platelets are essential for the mainte­ nance of the integrity of the vascular network. Platelet adhe­ sion, aggregation, and secretion are the processes involved in platelet activation, and these processes often occur simultane­ ously. Damaged endothelial exposes the sub‐endothelial colla­ gen that binds and activates blood platelets. Upon stimulation of platelets, granules release their contents into the extracel­ lular environment, contributing to further platelet activation and thrombus formation [18]. Several intracellular signal­ ing pathways involving collagen, thrombin, TxA2 and ADP are involved. The initial docking of platelets at sites of vascular injury is mediated by GPIb/V/IX, a structurally unique receptor complex expressed in megakaryocytes and platelets[19]. vWF is the major ligand for one component of this complex, GPIb, and the absence of vWF causes defects in primary hemostasis and coag­ ulation. Besides GPIb, several collagen receptors with a tether­ ing function are found on the platelet surface – notably GPVI and GPIa, members of the immunoglobulin superfamily [19]. After the initial adhesion of platelets to the damaged vessels, the repair process requires a rapid response to autocrine and paracrine mediators, including ADP, thrombin, epinephrine, and TxA2 [20]. These mediators amplify and sustain the initial platelet aggregation response. They recruit additional circulat­ ing platelets from the flowing blood, to form a growing hemo­ static plug. Most agonists that activate platelets operate through G‐protein‐coupled receptors [20]. The final pathway for all ago­ nists is the activation of the platelet integrins GPIIb/IIIa (αIIbβ3), the fibrinogen receptor, the main receptor for platelet adhesion and aggregation (Figure 1.4). Usually, platelet activation begins with the activation of one of the phospholipase C (PLC) isoforms expressed in platelets. PLC cleaves the phosphatidylinositol‐4,5‐bisphosphate (PIP2) to pro­ duce inositol‐1,4,5‐trisphosphate (IP3), the second messenger that raises the cytosolic Ca2+ concentration [21]. The raised level of Ca2+ activates integrin and, thus, activation of platelets starts. Different isoforms of PLC are activated by different agonists. Collagen activates PLCγ2 using a mechanism that depends on

Human Blood Platelets and Their Roles COLLAGEN THROMBIN

ADP

GpIIb/IIIa b

pl

G

Adrenaline Platelet

Aggregation Adhesion

vWF

Endothelium Exposed collagen

Figure 1.4  Platelet activation pathways. All the signaling events converge upon the final common pathway of platelet activation, the functional upregulation of integrin adhesion receptors. The most important is the activation of the GPIIb/IIIa receptor, which results in the cross‐linking of fibrinogen or vWF between receptors, leading to platelet aggregation. This further promotes the recruitment of additional platelets to the site of vascular injury, allowing subsequent thrombus formation. Thrombin is the most potent platelet agonist, and is also responsible for converting fibrinogen into fibrin to stabilize the platelet plugs.

scaffold molecules and protein tyrosine kinases, whereas throm­ bin, ADP and TxA2 activate PLCβ using Gq as an intermediary. The rise in the cytosolic Ca2+ concentration that is triggered by most platelet agonists is essential for platelet activation. In rest­ ing platelets, the cytosolic free Ca2+ concentration is maintained at approximately 0.1 μM, by limiting Ca2+ influx and pumping Ca2+ out of the cytosol, either out across the plasma membrane or into the dense tubular system. In activated platelets, the Ca2+ concentration rises tenfold to >1 μM, as Ca2+ pours back into the cytosol from two sources. The first is IP3‐mediated release of Ca2+ from the platelet dense tubular system. The second is Ca2+ influx across the platelet plasma membrane, an event triggered when depletion of the dense tubular system Ca2+ pool produces a conformational change in Stored Ca2+ Depletion‐induced Oligomerization of Stromal Interaction Molecule 1 (STIM1), a protein located in the dense tubular system membrane. Ultimately, it is the binding of fibrinogen or another bivalent ligand to αIIbβ3 that enables platelets to stick to each other.

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Nutraceuticals and Human Blood Platelet Function

Proteins that can substitute for fibrinogen include fibrin, vWF and fibronectin. Average expression levels of αIIbβ3 range from approximately 50 000 per cell on a resting platelet, to 80 000 on an activated platelet. Fibrinogen plays an important role in maintaining the stability of a thrombus, by bridging GPIIb/IIIa integrins between platelets. Quiescent platelets contain the pre‐ mRNA of the molecule termed TF, the primary initiator of the coagulation cascade that leads to the conversion of prothrombin to thrombin and fibrinogen to fibrin. Mutations in αIIbβ3 that suppress its expression or function produce a bleeding disorder (Glanzmann’s thrombasthenia), because platelets are unable to form stable aggregates.

1.4 ­Platelets and Vessel Wall Interactions The vascular endothelium controls platelet reactivity via differ­ ent mechanisms, such as ARA‐PGI2 (prostacyclin) pathway, the L‐arginine‐nitric oxide (NO) pathway, and the endothelial ecto‐ adenosine diphosphatase (ecto‐ADPase) pathway. Endothelial cells metabolize ARA into PGI2 with COX‐1 or COX‐2, with the involvement of PGI2 synthase. COX‐2 appears to be impor­ tant in prostacyclin synthesis by endothelium, on the basis of the effects of selective COX‐2 inhibitors on the excretion of PGI2 metabolites. PGI2 inhibits platelet function by elevating intracellular cyclic AMP levels via G protein‐linked receptor PGI2 [22]. PGE1 and PGI2 share the same receptor on platelet membrane [22]. NO produced by the L‐arginine‐NO pathway can stimulate the production of cyclic GMP in platelets, and regulates cyclic GMP‐dependent protein kinases, causing a secondary decrease in intracellular Ca2+ flux. This lowering of intracellular Ca2+ levels suppresses the conformational change in GPIIb/IIIa that is required for binding of the integrins to fibrinogen, thereby decreasing the number and affinity of fibrinogen binding sites on the platelet’s surface. Ecto‐ADPase, an integral component of the endothelial‐cell surface, limits the plasma level of ADP, ATP. The activity of this enzyme abrogates the critical recruit­ ment phase of platelet reactivity, as the availability of nucleo­ tides in the near environment is reduced.

Human Blood Platelets and Their Roles

1.5 ­Roles of Platelets in Atherosclerosis and Inflammatory Processes The important role of platelets in atherosclerosis development in humans has emerged. Several platelet‐derived chemokines and  growth factors are detectable in atherosclerotic plaques. Moreover, platelet activation and shedding of membrane parti­ cles is associated with increased wall thickness of the carotid artery. Persistent platelet hyperactivity, as reflected by enhanced excretion of thromboxane metabolites and other secretory com­ ponents, is associated with CVD risk factors. Different pathways are responsible for contribution of plate­ lets to atherogenesis, reduced blood flow, and hypertension, such as shedding of membrane particles, cytokines, and growth factors activating blood vessels. vWF mediates the recruitment of platelets at the site of vascular injury, and is also a determi­ nant of atherosclerotic plaque development. COX‐1‐dependent thromboxane synthesis has been demonstrated to accelerate atherogenesis in animal models, suggesting that platelet activa­ tion increases the rate of plaque formation [22, 24]. P‐selectin (CD62P) of platelets also stimulates monocytes and macrophages to release chemokines, and promotes the forma­ tion of platelet‐monocyte aggregates. A significant association has been reported between platelet hyperactivity and carotid artery wall thickness in diabetes and in hypertension [25]. The correlation between platelet reactivity and the extension of coronary atherosclerosis was observed in a study of more than 300 patients. Patients with more extensive coronary atherosclerosis have hyperactivity of platelets. Platelets adhere to the endothelium of carotid arteries in apolipoprotein E (apoE)−/− mice before atherosclerotic lesions are visible. vWF, when secreted in large amounts by endothelial cells in response to inflammatory stimuli, can recruit platelets to the site; the interaction between GPIb and vWF allows platelets to roll on endothelial cells. The acceleration of atherogenesis by COX‐1–dependent thromboxane in LDL receptor−/− mice suggests that platelet activation increases the rate of plaque formation. The inhibi­ tion of the synthesis of platelet thromboxane, as well as the antagonism or deletion of the thromboxane receptor, delays

13

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Nutraceuticals and Human Blood Platelet Function

atherogenesis in murine models. Activated platelets can also influence the progression of plaque formation by releasing adhesive ligands, such as P‐selectin expressed on the platelet membrane and mediate platelet‐endothelium interactions. P‐ selectin can stimulate monocytes and macrophages to produce chemoattractants or growth factors. Moreover, engagement by P‐selectin of the P‐selectin glycoprotein ligand 1 on the mono­ cyte surface initiates the formation of platelet‐monocyte aggre­ gates and outside‐in signaling, which induces the transcription of COX‐2. Prolonged adhesion‐dependent signaling promotes the expression of interleukin‐1β. This cytokine enhances the stability of COX‐2 mRNA, thereby promoting synthesis of the enzyme. All these factors render platelets to contribute to atherosclerosis. Platelets are increasingly recognized to be involved in the fields of wound healing, immune, and inflammatory process. At sites of injuries or infections, platelets are the first cells to be recruited to the vascular endothelium. There, platelets interact with vari­ ous cell types, including monocytes, neutrophils, and endothe­ lial cells and, thereby, regulate cellular adhesion and extravasation. Platelets are known to induce to pro‐ and anti‐inflammatory phenotypes, depending on the underlying pathology, site of inflammation, and experimental model employed [26]. Thrombin‐activated platelets bind monocytes, inducing the production of pro‐inflammatory cytokines. Depending on the experimental setting, membrane micro‐particles released from platelets can either enhance the pro‐inflammatory effects of macrophages, or inhibit pro‐inflammatory cytokine/chemokine secretion. Platelet granules contain a wide variety of signaling factors, such as chemokines, serotonin, histamine, nucleotides, and proteases involved in inflammatory processes. In addition, platelet eicosanoids, such as TxA2 or PGE2, can modulate inflammatory responses. Activated platelets enhance IL‐10 secretion and reduce TNFα secretion by monocytes, in order to counteract exaggerated pro‐inflammatory immune responses. Activated platelets alter the chemotactic and adhesive properties of endothelial cells by releasing these inflammatory molecules. These platelet‐induced alterations of endothelial‐ cell function can modulate the chemotaxis, adhesion, and

Human Blood Platelets and Their Roles

transmigration of monocytes to the site of inflammation. CD40 ligand (CD40L) released from platelets induces inflam­ matory responses in the endothelium. This ligand, originally identified on activated T cells, is a trimeric transmembrane protein in the TNF family. CD40L is stored in the cytoplasm of resting platelets, and rapidly appears on the surface after platelet activation. On the platelet membrane, the CD40L undergoes cleavage over a period of minutes or hours, gener­ ating a functional soluble fragment. Platelet‐derived CD40L induces production and release of pro‐inflammatory cytokines from vascular cells in the ather­ oma, to stabilize platelet‐rich thrombi and to inhibit the re‐endothelization of damaged vessels. CD40L can induce endothelial cells to produce reactive oxygen species, adhesion molecules, chemokines, and TF, all of which components con­ tribute to inflammatory and atherosclerosis processes. Blockade of the CD40L signaling pathway markedly inhibits the formation of atherosclerotic plaque and arterial lipid deposi­ tion in LDL‐receptor−/− mice. A prospective study of healthy women found that high plasma levels of soluble CD40L are associated with an increased risk of CVD events. Moreover, sev­ eral CVD risk factors, including cigarette smoking and diabetes, are associated with platelet activation and increased release of the CD4L. The combination of hyperinsulinemia and hypergly­ cemia upregulates the release of platelet CD40L and monocyte‐ derived tissue factor. In contrast to CD40L, which is stored in the platelet cyto­ plasm, interleukin‐1β is synthesized upon platelet activation. The amount of interleukin‐1β that activated platelets synthesize is enough to induce endothelial cells to express genes responsi­ ble for adhesion of leukocytes. Interleukin‐1β increases the release of chemokines and upregulates molecules that promote adhesion of neutrophils and monocytes to the endothelium. At sites of vascular injury, the expression of P‐selectin by acti­ vated endothelial cells or platelets can trigger the recruitment of micro‐particles bearing the P‐selectin glycoprotein ligand 1 and tissue factor. Micro‐particles have also been implicated in the upregulation of COX2‐mediated prostaglandins formation in monocytes and endothelial cells, and recruitment of monocytes,

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Nutraceuticals and Human Blood Platelet Function

or promoting their differentiation into macrophages. Platelet factor 4, a platelet‐specific chemokine released upon platelet activation, induces the expression of E‐selectin on endothelial cells. Activated platelets also release the matrix‐degrading enzymes matrix metalloproteinases‐2 and ‐9. All of these con­ tribute to the atherosclerosis process. The metabolism of ARA by the COX‐1 pathway contributes to the production of reactive oxygen species (ROS) by activated platelets. Agonists that induce platelet activation also activate the platelet isoform of NADPH oxidase. The production of O2– by platelets enhances the recruitment of platelets to the growing thrombus. The scavenging of NO by reactive oxygen species prevents its participation in the late disaggregation of a thrombus. Moreover, the accelerated removal of NO can occur through its reaction with COX‐1‐derived products. The increased generation of reactive oxygen species can induce enhance lipid peroxidation of cell‐membrane phospholipids or circulating LDL, leading to the increased generation of F2‐isoprostanes, a family of prostaglan­ din isomers produced from ARA. F2‐isoprostanes can modulate the adhesive reactions and activation of platelets induced by low levels of other agonists. The consistent relationship between the rates of formation of F2‐isoprostanes and thromboxane suggests that a low‐grade inflammatory state associated thromboxane‐dependent plate­ let activation. When activated, platelets alter the chemotactic properties of endothelial cells by inducing the secretion of monocyte chemoattractant protein. Similarly, transforming growth factor‐β released from activated platelet alpha granules augments the release of type‐1 plasminogen activator inhibitor from adipose tissue. Importantly, a recent report indicates that the recruitment of inflammatory cells in adipose tissue is facilitated by platelet adhesion along activated endothelium. Based on these obser­ vations, it seems that platelet activation, secondary to obesity, plays a causal role in triggering the pro‐inflammatory and pro‐ thrombotic state of obesity, creating a feedback loop involving adipose tissue, activated platelets and vascular endothelium that culminates in an environment favorable for atherothrom­ botic vascular events [27, 28]. Therefore, platelet activation

Human Blood Platelets and Their Roles

contributes to inflammatory, atherosclerosis, and thrombotic consequences.

1.6 ­Platelets and Their Role in the Development of Cardiovascular Disease Platelet activity is thought to play a major role in the devel­ opment, as well as the stability, of atherosclerotic plaques. Abnormal activation of blood platelets can represent a contribu­ tory risk factor for accelerated CVD, which occurs in hyperten­ sion, obesity, in smokers, and sedentary life style. Platelets thus play an important role in the development and progression of atherosclerosis [29]. In fact the pathophysiologic state of plate­ lets is the important underlying risk in diabetes, smoking, obe­ sity, and sedentary life style and other conditions (Table 1.2). Although the mechanism of the increase in platelet reactivity is uncertain in these conditions, it could be caused by sensitization of platelets to aggregation by elevated levels of agonists in vivo [30], or it could be due to the redistribution of young, more reactive plate­ lets that are concentrated in the spleen [31] and are released into the systemic circulation. Platelets are activated by a large number of agonists that are released in the circulation during some patho­ logic conditions, such as hypertension and diabetes mellitus [32]. Table 1.2  Hyperactive platelets associated with disease. Conditions that lead to hyperactive platelets:

Diabetes mellitus

Oxidative stress, inflammation and hyperlipidemia

Insulin resistance

Drugs, contraceptives

Obesity

Cancers

Ageing

Hypertension

Over‐nutrition, bad diets

Sedentary lifestyles

Platelets become hyperactive, or produce circulating micro‐aggregates, in the clinically defined conditions shown.

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Nutraceuticals and Human Blood Platelet Function

Several lines of evidence point to an important role for ­ latelets in the pathogenesis of sudden death, acute myocardial p infarction, and unstable angina [33]. In support of the ­pathophysiological role of platelets, platelet‐inhibitory drugs such as aspirin (an inhibitor of platelet cyclooxygenase‐1) have  been observed to reduce the incidence of myocardial infraction, stroke and death from CVD in secondary prevention trials [29, 34]. Recently, aspirin’s anti‐platelet limitations have progressively underscored the critical need for improved platelet aggregation inhibitor therapy which is not only effective but also safe and well‐tolerated [34–36]. Moreover, considering its side‐effects, use of aspirin as a primary preventive measure has been dis­ couraged. This underscores the need for a new regime for the primary prevention of platelet hyperactivity. Development of atherosclerosis is a gradual process, under­ stood to be influenced by well‐established traditional risk factors, including hypertension, cigarette smoking, diabetes mellitus, and dyslipidemia, where platelet hyperactivity is the major clinical feature. Activated platelets release different growth factors (e.g., PDGF and VEGF), membrane particles, and cytokines that participate in the development of atheroscle­ rosis by promoting vascular smooth muscle cells proliferation [37]. Stimulated platelets release VEGF [38], and elevated VEGF levels have been found in patients with atherosclerotic risk ­factors such as hypertension [39]. Platelet functional testing is being used to investigate a possible role of platelet hyper‐reactivity in the pathogenesis of vascular disorders and their complications. Platelet activation is  also involved in the development of hypertension in differ­ ent ways. Activated platelets release different mediators, such as 5‐hydroxytryptamine (5‐HT or serotonin), ADP, ATP and lysophosphatidic acid [37]. A number of these agents enhance the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC), which promotes vasoconstriction and increases catecholamines response. Furthermore, the number of platelet α‐adrenergic receptors increases in hypertensive persons [40], which may promote catecholamines responses. Catecholamines, β‐adrenoceptor agonist isoprenaline and an­­ giotensin II increase [Ca2+]i and promote contraction of VSMC,

Human Blood Platelets and Their Roles

platelet activation and aggregation [40], which may participate in  the development of hypertension. Moreover, angiotensin  II increases [Ca2+]i and pH in platelets, which may be associated with enhanced platelet aggregation [41]. In hypertension, plate­ lets showed spontaneous aggregation and increased sensitivity to agonists [42, 43]. Furthermore, platelets release more β‐thrombo­ globulin and P‐selectin, and have higher intracellular Ca2+ levels [44, 45]. Hypertensive platelets produce more reactive oxygen species, which enhance platelet activity by reducing the bioavail­ ability of NO and enhancing [Ca2+]i [41]. Platelets are also influenced by specific adipokines. Thus, in obesity, increased platelet aggregation, elevations in surface expression of markers of platelet activation, and heightened platelet micro‐particle formation, are observed. Activated plate­ lets alter the chemotactic properties of endothelial cells by inducing the secretion of monocyte chemoattractant proteins. Similarly, transforming growth factor‐β is released from acti­ vated platelet α‐granules, and has been shown to augment the release of type‐1 plasminogen activator inhibitor from adipose. Therefore, platelet activation contributes to the inflammatory and thrombotic consequences of obesity, and the importance of taming platelets in order to avoid CVD in obesity. CVD risk factors are associated with platelet hyperactivity, such as diabetes mellitus, obesity, hypertension, smoking, stress, sedentary life styles, and hypercholesterolemia [46]. Further­ more, excessive platelet activation is also attributed to the high mechanical shear forces in the circulation, reduced blood flow and vascular damage, which is observed in patients with hyper­ tension and diabetes [47]. Indeed, following blood vessel injury and/or atherosclerotic plaque erosion, platelets adhere by their surface receptors to the subendothelial matrix, which triggers their activation and subsequent aggregation [1, 48]. Activated platelets release prothrombotic mediators retained within their granules, such as ADP, serotonin, P‐selectin, fibrinogen, Ca2+, and TxA2, which further amplify platelet activation and throm­ bus formation [48]. The presence of a thrombus in an artery providing blood to a vital organ, such as heart or brain, is the most common cause of acute coronary disorders, including myocardial infarction and angina. Thus, the prevention of arterial thrombotic diseases has

19

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Nutraceuticals and Human Blood Platelet Function

a high priority. In this sense, the i­nhibition of platelet aggrega­ tion can provide protection against CVD that affect millions of people worldwide. A series of antiplatelet drugs have been used clinically to treat and prevent coronary syndromes and stroke, such as aspirin, clopidogrel, prasugrel, ticlopidine, dipyrida­ mole, and glycoprotein IIb‐IIIa antagonists (abciximab, eptifiba­ tide, and tirofiban) [49]. However, their actions are limited [50], and they are accompanied by a variety of side‐effects, such as hemorrhage, gastric ulcers, thrombocytopenia, increased risk for recurrent cardiovascular events, and therapeutic resistance [51, 52]. These drugs are mainly used as secondary prevention, not as primary preventive agents. In this sense, the search for more potent and safer antiplatelet agents as primary prevention has continued worldwide. The importance of the role of thromboxane A2 and ADP in amplifying platelet activation during hemostasis is supported by the twofold increase in the incidence of major bleeding compli­ cations (mostly in the upper gastrointestinal tract), associated with the use of low‐dose aspirin or the thienopyridines ticlopi­ dine and clopidogrel. The clinical relevance of adhesive interac­ tions with platelet GPIIb/IIIa in primary hemostasis is known largely from the study of Glanzmann’s thrombasthenia and the association of bleeding complications with the use of pharmaco­ logic blockers of GPIIb/IIIa. The impairment of primary hemostasis by antiplatelet drugs cannot be dissociated from their effects in the prevention of arterial thrombosis, which suggests that similar molecular mechanisms contribute to both processes. However, the tran­ sient incomplete blockade of platelet COX‐1 and of GPIIb/IIIa by some traditional non‐steroidal anti‐inflammatory drugs and oral inhibitors of GPIIb/IIIa, respectively, has been associated with an increased risk of bleeding and a lack of antithrombotic efficacy. This suggests that the extent and duration of platelet inhibition required to impair hemostasis may differ from that required to prevent atherothrombosis. The ex vivo measurement of platelet responses to various agonists provides an index of the functional capacity of plate­ lets, but such measurements by no means reflect the extent of platelet activation in vivo. The maximum capacity of platelets

Human Blood Platelets and Their Roles

to ­synthesize TxA2 in vitro is approximately 5000 times the basal rate of thromboxane biosynthesis in vivo, and only a frac­ tion of this biosynthetic capacity appears to contribute to plate­ let activation, as reflected by excretion of thromboxane metabolites. Platelet activation is one of the essential steps in the genesis and propagation of atherothrombosis. Accumulating clinical evidence suggests that an elevated platelet count, plate­ let activation, and platelet hyperactivity are associated with adverse cardiovascular events in patients with acute coronary syndromes. Several lines of evidences revealed that high consumption of fruit and vegetables may prevent CVD [53–55] by inhibiting platelet function. Recently, different studies around the world have been focused on medicinal plants with antiplatelet activ­ ity. However, it was reported that platelet hyperactivity, which is associated to different pathological situations, including hypertension, diabetes mellitus, and vascular diseases [46, 56], plays an important role in the development of thrombosis, and the incidence of cardiovascular disorders [32]. Accordingly, it has been reported that, in patients with diabetes and hyperten­ sion, platelets are more sensitive to agonists, and showed increased spontaneous platelet aggregation [57, 58], which pro­ motes thrombus formation. Furthermore, several studies suggest an important role for platelets in inflammation and atherosclerosis, not only by thrombus formation, but also as inducers of inflammation. Indeed, a range of molecules present on the platelet surface and/ or stored in platelet granules contributes to the cross‐talk of platelets with other inflammatory cells during the vascular inflammation involved in the development and progression of atherosclerosis. The roles of aspirin in prevention of CVD has been exten­ sively investigated. In primary prevention, aspirin reduces risks of first MI, but the evidence on stroke and CVD death remain inconclusive. In a recent study, primary prevention of CVD with low doses of aspirin in diabetic patients was found to be less efficient, compared with subjects with other CVD risk factors. Possible explanations for reduced aspirin efficacy in diabetic disease include increased platelet turnover (increased production

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Nutraceuticals and Human Blood Platelet Function

of platelets with non‐acetylated COX and/or expression of COX‐2 originating from megakaryocytes), upregulated i­nflammatory activity (inflammation‐induced COX‐2 expression in monocytes‐ macrophages as an additional source of endoperoxides and sub­ sequently TxA2 formation), and hyperglycemia (leading to glycosylated platelet membranes and/or increased production of thromboxane bypassing the COX step). Stress, hypercholester­ olemia and hypertension may also contribute to ‘aspirin resist­ ance’. Therefore, a better alternative to aspirin, in terms of safety and efficacy, is being investigated, so that it can be used as pri­ mary prevention.

1.7 ­Conclusions Platelets have emerged as a key cellular determinant of physio­ logic vascular integrity and repair, and its pathologic derange­ ment and CVD. Blood platelets act not only through the immediate release of a variety of lipids, ions, ADP and protein mediators, but also through previously unrecognized time‐ dependent events, such as signal‐dependent pre‐mRNA splic­ ing, and the translation of constitutively expressed mRNA. The relationship of platelet pathology to CVD is established now. New approaches are, however, required to bridge the gap between the large body of evidence supporting a role of platelets in the initiation and progression of atherogenesis, and the rela­ tively modest evidence for a role of platelets in the disease in humans. Studies of the use of new antiplatelet agents to reduce the risk for accelerated atherogenesis are needed. Given the growing concern over the CVD consequences of obesity, diabe­ tes, sedentary life styles, and smoking, many platelet inhibitors from dietary source or functional foods may constitute a suita­ ble primary prevention regime.

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Human Blood Platelets and Their Roles

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49 Park JY, Hong M, Jia Q, et al. (2012). Pistacia chinensis

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2 Epidemiology of Cardiovascular Disease ­Abbreviations Used in This Chapter Cardiovascular disease, CVD; Endoperoxide prostaglandin G2, PGG2; Polyunsaturated fatty acids, PUFA; glycoprotein, GP; particulate matter, PM; Diesel exhaust, DEP

2.1 ­Introduction Cardiovascular disease (CVD) is the largest single contributor to global mortality [1]. CVD data indicate that poor diet, physi­ cal inactivity, environmental pollution and social factors are the major contributors to the recent increase in the incidence of CVD worldwide [1]. Epidemiological evidence suggests that the increasing consumption of energy‐dense diets high in unhealthy fats, sodium, and sugars have contributed to an increase in CVD in many countries. The CVD risk factors that can be modified are life style, and also physical factors such as tobacco exposure, high blood pres­ sure, obesity, sedentary life styles, unhealthy diet, and harmful use of alcohol and tobacco. Abnormal blood lipid levels, such as high total cholesterol, high levels of triglycerides, free fatty acids, high levels of low‐density lipoprotein (LDL) or low levels of high‐density lipoprotein (HDL) cholesterol all increase the risk of CVD [2]. There are other many risk factors associated with CVD [3], such as impaired growth during fetal life, which also impacts the Nutraceuticals and Human Blood Platelet Function: Applications in Cardiovascular Health, First Edition. Asim K. Duttaroy © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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cardiovascular health of the next generation, implicating epige­ netic processes in the etiology of CVD that have their origins during fetal life and early development. Under all these condi­ tions, human blood platelets become hyperactive [4–7]. In fact, one of the main factors for CVD is blood platelet hyperactivity, which induces the alteration of blood platelet functions in the circulation. Hyperactive platelets can contribute to the develop­ ment of CVD risk factors, such as atherosclerosis, hypertension, vascular damage and inflammation. The main physiological function of blood platelets is to maintain hemostasis by the ini­ tiation and formation of a hemostatic plug, and by the secretion of various biologically active factors leading to the repair of vas­ cular injuries (Figure 2.1). Platelets’ secretion of several active molecules such as cy­­ tokines, ADP, growth factors, angiogenic factors, and shedding of platelet membrane particles, initiate vascular pathology. There is strong evidence for platelet hyperactivity in conditions such as diabetes, smoking, sedentary life styles, ageing, obesity and consumption of bad diet [7]. The platelet size distribution (mean platelet volume, MPV), altered in many diseases, corre­ lates positively with expression of platelet surface glycoprotein receptors (GPIb and GPIIb/IIIa), the thromboxane synthesizing capacity and protein contents in platelet granules.

Hemostasis: roles of platelets Platelets

Vessels

Vessels

Coagulation

Thrombosis

Fibrinolysis

Body

Bleeding

Figure 2.1  The haemostatic system: roles of human platelets.

Epidemiology of Cardiovascular Disease

Recent studies using modern techniques have confirmed the increased surface expression of activation markers of platelets in people with obesity, smokers, and diabetes. These active markers include several platelet membrane receptors responsi­ ble for platelet activation and aggregation, such as GPIIb/IIIa (fibrinogen receptor), GPIb‐IX (vWF receptor), GPIa/IIa (col­ lagen receptor) and CD62 (P‐selectin), and increased shedding of platelet membrane particles. Altered platelet membrane fluidity, results of membrane fatty acids compositional changes, or protein glycation also contribute to platelet hyperactivity. Changes in arachidonic acid, 20:4n‐6(ARA) metabolism, as observed in diabetes, smokers, and low intakes of n‐3 fatty acids, produces an increased TxA2 that contributes to platelet hyperactivity. During platelet activation, ARA is liberated from the platelet membrane phospholipids, and metabolized by COX‐1 to endop­ eroxides prostaglandin G2 (PGG2) and PGH2 [8]. PGH2 is then converted to TxA2, a potent platelet aggregating and vasocon­ stricting agent [8]. Isoprostane 8‐iso‐prostaglandin F2α (ARA peroxidation marker) is correlated with TxA2 biosynthesis, and may provide a link between glycaemic control, oxidative stress and platelet hyperactivity in diabetic patients. Diabetic platelets also have increased Ca2+‐ATPase activity, which results in ele­ vated intracellular Ca2+ levels and is responsible for platelet hyperactivity. CD40 ligand (CD40L), a member of the TNF‐family, is also expressed on activated platelets, indicating a link between hemo­ stasis and inflammation. CD40 ligand and its receptor CD40 have important roles in modulating immune responses and inflamma­ tion. CD40L is expressed in different cells present in or around atherosclerotic plaques, such as T cells, macrophages, smooth muscle cells, and endothelial cells. In addition, it is found on platelets, which are, on activation, the most important source of circulating, soluble CD40L. The endothelial cells synthesize both platelet‐inhibiting and vasodilating substances, NO and prostaglandin I2 (PGI2), or prostacyclin and platelet‐activating and constricting substances, such as endothelin‐1 and angiotensin II. Most of these com­ pounds are under the control of dietary intakes of nitrates and fatty acids so, therefore, diet may have an impact on platelet

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Nutraceuticals and Human Blood Platelet Function

function by producing an imbalance between these two groups of compounds with opposite effects on platelet function. There is growing interest in the possible beneficial effects of specific dietary components on cardiovascular health, par­ ticularly in the area of thrombosis and hemostasis, dyslipi­ demia, inflammatory diseases, obesity, insulin sensitivity, oxidative stress, and endothelial function. The impact of these dietary components on platelet function tests needs to be determined. Therefore, this chapter focuses on diet and nutri­ ents that have the potential to affect platelet function tests, particularly platelet aggregometry, flow cytometry, and the PFA‐100 assay. Diets and nutrients play a potential role in modifying CVD progression, particularly in platelet function, and have the potential of altering platelet function tests. Effects of different nutrients are discussed in elsewhere in this book. Diets such as a Mediterranean diet, high in n‐3 polyunsaturated fatty acids (PUFA), and vegetarian diets have inverse relationships with CVD. Dark chocolate, foods with low glycemic index, garlic, ginger, n‐3 PUFA, onion, purple grape juice, tomato, and wine all reduce platelet aggregation. This chapter briefly describes the epidemiology of diet and CVD with emphasis on platelets.

2.2 ­Dietary Lipids and Cardiovascular Disease The relationship between CVD and diet is extensively studied in epidemiology. Dietary lipids are thought to influence the development of CVD via a number of processes, such as the hemostatic system and platelet function. The amounts and composition of dietary fatty acids modulate the different limbs of the cardiovascular system, such as platelet function, fibrino­ lytic system, and coagulation cascade (Figure 2.2). Epidemiological data provide the convincing evidence that CVD risk reduction is associated with the lower consumption of saturated fat, low‐to‐moderate intake of alcohol and salt, and increasing the consumption of linoleic acid, 18:2n‐6, marine fish and marine fish oils, vegetables and fruits [9, 10].

Epidemiology of Cardiovascular Disease Amount of fat

Fatty acid composition

Dietary Lipids

Coagulation cascade

Fibrinolytic activity Platelet activity

Figure 2.2  Effects of composition and amounts of fatty acids on three limbs of hemostasis – platelet, blood coagulation and fibrinolysis.

The Mediterranean diet is characterized by: an abundance of fruits, vegetables, whole grain cereals, nuts, and legumes; olive oil as the principal source of fat; moderate consumption of fish; lower consumption of red meat; and moderate consumption of alcohol. Components present in these food items directly or indirectly influence different aspects of the cardiovascular system, including platelet function, atherosclerosis, LDL oxi­ dation, vascular function and inflammation. Vegetable oils are primarily comprised of oleic acid, 18:1n‐9 (OA), linoleic acid, 18:2n.6 (LA), and other saturated fatty acids. Among these fatty acids, monounsaturated fatty acids, LA acid containing oils, seem to lower the CVD risk. The n‐3 alpha‐lino­ lenic acid, 18:n‐3(ALA)‐containing oils, such as rapeseed or canola oil, are cardio‐protective. Replacing saturated fat with monounsaturated or polyunsaturated fat reduces low‐density lipoprotein cholesterol and preserves HDL cholesterol. Trans‐ fatty acids increase CVD risk, compared with other macronutri­ ents, with strong evidence of adverse effects of small amounts of trans‐fats on lipids and CVD risk. Saturated fats have not been consistently associated with CVD in meta‐analyses of cohort studies [11]. However, in most of these studies, the association of high saturated fat intake largely represents replacing highly refined carbohydrates. Replacing saturated fat with highly refined carbohydrate is not associated with reduced CVD risk, whereas replacing saturated fat with polyunsaturated fat reduces CVD risk [12, 13]. Emerging

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Nutraceuticals and Human Blood Platelet Function

evidence suggests that the effect of a saturated fat on CVD may depend on the type of fatty acid and the specific food source. Consumption of non‐hydrogenated vegetable oils appears to be superior to consumption of animal fats. Consumption of plant oils in a Mediterranean diet reduced CVD in two random control trials. The Lyon Diet Heart Study found regular consumption of ALA (canola oil) significantly reduced cardiac deaths and nonfatal CHD [14], while the PREDIMED RCT found that a Mediterranean diet (50 grams/ day extra virgin olive oil) reduced CVD events by 30% over a five‐year period. Palm oil is the dominant fat globally, and is relatively high in saturated fat. Based on controlled‐feeding studies examining changes in blood lipids, replacing palm oil with unsaturated fatty acids is expected to lower CVD risk [15], but palm oil would be preferred to partially hydrogenated oils high in trans‐fatty acids. Few studies have directly compared palm oil with other oils for CVD risk. One large case‐control study in Costa Rica found that soybean oil consumption was associated with lower acute MI risk, compared with a palm oil consumption‐based diet. Given that the components of the diet are sufficiently under­ stood, it is possible to test their effects on different pathways involved in platelet function, blood coagulation and inflamma­ tion. Despite significant advances in our understanding of opti­ mal dietary patterns to prevent CVD, additional research is required to find out how these dietary components affect differ­ ent aspects of CVD, such as platelet function, blood coagulation factors, vascular function, inflammation, and atherosclerosis. The dietary lipids affect platelet function, blood coagulation, and fibrinolysis, depending on their composition, triglyceride structure, and amount.

2.3 ­Environmental Factors and Cardiovascular Disease Over the past 20 years, there has been a growing body of evi­ dence linking air pollution to increased CVD incidence and mortality. Air pollution is composed of a mix of gaseous and particulate matter, and is created largely as a result of fossil fuel

Epidemiology of Cardiovascular Disease

combustion. Numerous epidemiological studies in both devel­ oped and developing regions of the world have found that both short‐term (several hours to a few days) and long‐term exposure to particulate matter air pollution significantly increases cardio­ vascular events and CVD related deaths [16. 17]. The association between air pollution and CVD is well estab­ lished, with exposure to traffic‐derived pollutants implicated in acute myocardial infarction. The adverse cardiovascular effects of airborne pollution have been attributed to combustion‐ derived particulate matter (PM), especially ultrafine particles from diesel exhaust (DEP), which have a large reactive surface area and can penetrate deep within the lung. Recent investiga­ tions suggest that DEP and ambient particulate matter increase cardiovascular events by accelerating platelet activation. Diesel exhaust inhalation in humans produces endothelial cell dysfunc­ tion 2–24 hours after exposure, and produces impaired endoge­ nous fibrinolysis, increased thrombogenicity, and platelet activation [18]. Several different mechanisms for enhanced coag­ ulation in response to particles have been implicated, including platelet activation. However, the mechanisms responsible for PM‐induced enhancement of thrombosis remain controversial.

2.4 ­Genetic Basis of Cardiovascular Disease Incidence Researchers have recognized for decades that family history of CVD is associated with increased atherosclerotic risk of CVD, which has led to the presumption of a genetic component to CVD. Large‐scale genome‐wide association studies have identi­ fied many single‐nucleotide polymorphisms (SNPs) and multi‐ SNP genetic risk scores that are independently associated with CVD events [19]. Recent guidelines and reviews do not address genetic risk factors, and few cost‐effectiveness or decision anal­ yses have been performed to quantify the potential benefits, in terms of clinical outcomes, that might result from use of genetic information for cardiovascular risk assessment. There are several well‐characterized single‐gene disorders that contribute to CVD, such as certain forms of familial

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Nutraceuticals and Human Blood Platelet Function

hypercholesterolemia linked to mutations of the apolipopro­ tein B  gene and, during the past few years, there have been major advances in the identification of genetic risk factors for CVD risk factors, such as blood pressure, blood lipids, obesity, and diabetes. The identification of genetic loci associated with CVD, such as 9p21 [20], has led to major advances in under­ standing the pathophysiology of CVD, but genetic variants identified to date have explained only a fraction of heritability, and do not appear to have substantial added value in predict­ ing CVD beyond traditional CVD risk factors. The prevailing view within the research community is that the genetic under­ pinnings of most common forms of CVD involve a complex interplay of many different genes, and much work remains to be done to develop a more thorough understanding of the complex gene‐gene and gene‐environment interactions involved in the development of CVD. Indeed, in addition to the investigation of genes that influence CVD and its risk factors, there has recently been a surge in research examining how environmental factors affect gene expression. Although research indicates that gene expression is most sensitive to environmental influence from conception to early life, there is also evidence that environmentally related gene expression changes can occur throughout life [21]. Future findings could have implications to help elucidate the physiolog­ ical processes by which individuals with similar CVD risk ­profiles have different outcomes. There are several genetic disorders whose relationship to platelet dysfunction have been established [22]. Over recent years, the pathogenesis of congenital platelet disorders has been better understood. The genes responsible for various congenital platelet diseases have been identified, and advances are being made in molecular characterization of these disorders. However, despite recent gains in knowledge, the underlying molecular mechanisms remain unknown in most patients with a congeni­ tal bleeding disorder and impairment of platelet function. The challenge for the future is to increase our understanding of congenital platelet disorders, in order to obtain effective and reliable strategies for the prevention, diagnosis and therapy of bleeding.

Epidemiology of Cardiovascular Disease

2.5 ­Fruits and Vegetables Consumption and Cardiovascular Disease Risk Reduction Consumption of sufficient amounts of fruit and vegetables is recommended as part of a healthy diet. Fruit and vegetables may reduce chronic diseases and, more specifically, CVD, by means of their protective constituents, such as potassium, folate, vita­ mins, fiber, and different phenolic compounds. These nutrients act through a variety of mechanisms, such as reducing antioxi­ dant stress, improving lipoprotein profile, lowering blood pres­ sure, increasing insulin sensitivity, and improving hemostasis regulation [23, 24]. However, the recommendation for con­ sumption of fruit and vegetables to prevent chronic diseases is mainly based on observational epidemiological studies, which leaves much uncertainty regarding the causal mechanism of this association. Several cohort studies have examined the relationship between fruit and vegetable intake and coronary heart disease. In gen­ eral, these studies report a favorable relation between fruit and vegetable consumption and CVD occurrence although, some­ times, the results are inconsistent. Furthermore, the magnitude of the favorable association remains uncertain, because of dif­ ferences in methodological approaches, analytical techniques, and outcome definitions. Several clinical and biological investigations support the ­protective effect of fruit and vegetables against CVD. Firstly, the relationship is biologically plausible, with abundant clinical and laboratory data demonstrating that the micro‐ and macro‐­ constituents of fruit and vegetables improve important risk factors of CVD, such as hypertension, dyslipidemia, and diabe­ tes mellitus. Secondly, the association persists after adjustment of these risk factors, suggesting a specific effect of fruit and vegetables. In contrast, other facts are not in favor of a causal relation. In population studies, fruit and vegetable intake correlates with healthy lifestyles, which may explain the lower CVD rates. High intakes of fruit and vegetables are associated with a pru­ dent diet pattern, and inversely related to the consumption of saturated fat‐rich food, which may also contribute to the lower

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Nutraceuticals and Human Blood Platelet Function

CVD risk. A beneficial association between fruit and vegetable consumption and CVD risk supports the recommendation to eat a sufficient amount of fruit and vegetables to lower CVD risk. Moreover, fruits and vegetables are rich sources of vita­ mins and minerals, dietary fiber, and a host of beneficial non‐ nutrient substances, including plant sterols, flavonoids and other antioxidants, so consuming a variety of fruits and vege­ tables helps to ensure an adequate intake of many of these essential nutrients. Several epidemiological studies have suggested protective effects of flavonoids against cardiovascular diseases [25]. These effects may be linked to their influence on platelets, as can be implied from the lower incidence of ischaemic stroke observed with increasing flavonoid consumption [25]. Although positive effects of flavonoids in experimental models of thrombosis have been documented in animal studies, clinical data are still miss­ ing, and the few available small human studies have reported no effect of flavonoids on platelet aggregation in healthy volunteers [26]. Epidemiological data are based on a food questionnaire, and such estimates of flavonoid consumption represent a very imprecise indicator of flavonoid plasma levels. There is high variability in pharmacokinetics and pharmacodynamic effects of flavonoids. Variation in response to the thromboxane recep­ tor agonists and inhibition of platelet aggregations has been reported. Nevertheless, various studies have reported that polyphenols present in red grape juice, purple grape juice and sea buckhorn juice inhibited platelet aggregation. The major berry phenolic compounds that were shown to have the greatest beneficial impact on CVD and platelet functions may be anthocyanidins, phenolic acids, flavonols and procyanidins. However, the exact mechanisms of their anti‐platelet activities are not well known. These compounds reduce platelet activity by changing the ROS level and modulating different signaling pathways, as well as eicosanoid pathways in platelets. Their possible effects on bio­ chemical processes in platelets and platelet response may be mediated via reduction of ROS and thromboxane synthesis. Recently, it was shown that the functional groups of phenolics identified as relevant for the potent inhibition of platelet signal­ ing include, at least, benzene rings.

Epidemiology of Cardiovascular Disease

Epidemiologic studies have focused on tomato and tomato products associated their intake with a reduced risk of CVD [27–29]. However, tomatoes and tomato‐based products are important dietary sources of lycopene in observational studies, and most human lycopene trials are performed using tomato‐ based interventions. Studies have shown that increased plasma lycopene levels were associated with reductions in CVD risk factors [30–32]. The strongest population‐based evidence for the beneficial effects of tomato lycopene came from a multi‐centre case‐con­ trol study (EURAMIC) [33]. However, a dietary intervention study observed that consumption of a carotenoid‐rich diet did not have an effect on plasma antioxidant status or markers of oxidative stress [34]. Several [32, 35], but not all [35–39] pro­ spective studies relating circulating lycopene concentrations and CVD risk have reported inverse associations, while studies based on dietary intake have generally failed to detect signifi­ cant independent associations between lycopene and CVD risk [39–43]. In fact, an inverse association was observed between the consumption of tomatoes and tomato products and CVD incidence, but found no significant association between lyco­ pene intake and CVD risk [40]. Thus, it is difficult to separate out the potential lycopene contribution to cardiovascular health from the overall contribution from tomato products and other components present in tomatoes. As described in an earlier section, tomatoes contain several known and unknown compounds that might affect platelet function, lipid metabolism, blood pressure, and endothelium function. Epidemiological data underscore the requirements of further studies to unravel other non‐lycopene components in tomato and their roles in CVD risk reduction. LDL oxidation is considered an important early event in the development of ath­ erosclerosis, although there are CVD risk factors, such as plate­ let hyperactivity, endothelium relaxation, and blood pressure. Plasma lipids and platelet hyperactivity play very important roles in the development of CVD. Platelet hyperactivity is associ­ ated with hypertension, hypercholesterolemia, insulin resist­ ance, smoking, obesity and diet, which are also major risk factors for CVD. Therefore, inhibition of only LDL oxidation by lyco­ pene may only partly contribute to the CVD risk reduction.

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Nutraceuticals and Human Blood Platelet Function

Moreover, this explains why, despite an inverse association found for high intakes of tomato‐based products, dietary lycopene is not strongly associated with the risk of CVD. This indicates that other, unidentified compounds (non‐lycopene compounds) may also contribute to the cardio‐protective effects of tomatoes, as observed in epidemiological and interventional studies. The water‐soluble non‐lycopene compounds in tomatoes have been shown to be a strong inhibitor of platelets [44, 45].

2.6 ­Conclusions Dietary factors play an important role in modifying the progres­ sion of CVD, particularly in blood platelet functions and lipo­ protein profiles, and endothelium function. Fruits and vegetables contain a range of bioactive compounds that may modulate platelet activation. These fruits and vegetables have several inhibitors of COX involved in TXA2 biosynthesis. In addition to the various health benefits associated with the consumption of several fruits and vegetables reported in epidemiological stud­ ies, a number of articles have found them to bestow beneficial effects on CVD. In vivo and in vitro studies have clearly indi­ cated that polyphenolic extracts from fruits and vegetables exert a plethora of effects on cellular functions, due to their strong anti‐inflammatory, anti‐oxidative, anti‐aggregatory, vasorelax­ ant, hypolipemic, and hypoglycemic properties. This chapter has described the epidemiological evidence for intervention with fruits, vegetables and n‐3 fatty acids to lower the future development of CVD. Saturated fats, red meat, tobacco use, and sedentary life styles affect platelet function, and are major contributors to the CVD epidemic. Therefore, prevention through promoting healthy diet and life style should remain one of the cornerstones of CVD reduction efforts.

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Epidemiology of Cardiovascular Disease

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Epidemiology of Cardiovascular Disease

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Epidemiology of Cardiovascular Disease

45 O’Kennedy N, Crosbie L, van Lieshout M, Broom JI, Webb DJ,

Duttaroy AK (2006). Effects of antiplatelet components of tomato extract on platelet function in vitro and ex vivo: a time‐course cannulation study in healthy humans. American Journal of Clinical Nutrition 84(3): 570–579.

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3 n‐3 Fatty Acids and Human Platelets ­Abbreviations Used in This Chapter Linoleic acid, 18:2n‐6, LA; α‐linolenic acid, 18:3n‐3, ALA; Long chain polyunsaturated fatty acids, LCPUFAs; Arachidonic acid, 20:4n‐6, ARA; Eicosapentaenoic acid, 22:5n‐3, EPA; Docosahex­ aenoic acid, 22:6n‐3, DHA; Prostacyclin, PGI2, Phosphoinositide 3 kinase, PI3K; Thromboxane A2, TxA2

3.1 ­Introduction Linoleic acid, 18:2n‐6 (LA), and α‐linolenic acid, 18:3n‐3 (ALA) are the two main dietary essential fatty acids (EFAs) that are readily available from the dietary sources such as vegetable oils, seeds, and dark green leaves [1]. These EFAs can be metabolized to their long chain polyunsaturated fatty acids (LCPUFAs) derivatives, and also can be obtained from sea foods and animals [1]. Both EFA and their LCPUFA derivatives are of critical importance in cell homeostasis, growth, and development [2]. Dietary LA and ALA must be converted in the body to their further metabolites, LCPUFAs, to exert the full range of biologic actions (Figure 3.1). Arachidonic acid, 20:4n‐6 (ARA) is a precursor for bioactive eicosanoids and leukotrienes [2]. Human blood platelet activation is accompanied by the synthesis and release of pro‐­aggregatory molecules, such as thromboxane A2 (TxA2 ) and other eicosanoids from ARA, which amplify platelet aggregation responses [3]. Nutraceuticals and Human Blood Platelet Function: Applications in Cardiovascular Health, First Edition. Asim K. Duttaroy © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

Seed oils Sunflower, Safflower, Peanut

N-6 fatty acids

N-3 fatty acids

Linoleic acid, 18:2n-6

Flaxseed oils Green leaves

α Linolenic acid, 18:3n-3

Delta 6 desaturase

Evening Primrose, Borage oil

γ Linolenic acid, 18:3n-6

Octadecatetraenoic acid, 18:4n-3

Elongase Dihomo γ Linolenic acid, 20:3n-6

Meat, Egg

Sea Foods, Meat, Egg

Delta 5 desaturase Eicosapentaenoic acid, 20:5n-3

Arachidonic acid, 20:4n-6 Prostaglandins-1 series leukotrienes-3 series

Eicosatetraenoic acid, 20:4n-3

Elongase Delta 6 desaturase Beta oxidation (peroxisome)

Docosahexaenoic acid, 22:6n-3 Prostaglandins-2 series Leukotrienes-4 series

Prostaglandins-3 series Leukotrienes-5 series

Figure 3.1  Classical n‐6 and n‐3 fatty acid LCPUFA synthesis pathways and their dietary sources. For details, please see the text.

n‐3 Fatty Acids and Human Platelets

To  serve as the substrate for synthesis of TxA2, ARA must be l­iberated from membrane phospholipids by phospholipases. The concerted action of collagen, ADP, and TxA2 activates specific signaling pathways, generating a number of second mes­ sengers, and leading to the functional expression of GPIIb/IIIa complex, the fibrinogen receptor on platelets. Pro‐hemostatic mechanisms are counterbalanced and regulated by a number of physiological anti‐hemostatic molecules that work in a con­ certed and redundant manner, including the release of prostacy­ clin (PGI2), nitric oxide (NO), and endothelium‐dependent hyperpolarizing factor by the endothelium [4]. The aggregation of platelets by agonists is mediated, in part, through the intracel­ lular formation of prostaglandin (PG) G2, PGH2, and TxA2 from ARA. In contrast, PGI2, an ARA metabolite of endothelial cells, is the most potent inhibitor of platelet aggregation [4]. An important mechanism of platelet aggregation is the synthesis of TXA2 from ARA via the cyclooxygenase‐mediated pathway. TXA2 is a potent platelet agonist [5]. Inhibition of TXA2 synthesis decreases ischemic events in clinical trials, suggesting an important role for TXA2 in the regulation of hemostasis and thrombosis [5]. LCPUFA provide adequate substrates for syn­ thesis of circulating vasoactive factors such as PGI2 (produced by endothelium) and TxA2 (produced by platelets). The ratio of TxA2 : PGI2 is an index of the relative activity of the opposing stimuli that modulate vascular tone and platelet activation. The ratio of TxA2 : PGI2 in inter‐villous space could be involved in the mechanism of initiating platelet function. During activation, exogenous ARA can also readily taken up by platelets. Fatty acid translocase (FAT or CD36) is involved in ARA uptake by human platelets. Both total and surface expres­ sion of CD36 are increased in patients with myeloproliferative disorders (MPD), consistent with an enhanced capacity for uptake of ARA by platelets [6]. Increased expression of CD36 in platelets may play a role in the vaso‐occlusive manifestations of MPD. In contrast, PGI2 is synthesized by endothelial cells by the action of prostaglandin I synthase‐1. A number of endogenous substances, including thrombin, ARA, PGH2, bradykinin and adenosine, promote the release of PGI2. Platelets can also contribute to PGI2 formation in the endothelium, by supplying

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Nutraceuticals and Human Blood Platelet Function

preformed PGH2. PGI2 strongly inhibits aggregation of plate­ lets, an effect mediated by stimulation of receptor‐linked ade­ nylate cyclase, which results in accumulation of cAMP. In general, it seems that, in diseases where there is a tendency for thrombosis to develop, TxA2 production is elevated, while PGI2 production may be either elevated or reduced. The oppo­ site is found in some diseases associated with increased bleeding tendency. Because both n‐3 and n‐6 fatty acids owe their pres­ ence in vertebrate tissues to dietary intake, important physio­ logical consequences follow the inadvertent selection of different average daily dietary supplies of these two types of EFAs and their LCPUFA derivatives (Figure 3.2). The abundance of preformed ARA in Western diets ensure that this is the most common fatty acid in tissue phospholipids, TXA2

Thromboxane receptor (TP) P-selectin

PLC IP3 + DAG

PIP2 PI3K

Release of alpha granules

PKC activation

Akt1/2

Ca2+ release from dense granules

GPIIb/IIIa complex activation

Dense granule release

MLCK

PLA2 ARA

ATP, ADP MLC

COX-1 GPIIb/IIIa (fibrinogen receptor)

Shape change

TxA2

Figure 3.2  Thromboxane receptors of human platelets. The receptor for TxA2 (designated TP), is expressed as two isoforms, designated TPα and TPβ. The TP receptors mediate phospholipase C activation. The regulation of myosin light chain (MLC) phosphorylation, which has been suggested to be involved in the induction of platelets, can be controlled through a Ca2+/calmodulin‐dependent regulation of MLC kinase and through a Rho/Rho‐kinase‐mediated regulation of myosin phosphatase.

n‐3 Fatty Acids and Human Platelets

and that the eicosanoids (prostaglandins of 2 series and leukot­ rienes of 4 series) derived from this fatty acid predominate in human tissues. When the eicosanoids from ARA are produced in large excess, they shift the physiological state to pathophysi­ ological conditions. It has been postulated that platelets could be one of the mechanisms by which dietary fatty acids influ­ ence the development of CVD, and particularly atherogenesis. Indeed, since the 1970s, there has been compelling evidence to suggest that dietary fatty acids are able to affect human platelet function in vitro [7–10] and in vivo [11–13]. Fatty acids (monounsaturated, polyunsaturated) have been shown to inhibit agonist‐induced platelet aggregation, while saturated fatty acids and trans fatty acids tend to enhance plate­ let aggregation. EPA and DHA are the most studied dietary anti‐ platelet compounds in human intervention studies. The dietary intervention of platelet function is important, as platelets play a central role in the athero‐thrombotic process [14, 15]. There are several reviews available on the effects of ALA, EPA, and DHA on some on platelet functions and other CVD risk factors, including oxidative modification of LDL [16–18]. In this chapter, the role of LCPUFA metabolites of n‐3 and n‐6 series and their regulation of platelet function are discussed.

3.2 ­Epidemiology of n‐3 Fatty Acids Intake and Cardiovascular Disease Epidemiological data suggest that a high intake of marine fish oil or fish products is correlated with a low incidence of CVD. This finding is frequently related to the level of n‐3 LCPUFAs in platelet membrane phospholipids. The low frequency of ischae­ mic heart diseases in Inuits has been known for about a century [19], and a diet containing n‐3 LCPUFAs has been thought to be the reason for the low incidence of thromboembolic diseases in Inuits in the 1970s [19]. The increase in CVD mortality attribut­ able to atherosclerotic diseases and cerebral vascular accidents with age has been associated with enhanced platelet hyperactiv­ ity. N‐3 fatty acids lower platelet reactivity, thus reducing CVD mortality in marine fish‐consuming populations.

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Nutraceuticals and Human Blood Platelet Function

Dyerberg and Bang showed that Inuits had attenuated platelet reactivity [20] as they consumed n‐3 LCPUFAs such as eicos­ apentaenoic acid, 20:5n‐3 (EPA) and docosahexaenoic acid, 22:6n‐3 (DHA). EPA and DHA compete with ARA for acylation into cellular phospholipids. These effects can be explained by changes in eicosanoid metabolism, shifting it from the ARA‐ derived series 2 prostanoid metabolites to the EPA‐derived series 3 protsanoid metabolites. The 3 series of prostanoids produced from n‐3 LCPUFAs have very low or no activity on platelet activation. Activated platelets contribute to plaque formation within blood vessels in the early and late stages of atherogenesis and, therefore, they have been proposed as a risk factor for cardio­ vascular disease. Numerous epidemiological studies and clinical trials have reported the health benefits of different n‐3 fatty acids, including a lower risk of coronary heart diseases [16]. There are several observations that indicate diminished platelet aggregation response with EPA and DHA present in marine fish or fish product consumption [21–23].

3.3 ­n‐3 Fatty Acids and Platelet Function The role of platelets in hemostasis and thrombosis has been known for a long time and is well defined but, more recently, a new concept has emerged, stating that platelets play a central role in the atherothrombotic process [14, 15]. Nutritional modi­ fication of cellular functions by dietary lipids and other nutritive and non‐nutritive factors offers an attractive avenue to correct, modify or prevent many pathophysiological processes, includ­ ing platelet hyperactivity [24]. The mediation of such effects may primarily be achieved through alterations of cell membrane composition and other endogenous lipid stores, with a conse­ quent reduction in ARA‐derived eicosanoid production and modification of the functional activity of various receptors on platelet membranes [24]. Dietary fatty acids have been postulated as a reason that plate­ lets could be one of the mechanisms by which dietary fatty acids influence the development of CVD. Indeed, since the 1970s,

n‐3 Fatty Acids and Human Platelets Diet Linoleic acid, 18:2n-6 γ-Linolenic acid, 18:3n-6 Dihomo γ-Linolenic acid, 20:3n-6

Sea foods Meat, egg Eiocosapentaenoic acid, 20:5n-3 Alpha Linolenic acid, 18:3n-3 Diet

Group-1 Prostanoids PGE1 PGF1α TXA1

Meat, egg Group-2

Arachidonic acid, 20:4n-6

Leukotrienes LTA3 LTC3 LTD3 Group-3 Prostanoids leukotrienes PGE3 LTA5 PGF3α LTC5 TXA3 LTB5 PGI3 PGD3

Prostanoids PGE2 PGF2α TXA2 PGI2 PGD2 Leukotrienes LTA4 LTC4 LTD4 LTB4

Figure 3.3  Eicosanoids synthesis from different fatty acids series. For details, please see the text.

there has been compelling evidence to suggest that dietary fatty acids are able to affect human platelet function [25]. The three main types of n‐3 long chain polyunsaturated fatty acids include 20‐carbon eicosapentaenoic acid, 22:5n‐3 (EPA), docosahexaenoic acid, 22:6n‐3 (DHA) obtained from fish and fish oil, and ALA from plants. Some of the eicosanoids formed from ARA, such as TxA2, leukotrienes (LTs) of 4 series and isoprostanes (non‐cyclooxygenase product) have deleterious effects, while those derived from n‐3 fatty acids are generally less potent, or have beneficial actions (Figure 3.3). Interference with eicosanoid biosynthesis is character­ istic  of  many anti‐inflammatory, anti‐thrombotic and anti‐­ hypertensive agents and diuretics, indicating that eicosanoids are involved in a broad spectrum of prevailing diseases [26]. The production of biologically active eicosanoids can be reduced by pharmaceutical intervention – for example, by the use of steroid anti‐inflammatory agents, such as cortisone, which inhibits release of ARA from phospholipids. EPA and DHA substitute biologically less potent eicosanoids, while the non‐nutritive compounds act to reduce all eicosanoid formation, especially ARA derived TxA2, an important platelet‐aggregating agent (Figure 3.4).

53

Dietary intervention Flavonoids

Platelets

Pharmacological intervention

Lipases

Steroid

Eicosapentaenoic acid, 20:5n-3 Arachidonic acid, 20:4n-6 Flavonoids

TxA2 synthetase inhibitor TxA2

GPIIb/IIIa receptor antagonist

Vasoconstriction platelet aggregation platelet recruitment

Figure 3.4  Mode of actions of different dietary compounds at different steps of human platelet activation/aggregation. Modulation of platelet activation/aggregation response can be modulated by EPA by replacing ARA. Additionally, Flavonoids can lower the activity of lipases and cyclooxygenase via reduced formation of TxA2.

n‐3 Fatty Acids and Human Platelets

An increased amount of dietary n‐3 fatty acids reduces ARA in tissue lipids by inhibiting its synthesis from its parent mole­ cule, linoleic acid. While n‐6 LCPUFAs are vigorously converted to potent eicosanoids that exert intense agonist actions at eicos­ anoid receptors, the n–3 LCPUFAs‐derived eicosanoids are formed more slowly, and often produce less intense actions. Ingestion of fish oil results not only in the reduction of tissue ARA, but also in a concomitant decrease in the capacity of the tissues to synthesize ARA‐derived eicosanoids and their substi­ tution with eicosanoids of lesser potency. The replacement of TxA2 from ARA with the less potent TxA3 from EPA leads to a marked shift in the TxA2/PGI2 balance,  which may act to produce an anti‐aggregatory state. Modifying platelet aggregation and subsequent adhesion to a blood vessel affected by TxA2/PGI2 interactions suggests a mechanism, already partially realized, for preventing and treat­ ing circulatory diseases. There is growing evidence that a reduc­ tion in TxA2 synthesis by incorporation into the diet of n‐3 LCPUFAs may reduce the risk of ischaemic heart disease. However, dietary interventions have to be treated with some caution, in view of the potential of n‐3 fatty acids to enhance the oxidative modification of apo‐B‐containing lipoproteins, with a concurrent increase in cellular uptake by scavenger receptors present in macrophages. Though EPA and DHA are the most studied dietary anti‐ platelet compounds in human intervention studies, Fruitflow® (a specially isolated aqueous tomato extract) has emerged as a new dietary means for a long‐term strategy to modify human blood platelet activity favorably [24]. A combination of this tomato extract, containing anti‐platelet factors [27] and n‐3 LCPUFAs on platelet aggregation in vitro, was investigated. The combination of tomato extract and n‐3 LCPUFAs inhibited in vitro platelet aggregation to a greater extent than either alone, and this inhibition was correlated with intracellular platelet cAMP levels [28]. It seems that the combinations of Fruitflow® and n‐3 fatty acids, and other non‐nutritive compounds such as resveratrol and other polyphenols, may be effective in improving platelet func­ tion. In the case of excess n‐6 fatty acid consumption, typical of Western diets, the effects of increasing n‐3 fatty acid consumption

55

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Nutraceuticals and Human Blood Platelet Function Fatty acids

Cell types

ARA

EPA

ARA

EPA

Cycloxygenase Endothelial cells Platelets TXA2 TXA3 PGI2 PGI3

ARA

EPA

Lipoxygenase leukocytes LTB4 LTB5

Biological effects Aggregation

+++

Anti-aggregation Vasoconstriction Vasodilatation chemotaxis

+

+++

+++

+++

+++

+++ +++

+

Figure 3.5  Biological effects of arachidonic acid, 20:4n‐6 (ARA) and Eicosapentaneoic acid, 20:5n‐3 (EPA). For details, please see the text.

and non‐nutritive compounds present in plants, vegetables and fruits can be seen as acting in concert (Figure 3.5). EPA substitutes biologically less potent eicosanoids, while the non‐nutritive compounds act to reduce all eicosanoid forma­ tion. Excessive production of eicosanoid derived from the much more rarely encountered diets rich in n‐3 fatty acids can also be counteracted by non‐nutritional compounds present in the diet. An overall reduction in eicosanoid biosynthesis may prevent the bleeding disorders caused essentially by an imbalance in the TxA2/PGI2 system, while the anti‐oxidant properties of most non‐nutritional compounds counters the inherently greater sus­ ceptibility of n‐3 fatty acids and their derivatives to oxidative damage. This may explain why, for example, the traditional diet of the Inuit is inherently less healthy than the traditional Japanese diet, in which a high fish intake is balanced by an equally high consumption of plant foods not available to the Inuits.

3.4 ­Platelet Function and Eicosanoids Prostaglandins and thromboxane are the major metabolites of ARA that modulate platelet function through their respective membrane receptors. Activated platelets release ARA/EPA/

n‐3 Fatty Acids and Human Platelets

dihomo gamma linolenic acid, 20:3n‐6 (DGLA) from membrane phospholipids, which is metabolized by COX and LOX to ­produce different eicosanoids. The liberated ARA is primarily converted to TXA2, but is also converted to a very small amount of PGE2. EPA produces 3 series of prostanoids, while DGLA is mostly converted to PGE1. TXA2 exerts its actions via heterotrimeric G protein‐coupled thromboxane receptors (TP receptors), which activate Gαq, Gαi and Gα12/13. Gαq‐mediated activation of phospholipase Cβ‐­isoforms plays an essential role in agonist‐induced platelet aggregation and secretion. The Gα12/13‐mediated signal path­ way is also important in TXA2‐induced platelet shape change and platelet aggregation [29]. TXA2 directly activates the Gαi signaling pathway via its receptor, whiles recent studies from several groups indicate that TXA2‐induced Gαi activation requires ADP secretion and ADP‐induced activation of P2Y12 receptor. In addition, dissociation of Gα subunits from Gβγ sub­ units induced by receptor occupancy allows Gβγ subunits to activate phosphoinositide 3 kinase (PI3K), resulting in increases in phosphatidylinositol 3,4‐­bisphosphate and phosphatidylinosi­ tol 3,4,5‐trisphosphate, and activation of Akt (Figure 3.6). Activation of adenylate cyclase is initiated by binding of prostacyclin (PGI2) or PGE1 through specific platelet surface receptors [4]. cAMP level in platelets may also be increased by inhibiting cAMP phosphodiesterase activity. Each of the eicosa­ noids acts via interactions with cell‐surface receptors that are members of the G‐protein coupled receptor (GPCR) family. Cell membrane receptors that bind the PGD family of lipids are called the DP receptors; those that bind E family prostaglandins are called the EP receptors; those that bind F family prostaglan­ dins are called the FP receptors; those that bind PGI2 are called the IP receptors; and those that bind the thromboxanes are called the TP receptors. PGE1 stimulates adenyl cyclase activity and is a potent inhibi­ tor of platelet aggregation in vitro [4]. Specifically, PGE1 binds to  prostacyclin receptor (IP) on platelets, raises intracellular cAMP, which inhibits phospholipase C activation, and reduces Ca2+ mobilization from intracellular stores. PGE1 thus antago­ nizes the effects of P2Y1 receptor activation. Activation of P2Y1, which is a Gq‐coupled receptor linked to phospholipase  C,

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Nutraceuticals and Human Blood Platelet Function

N-3 fatty acids

Liver lipid metabolism

Triglycerides free fatty acids

Platelets

Platelet activation platelet aggregation TxA2 production membrane shedding

Blood vessels

Inflammation endothelial function vasodialation

Atherosclerosis, plaque rupture, thrombosis

Cardiac events

Figure 3.6  Overall effects of n‐3 fatty acids in the body. For details, please see the text.

generates IP3 and is followed by release of Ca2+, resulting in platelet activation and aggregation. It is thus possible that the anti‐atherosclerotic effects of DGLA may be mediated by PGE1 action on platelets, whereas PGE2 acting via EP3 receptors is involved in platelet thrombus formation [30]. Activated human platelets synthesize PGE2, although at lower rate than TxA2. PGE2 acts through different receptors, but its role in human platelet function remains poorly characterized, compared with thromboxane. PGE2 at nanomolar concentra­ tions, dose‐dependently increases ADP‐induced platelet aggre­ gation without affecting final maximal aggregation. Activated human platelets synthesize and release PGE2 during whole‐ blood clotting, although at concentrations approximately 30‐ fold lower compared with TxA2. Platelet‐type 12‐lipoxygenase is an enzyme which oxidizes the free fatty acid in the platelet, resulting in the production of the stable metabolite 12‐hydrox­ yeicosatetraenoic acid (12‐HETE). The role of 12‐HETE in the platelet has been controversial, with reports associating its func­ tion with being both anti‐ and pro‐thrombotic.

n‐3 Fatty Acids and Human Platelets

3.5 ­Clinical Trials with n‐3 Fatty Acids The low incidence of myocardial infarction in Greenland Inuits was thought to be linked to their intake of marine food with a high content of n‐3 LCPUFAs. In Inuits, the platelet count is lowered, platelet aggregation is inhibited, bleeding time is pro­ longed, and the ratio between pro‐aggregatory TxA2 and anti‐ aggregatory PGI2 is decreased when compared to age‐ and sex‐matched Danes. The well established in vitro anti‐platelet effect of n‐3 fatty acids is, however, required to investigate whether their bio‐activity is expressed in vivo after they are consumed. Various studies have shown that the fatty acid composition of dietary fat has differential effects on platelet function [11]. There have been several placebo‐controlled trials in healthy subjects, and seven placebo‐controlled trials in patients with CVD, that assessed the effects of these fatty acids on platelet function. EPA and DHA are one of the most studied dietary fatty acids, with anti‐platelet effect in human trials [25]. Generally, supplementation with n‐3 fatty acids for an average period of 4–12 weeks inhibits collagen‐ and ADP‐induced plate­ let aggregation, and effects are more pronounced in healthy sub­ jects [31–33] than in volunteers with existing CVD on anti‐platelet medication [34,35]. A similar inhibitory effect of n‐3 fats sup­ plementation on ADP‐induced platelet aggregation was found in a recent meta‐analysis, using data from placebo‐controlled trials between 1966 and 2011 [36]. Interestingly, acute inter­ ventions with fish oils resulted in sex‐specific responses after 24 hours, indicating that EPA is more effective in males in the short term, whereas DHA inhibited collagen‐induced platelet aggre­ gation in females only [37]. These effects were subsequently confirmed in a chronic intervention study [23]. In another double‐blind, placebo‐controlled random trial, the  potential benefits of n‐3 LCPUFAs supplementation, in individuals with multiple CVD risk factors without history of myocardial infarction, were investigated. After a follow‐up of five years, the primary end point of time to death from CVD, or admission to hospital from CVD‐related causes, was not signifi­ cantly different between the two groups consuming 1 g/day of n‐3 LCPUFAs (6244 subjects) or olive oil (n = 6269).

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A double‐blind, placebo‐controlled random trial conducted to investigate the effects of n‐3 LCPUFAs supplementation on  major CVD events, among individuals with a history of myocardial infarction, on state‐of‐the art antihypertensive, antithrombotic, and lipid‐modifying therapy. A total of 4837 subjects were assigned to one of the four different groups con­ suming ALA (2 g/day) or EPA/DHA (400 mg/day). There was no significant reduction in the rate of major CVD events between the groups. A meta‐analysis of random‐controlled trials (n = 14) found no significant effects of n‐3 LCPUFA supplementation on major CVD outcomes and mortality among subjects with prevalent disease. However, a reduced risk of cardiac mortality, sudden cardiac death, and all‐cause mortality was observed. In another meta‐analysis of 11 random controlled trial among patients with prevalent CVD, Casula et al. [38] found that n‐3 LCPUFAs sup­ plementation was associated with a significant reduction of myocardial infarction, cardiac death, and sudden death; how­ ever, there was no significant reduction noted in all‐cause mor­ tality or stroke. The optimal dose of n‐3 LCPUFAs, and the patient groups most likely to profit from supplementation, need to be defined. The safety and the clinical effect of the supplementation should be investigated in long‐term studies. Although some epidemiologic studies clearly indicate that a low intake of fish may beneficially influence coronary heart diseases, few data are available from experimental studies on low intake of n‐3 LCPUFAs. Most studies have demonstrated that supplementation with n‐3 LCPUFAs can cause inhibition of platelet behavior. A meta‐ analysis conducted by Gao et al. [36] has demonstrated that n‐3 LCPUFAs are associated with a significant reduction of platelet aggregation. In addition, low intake of EPA may reduce platelet aggregation without changing the fatty acid platelet composi­ tion [39,40]. In the same way, following supplementation with DHA [41], platelet function was reduced and a retroconver­ sion of DHA into DPA and EPA was evidenced. More recently, a  dose–response study with middle‐aged healthy volunteers ingesting increasing amounts of DHA, indicated that platelet reactivity was decreased after 400 and 800 mg DHA/day [42].

n‐3 Fatty Acids and Human Platelets

3.6 ­Dietary Recommendation and Sources of n‐3 Fatty Acids In general, the tendency of platelets to aggregate in response to different agonists is significantly lowered after dietary intake of n‐3 fatty acids, and might be explained by the enrichment of membrane phospholipids with EPA and DHA at the expense of ARA. The American Heart Association (AHA) recommends that all adults eat fish at least twice per week for primary preven­ tion, about 1 g/day of combined EPA and DHA, preferably from oily fish for secondary prevention, and 2–4 g/day of combined EPA + DHA for those with hypertriglyceridemia. Prior studies have demonstrated a beneficial role of n‐3 FA among people with CVD. Although it is possible for the body to convert some ALA to EPA and DHA by elongase and desaturase enzymes, conversion to DHA is pretty small [43]. Conversion of ALA is only 0.7% for EPA, and only 0.013% for DHA. Seafood sources, such as fish and fish‐oil supplements, are the primary contributors of EPA and DHA in humans. However, higher intake of n‐6 fatty acids can deplete DHA from membrane phos­ pholipids. Thus, a proper balance in the ratio between n‐3 and n‐6 is necessary. However, our present‐day food habits and preferences have given an abnormal tilt towards n‐6, with n‐3 content getting greatly reduced. This has to be corrected, and a healthy ratio of 2 : 1 between n‐3 and n‐6 must be aimed for in our food intake. DHA is a unique nutrient that should be regularly consumed as oily sea fish, or supplemented as fish oil or algal supplements. France is the only country where recommendations specifi­ cally for DHA are provided by health bodies, at 120 mg for men and 100 mg for women per day. Driven by consumer demand, and despite additional production costs, modern food proces­ sors are increasingly fortifying manufactured food products, particularly milk, bread and eggs, with DHA from algae and fish oil. Meat from wild animals contains significantly higher DHA than meat from domesticated animals, be they free‐range, pas­ ture‐fed, and/or organically‐reared animals. Food sources of ARA are meat, seafood and poultry. Most of the seed oils contain a high concentration of LA, a precursor for synthesis of ARA in the body. ARA is very important for many

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Nutraceuticals and Human Blood Platelet Function

biochemical processes, such as production of anti‐aggregatory prostacyclin by endothelium. However, its excess presence can precipitate many inflammatory disorders and platelet hyperac­ tivity. The intake of sufficient n‐3 fats attunes the presence of ARA. Selection of foods from various sources for balancing the presence of n‐3 and n‐6 fats will negate the harmful effects of excess of ARA.

3.7 ­Conclusions N‐3 fatty acids undoubtedly modify biochemical function in many systems, including human blood platelets. Dietary n‐3 fatty acids are incorporated into membrane phospholipids and may alter membrane function. N‐3 LCPUFAs can decrease the arachidonic acid content of platelet cell membranes, which reduces n‐6 eicosanoid production. Consequently, n‐3 fatty acids reduce aggregational properties of blood platelets by reducing thromboxane synthesis, thereby interfering with thrombosis formation. N‐3 fatty acids can inhibit the synthesis of pro‐inflammatory cytokines. Finally, in high doses, they reduce serum triglyceride levels, which could further reduce risk for CVD. Prospective epidemiological studies bolster the evidence that n‐3 fatty acids offer protection against CVD. Since the dietary intake of n‐3 LCPUFAs is not optimum in many countries, it is therefore recommended to have a supplement of n‐3 fats. Evidences from epidemiology, observational, and interventional studies indicate that the intake of these fatty acids may reduce CVD risk via different mechanisms, including lowering platelet activity.

­References 1 Brenna JT, Salem N, Jr., Sinclair AJ, Cunnane SC, International

Society for the Study of Fatty Acids and Lipids (2009). alpha‐ Linolenic acid supplementation and conversion to n‐3 long‐ chain polyunsaturated fatty acids in humans. Prostaglandins, Leukotrienes and Essential Fatty Acids 80(2–3): 85–91.

n‐3 Fatty Acids and Human Platelets

2 Duttaroy AK (2009). Transport of fatty acids across the human

placenta: a review. Progress in Lipid Research 48(1): 52–61.

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23 Phang M, Lincz LF, Garg ML (2013). Eicosapentaenoic and

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docosahexaenoic acid supplementations reduce platelet aggregation and hemostatic markers differentially in men and women. Journal of Nutrition 143(4): 457–463. Dutta‐Roy AK (2002). Dietary components and human platelet activity. Platelets 13(2): 67–75. Bachmair EM, Ostertag LM, Zhang X, de Roos B (2014). Dietary manipulation of platelet function. Pharmacology & Therapeutics 144(2): 97–113. Yamagishi K, Folsom AR, Steffen LM, for the ARIC Study Investigators (2013). Plasma fatty acid composition and incident ischemic stroke in middle‐aged adults: the Atherosclerosis Risk in Communities (ARIC) Study. Cerebrovascular Diseases 36(1): 38–46. Dutta‐Roy AK, Crosbie L, Gordon MJ (2001). Effects of tomato extract on human platelet aggregation in vitro. Platelets 12(4): 218–227. Lazarus SA, Garg ML (2003). The effects of tomato extract (TE) and omega‐3 fatty acids on platelet cAMP levels and inositol triphosphate (IP(3)) release. Asia Pacific Journal of Clinical Nutrition 12 Suppl: S20. Dorsam RT, Kim S, Jin J, Kunapuli SP (2002). Coordinated signaling through both G12/13 and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets. Journal of Biological Chemistry 277(49): 47588–47595. Vezza R, Roberti R, Nenci GG, Gresele P (1993). Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C. Blood 82(9): 2704–2713. Agren JJ, Vaisanen S, Hanninen O, Muller AD, Hornstra G (1997). Hemostatic factors and platelet aggregation after a fish‐enriched diet or fish oil or docosahexaenoic acid supplementation. Prostaglandins, Leukotrienes and Essential Fatty Acids 57(4–5): 419–421. Freese R, Mutanen M (1997). Alpha‐linolenic acid and marine long‐chain n‐3 fatty acids differ only slightly in their effects on hemostatic factors in healthy subjects. American Journal of Clinical Nutrition 66(3): 591–598. Mori TA, Beilin LJ, Burke V, Morris J, Ritchie J (1997). Interactions between dietary fat, fish, and fish oils and their effects on platelet function in men at risk of cardiovascular

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disease. Arteriosclerosis, Thrombosis, and Vascular Biology 17(2): 279–286. Gajos G, Rostoff P, Undas A, Piwowarska W (2010). Effects of polyunsaturated omega‐3 fatty acids on responsiveness to dual antiplatelet therapy in patients undergoing percutaneous coronary intervention: the OMEGA‐PCI (OMEGA‐3 fatty acids after pci to modify responsiveness to dual antiplatelet therapy) study. Journal of the American College of Cardiology 55(16): 1671–1678. Serebruany VL, Miller M, Pokov AN, et al. (2011). Early impact of prescription Omega‐3 fatty acids on platelet biomarkers in patients with coronary artery disease and hypertriglyceridemia. Cardiology 118(3): 187–194. Gao LG, Cao J, Mao QX, Lu XC, Zhou XL, Fan L (2013). Influence of omega‐3 polyunsaturated fatty acid‐ supplementation on platelet aggregation in humans: a meta‐ analysis of randomized controlled trials. Atherosclerosis 226(2): 328–334. Phang M, Sinclair AJ, Lincz LF, Garg ML (2012). Gender‐ specific inhibition of platelet aggregation following omega‐3 fatty acid supplementation. Nutrition, Metabolism & Cardiovascular Diseases 22(2): 109–114. Casula M, Soranna D, Catapano AL, Corrao G (2013). Long‐ term effect of high dose omega‐3 fatty acid supplementation for secondary prevention of cardiovascular outcomes: A meta‐ analysis of randomized, placebo controlled trials [corrected]. Atherosclerosis. Supplements 14(2): 243–251. Driss F, Vericel E, Lagarde M, Dechavanne M, Darcet P (1984). Inhibition of platelet aggregation and thromboxane synthesis after intake of small amount of icosapentaenoic acid. Thrombosis Research 36(5): 389–396. Croset M, Vericel E, Rigaud M, et al. (1990). Functions and tocopherol content of blood platelets from elderly people after low intake of purified eicosapentaenoic acid. Thrombosis Research 57(1): 1–12. von Schacky C, Weber PC (1985). Metabolism and effects on platelet function of the purified eicosapentaenoic and docosahexaenoic acids in humans. Journal of Clinical Investigation 76(6): 2446–2450.

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42 Guillot N, Caillet E, Laville M, Calzada C, Lagarde M, Vericel E

(2009). Increasing intakes of the long‐chain omega‐3 docosahexaenoic acid: effects on platelet functions and redox status in healthy men. FASEB Journal 23(9): 2909–2916. 43 Neff LM, Culiner J, Cunningham‐Rundles S, et al. (2011). Algal docosahexaenoic acid affects plasma lipoprotein particle size distribution in overweight and obese adults. Journal of Nutrition 141(2): 207–213.

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4 Effects of Garlic, Onion, Ginger, and Turmeric on Platelet Function ­Abbreviations Reactive oxygen species (ROS); Onion peel extract, OPE; Aged garlic extract, AGE; Spleen tyrosine kinase, Syk

4.1 ­Introduction The regulation of platelet inhibition is one of the primary strate­ gies for preventing CVD and treating diseases. Therefore, the inhibitors of platelet aggregation show promise as preventive or therapeutic agents for various vascular diseases, with limited adverse effects. During the last two decades, there has been a growing interest in the field of herbs and spices, as reflected by a number of studies concerning the anti‐platelet and antithrom­ botic properties of natural products. Herbs and spices are an important part of human nutrition, used as food additives mainly to impart color and flavor, stimulation of appetite, and carminative action, as well as preservation of foods [1]. Research on human nutrition has led to an awareness of the health benefits of foods associated with non‐nutrient compo­ nents, as in herbs and spices. Specifically, it has been recog­ nized that a complex array of naturally occurring bioactive non‐­nutrients may confer significant long‐term health benefits via different mechanisms, including well‐established antioxi­ dant activity. The type of herbs used depends on race, ethnicity,

Nutraceuticals and Human Blood Platelet Function: Applications in Cardiovascular Health, First Edition. Asim K. Duttaroy © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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Nutraceuticals and Human Blood Platelet Function Prevention of LDL oxidation (Turmeric)

Inhibition of platelet aggregation (Garlic, onion, ginger, Turmeric) Cardioprotective property

Cholesterol lowering activity (Garlic, onion, Ginger, Turmeric)

Hypotensive propertry (Garlic)

Figure 4.1  Cardio‐protective property of spices.

geographical locations, family history, and herbal use by other family members. Turmeric, ginger garlic, onion, pepper, and cloves are some of the most commonly used spices in culinary preparations. A large body of evidence supports the fact that consumption of spices may exert some beneficial effects on the cardiovascular system, and thereby reduces the risk for CVD [2]. Figure 4.1 summarizes the cardio‐protective properties of spices. Platelet hyperactivity plays a significant role via multiple mechanisms in the development of CVD [3]. Platelets are involved in atherothrombosis through the ability to adhere and accumulate to injured vessel wall and release bioactive media­ tors. In fact, hyperactivity of platelets, and their adhesion and aggregation at the site of injury in atherosclerotic vessel walls, is critically important in the pathogenesis of CVD, as described in Chapter 1. On platelet activation, there is a transient increase in cytosolic Ca2+, TxA2 synthesis, and surface expression of fibrinogen receptors (GPIIb/IIIa). Other modulators involved in platelet aggregation include lipoxygenase metabolites, protein kinase C, cAMP, cGMP and NO. Several bio‐active compounds present in herbs and spices intervene in these processes involved in plate­ let activation/aggregation. Spices also protect the cardiovascu­ lar system via several other mechanisms, such as lowering of

Effects of Garlic, Onion, Ginger, and Turmeric on Platelet Function

LDL cholesterol levels, and inhibition of reactive oxygen species (ROS) [4]. Many extracts of food spices influence production of TXA2 directly, by inhibition of thromboxane synthase, and indirectly, by inhibition of enzymes responsible for its precursors’ forma­ tion. A range of compounds from spices and from the labiate herbs inhibit PGH synthase, thus affecting prostaglandins syn­ thesis. A number of chalcones, including licochalcone A, and the sweet‐tasting triterpenoid from licorice, glycyrrhizin, or its degradation product glycyrrhetic acid, inhibit prostaglandin‐­ endoperoxide synthase activity. Thus, the non‐nutritive com­ pounds may have an important role in moderating ARA metabolism and maintaining healthy physiological platelets. Spices also contain a diverse array of natural phytochemicals that have complementary and overlapping actions, including antioxidant effects, modulation of detoxification enzymes, stimulation of immune system, and reduction of inflammatory responses. The spices contain phenolic compounds, including flavonoids and non‐flavonoids, that possess anti‐platelet prop­ erties. The active principles of spices such as curcumin (tur­ meric), quercetin (onion, garlic), capsaicin (capsicum), piperine (pepper), eugenol (cloves) and allyl sulfide (garlic), have a ­protective effect on the cardiovascular system. This chapter describes the effects of some herbs and spices on human blood platelet aggregation.

4.2 ­Effects of Garlic (Allium Sativum) on Platelet Function Garlic (Allium sativum) has been used in the human diet for millennia. Epidemiological studies show an inverse relationship between the consumption of garlic and progression of CVD. Recently, garlic has been used as a food or herbal supplement for reducing CVD, particularly for hypertension, hypercholester­ olemia, and also for the inhibition of platelet aggregation [5, 6]. Inhibition of platelet aggregation by garlic has been extensively investigated in vitro, although in vivo studies are limited. In vitro studies indicate that garlic prevents inhibition of  platelet aggregation via inhibiting several mechanisms,

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including by inhibiting TxA2 formation, by suppressing mobi­ lization of intra‐platelet Ca2+, and by increasing levels of cAMP and cGMP. Garlic also displays strong antioxidant pro­ perties, and activates nitric oxide synthase (NOS), leading to an increase in platelet‐derived NO. It also interacts directly with the GPIIb/IIIa receptors of platelets. In vitro studies sug­ gest that various bioactive constituents of garlic inhibit plate­ let function. The extent, however, to which dietary doses of garlic influence platelet function in vivo remains unknown. Aged garlic extract (AGE), a garlic preparation rich in water‐ soluble cysteinyl moieties, has been reported to have multiple beneficial effects on CVD. Dietary supplementation with AGE significantly inhibited both the total percentage and initial rate of platelet aggregation induced by ADP. However, no significant changes in plasma TxB2 and 6‐ketoPGF1α concentrations or serum lipid profiles were observed. Thus, AGE may be benefi­ cial in protecting against CVD, as a result of inhibiting platelet aggregation and lowering of plasma cholesterol. Conversely, methylallyltrisulfide from garlic oil, a potent inhibitor of platelet aggregation, promoted release of ARA from rabbit platelets, implying stimulation of PLA2. However, ARA was not converted to prostanoids, suggesting an inhibition of COX pathway by this compound. Quercetin, a flavonol present in plants and a potent inhibitor of platelet aggregation is the only agent of dietary origin thought to directly inhibit phospho­ lipase C. Organosulfur compounds from Allium species have been shown to be potent inhibitors of platelet aggregation, and to have antiasthmatic and antiallergic effects. Most of the sulfur‐ containing compounds detected are formed from highly reac­ tive sulfenic acids, released from naturally‐occurring S‐alk(en) yl‐L‐cysteinesulfoxides by the action of alliin lyase (EC 4.4.1.4.‐ alliin alkyl‐sulfenate‐lyase) when tissues are damaged or disrupted. Two groups of compounds predominate: the thio­ sulfinates, lachrymatory factors with demonstrable antiasth­ matic activity in vivo, and the sulfinyldisulfides (cepaenes), formed from the thiosulfinates by a mechanism similar to that described for the formation of the structurally‐related (E,Z)‐ ajoene from allicin in garlic extracts. Both groups of com­ pounds have been shown to inhibit PGH synthase to varying

Effects of Garlic, Onion, Ginger, and Turmeric on Platelet Function

extents. Studies of the structure‐activity relationships of the alpha‐sulfinyldisulfides have confirmed the requirement for sulfinyl and disulfide groups, and have shown that the potency of platelet inhibition is directly related to alkyl chain length, the propyl derivative showing greatest activity. A 10‐month double‐blind crossover study compared the effects of 7.2 g of AGE with placebo on ADP‐, adrenaline‐, and  collagen‐induced platelet aggregation in moderately hyper‐­cholesterolemic men. The AGE significantly reduced adrenaline‐­ induced platelet aggregation. AGE had a lesser effect on collagen‐induced platelet aggregation, while there was no effect on ADP‐induced platelet aggregation [7]. ADP‐, adrenaline‐, and collagen‐induced platelet aggregation was tested before, and four hours after, the consumption of sepa­ rate doses of garlic (6, 8, 10, and 14 g), with washout periods of five days. Adrenaline‐induced platelet aggregation was inhib­ ited by garlic extract, though there was no change in ADP‐ or collagen‐induced platelet aggregation [8]. In a four‐week study, five healthy males and 10 patients (40– 60 years) with coronary artery disease took a dose of 2 g garlic three times a day. At the end of the four‐week study, garlic sig­ nificantly reduced platelet aggregation induced by ADP and adrenaline in the healthy subjects whereas, in CAD patients, only adrenaline‐induced platelet aggregation was inhibited [8]. The extent and rate of platelet aggregation in response to ADP was significantly reduced in healthy subjects after supplementa­ tion with 5 mL of AGE per day for 13 weeks [8]. In another study, platelet function was measured before, and then four hours after, subjects consumed a capsule containing 9.9 g of garlic or a placebo. Adrenaline‐induced platelet aggrega­ tion was found to be significantly reduced. However, no change in platelet aggregation in response to ADP or collagen was observed [10]. AGE inhibited ADP‐induced platelet aggregation and reduced intracellular calcium ion mobilization [11]. The effect of AGE on the functional property of platelets was investigated, administering AGE to rats and evaluating the platelet aggregation in response to collagen in vitro [12]. AGE significantly reduced the ability of platelets to aggregate, and this effect of AGE was expressed in the 14‐day, but not the seven‐day, treatment. AGE treatment also dose‐dependently

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inhibited the phosphorylation of collagen‐induced ERK, p38 and JNK [12]. The effect of raw garlic on platelet function was investigated in whole blood in 18 healthy volunteers, before and five hours after ingestion of Greek tsatsiki with 4.2 g of raw garlic (verum), or Greek tsatsiki without garlic (placebo), in a randomized, crossover, single blind placebo‐controlled study. Platelet func­ tion was not impaired by single and repeated oral consumption of Greek tsatsiki containing raw garlic. Platelet function was also not impaired by single and repeated oral consumption of a die­ tary dose of garlic in healthy volunteers. Also, in rats treated with aqueous extracts of garlic and onion (500 mg/kg of body weight) for four weeks, TXB2 levels were significantly inhibited compared with that of control in serum, without affecting plate­ let function [13]. In another human trial, the anti‐platelet effects of garlic were evaluated in a randomized controlled clinical trial, using four known agonists in the platelet aggregation pathway, including ADP, collagen, epinephrine and ARA. An attempt was also made to find out whether garlic is a proper alternative to aspirin (at its anti‐platelet dose (80 mg/day)) or no garlic tablet [14]14. After a one‐month washout period, volunteers were randomized into three groups, each receiving one, two or three tablets/day for one month, and PA was examined in all groups. The garlic tablet did not have a significant effect on platelet aggregation response at any dose. However, 30% of volunteers in the group that used three garlic tablets/day reported adverse effect (i.e., bleeding).

4.3 ­Effects of Onion (Allium Cepa L.) on Platelet Function Onion (Allium cepa L.) has been reported to have beneficial effects cardioprotective effects, including preventing stroke, thrombosis, atherosclerosis, hyperlipidemia and hypertension [13]. Onion has been shown to inhibit platelet aggregation induced by various agonists, both in vitro and ex vivo [15]. In dogs, onion juice reduced collagen‐induced whole‐blood plate­ let aggregation. These results may be linked by quercetin, which is known to be one of the most abundant flavonoids in onion. Epidemiological data suggest that those who consume a diet

Effects of Garlic, Onion, Ginger, and Turmeric on Platelet Function

rich in quercetin‐containing foods may have a reduced risk of CVD. Indeed, collagen‐stimulated platelet aggregation was inhibited after ingestion of onion soup high in quercetin [16]. Both in vitro and in vivo studies showed inhibition of platelet aggregation and activation [15, 17, 18]. However, while those studies investigated the effects of anti‐platelet aggregation of onion bulb extract, the signaling for anti‐platelet effects of the outer skin extract of onion was barely analyzed. The onion peel extract contained quercetin, one of the major flavonoids, which has anti‐platelet effect [19]. Onion peel extract is an effective inhibitor of collagen‐­ stimulated platelet aggregation in vitro [19]. Onion peel extract inhibitory effects are mediated via downregulation of TXA2 and lowering the level of intracellular [Ca2+]i, and COX‐1 activity, as well as upregulation of cAMP levels.

4.4 ­Effects of Ginger (Zingiber Officinale) on Platelet Function The rhizome of ginger (Zingiber officinale) is a traditional spice with anti‐platelet properties [20]. In addition to anti‐platelet properties, the rhizome of ginger has anti‐­inflammatory, car­ minative, and anti‐nausea effects. Ginger consists of numer­ ous complex combinations of biologically active constituents, including gingerols, shogaols, and paradols [20, 21]. In one study, ginger was administered to patients with CAD in two different doses, 4 g daily for three months, and as a single dose of 10 g. The daily dose of 4 g ginger did not affect ADP‐ or adrenaline‐induced platelet aggregation measured at 1.5 and three months of administration. However, a single dose of 10 g powdered ginger produced a significant reduction in platelet aggregation induced by the ADP and adrenaline four hours after ingestion [8]. In a randomized crossover study, conducted in healthy males, ginger did not alter ARA‐induced whole blood platelet aggrega­ tion or the pharmacokinetics and pharmacodynamics of warfa­ rin [22]. Compounds of ginger were measured for their in vitro anti‐platelet activity, using blood from 10 healthy volunteers. Gingerol, gingerol analogues (1 and 5), shogaol, and paradol exhibited anti‐platelet activities, with IC50 values ranging from

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3–7 μM. Paradol exhibited the strongest COX‐1 inhibitory activity, with IC50 values of 4 ± 1 μM, whereas the IC50 value for aspirin was 20 ± 11 μM20. Ginger extracts are powerful inhibitors of human platelet aggregation and eicosanoids formation. However, the two effects do not appear directly related, as [6]‐gingerol and its analogue reduce prostaglandin formation, but are only weak inhibitors of platelet aggregation, while the labdane‐type diterpenes from ginger strongly inhibit aggregation without inhibition of the arachidonic acid cascade. Evidently, inhibition of aggregation requires a different mechanism from that of the organosulfur compounds derived from Allium sp., and it is suggested that the diterpenes block the ADP binding site on platelets and, thus, inhibit platelet aggregation and activation. The mechanism of inhibition of prostaglandin‐endoperoxide synthase can be deduced from the relative effects of gingerol derivatives. Only methylgingerol, in which the phenolic hydroxyl is methylated, proved non‐inhibitory, confirming that the pres­ ence of a phenol group is essential. It is suggested that the phe­ nol group inhibits radical formation, or quenches radical generation in the prostaglandin‐endoperoxide synthase reac­ tion. The hydrophobic alkyl side chains also play an important role, by binding to the enzyme by hydrophobic interaction. Zingerone, a phenolic degradation product of gingerols, in which the alkyl group is lost, was only weakly inhibitory, com­ pared with the parent gingerols. Gingerols with six or more alkyl carbons, in addition to inhibiting prostaglandin‐endoperoxide synthase activity, were potent inhibitors of lipoxygenase. [6]‐ Gingerol, the main constituent responsible for the pungent taste of ginger, had an IC50 of 3.0 mM, while the corresponding value for [10]‐gingerol was approximately 0.05 mM. Further increase in alkyl chain‐length up to 16 carbons did not improve potency.

4.5 ­Effects of Turmeric (Curcuma Longa) on Platelets Turmeric (Curcuma longa) is a common oriental spice fre­ quently used in cooking, particularly Indian and Thai cooking, which gives curry powder its yellowish color. Turmeric has been

Effects of Garlic, Onion, Ginger, and Turmeric on Platelet Function

used in Ayurvedic medicine for aches, pains, wounds, sprains, and liver disorders. In addition, turmeric has been used to treat conditions of the skin, respiratory, and gastrointestinal systems. Turmeric shows potential in reducing platelet aggregation. Curcumin (diferuloylmethane), a polyphenol, is an active principle of the perennial herb Curcuma longa (commonly known as Turmeric). Curcumin has a wide range of beneficial actions in CVD, including anti‐inflammatory and antioxidant actions, as well as antibacterial properties [23]. The yellow‐pig­ mented fraction of turmeric contains curcuminoids, which are chemically related to curcumin. The major curcuminoids present in turmeric are demethoxy­ curcumin (curcumin II), bisdemethoxycurcumin (curcumin III), and the recently identified cyclocurcumin. The major com­ ponents of commercial curcumin are curcumin I (77%), cur­ cumin II (17%), and curcumin III (3%). The active principles, which constitute about 1–4% of the raw spice, thus impart ben­ eficial properties as natural, dietary antioxidants on oxidation of human LDL. There is limited clinical data on the effect of turmeric on platelet function. However, in vitro studies showed inhibition of ADP‐, adrenaline‐, ARA‐, collagen‐, GPVI‐, and PAF‐induced platelet aggregations [23–25]. Furthermore, in vitro studies have found that curcumin inhibition of dense granule secretion is induced by GPVI [24]. Curcumin inhibited human platelet aggregation and dense granule secretion, induced by GPVI ago­ nist convulxin, in a concentration‐dependent manner. Curcumin also strongly inhibited the activation‐dependent tyrosine phos­ phorylation of Y753 and Y759 on PLCγ2, but did not affect the phosphorylation of Y145 residue on the cytosolic adaptor pro­ tein SLP‐76. Interestingly, curcumin had no significant effect on the phosphorylation of Y525/Y526 present on the activation loop of Syk (spleen tyrosine kinase), but had a significant inhibi­ tory effect on in vitro Syk kinase activity. The inhibitory action of curcumin is independent of the thromboxane pathway. Curcumin inhibits platelet activation induced by GPVI agonists through interfering with the kinase activity of Syk, and the subsequent activation of PLCγ2. Future clinical trials are needed to substantiate their cardio‐protective effects.

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4.6 ­Conclusions Platelets play a pivotal role in both health and disease, through their central involvement in hemostasis and thrombosis. The process of primary hemostasis is initiated through platelet adhe­ sion at sites of vascular injury. The next step is platelet activa­ tion, which is initiated by several stimuli, such as ADP, collagen, thrombin, and so on. Platelets have long been implicated in the pathogenesis of atherosclerosis. With increasing interest in alternatives in the preventive of CVD, research is emerging on the use of spices and herbs. In addition to delivering antioxidant and other properties, spices and herbs can be used in partially or wholly and make foods cardio‐protective. As several metabolic diseases, such as diabe­ tes, CVD, and age‐related degenerative disorders, are closely associated with oxidative processes in the body, the use of spices as a source of antioxidants to combat oxidation warrants further attention. Figure 4.2 shows the mode of actions of different herbs and spices in platelet activation/ aggregation pathways. Clinical trials with the spices should be carried out to establish their roles in the prevention of CVD. From a dietary perspective, the functionality of spices will be exposed through considera­ tion of their properties as foods. As with most foods, the real benefits of including these in the diet are likely to emerge, with

COX inhibitors

ADP binding sites inhibitors Spices and S herbs

Phosphodiesterase inhibitors

GPIIb/IIIainhibitors

Figure 4.2  Effects of spices/herbs at different platelet‐activating pathways.

Effects of Garlic, Onion, Ginger, and Turmeric on Platelet Function

a better understanding of the attributes of health that are best supported by food, and in methodological developments addressing their effects. At present, recommendations are war­ ranted to support the consumption of foods rich in bioactive components, such as herbs and spices. A greater body of scien­ tific evidence, however, is needed in supporting the benefits of spices in the overall maintenance of cardiovascular health.

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Potential In Cardiovascular Health. Current Pharmaceutical Design 23(7): 989–998. Mahmood ZA, Sualeh M, Mahmood SB, Karim MA (2010). Herbal treatment for cardiovascular disease the evidence based therapy. Pakistan Journal of Pharmaceutical Sciences 23(1): 119–124. Dey S, Mukherjee D (2003). Clinical perspectives on the role of anti‐platelet and statin therapy in patients with vascular diseases. Current Vascular Pharmacology 1(3): 329–333. Lv J, Qi L, Yu C, et al. (2015). Consumption of spicy foods and total and cause specific mortality: population based cohort study. BMJ 351: h3942. Khatua TN, Adela R, Banerjee SK (2013). Garlic and cardioprotection: insights into the molecular mechanisms. Canadian Journal of Physiology and Pharmacology 91(6): 448–458. Rahman K, Lowe GM (2006). Garlic and cardiovascular disease: a critical review. Journal of Nutrition 136(3 Suppl): 736S–740S. Steiner M, Lin RS (1998). Changes in platelet function and susceptibility of lipoproteins to oxidation associated with administration of aged garlic extract. Journal of Cardiovascular Pharmacology 31(6): 904–908. Bordia A, Verma SK, Srivastava KC (1996). Effect of garlic on platelet aggregation in humans: a study in healthy subjects and patients with coronary artery disease. Prostaglandins, Leukotrienes and Essential Fatty Acids 55(3): 201–205. Rahman K, Billington D (2000). Dietary supplementation with aged garlic extract inhibits ADP‐induced platelet aggregation in humans. Journal of Nutrition 130(11): 2662–2665.

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inhibitors from pungent constituents of ginger. Thrombosis Research 111(4–5): 259–265. Bartels EM, Folmer VN, Bliddal H, et al. (2015). Efficacy and safety of ginger in osteoarthritis patients: a meta‐analysis of randomized placebo‐controlled trials. Osteoarthritis and Cartilage 23(1): 13–21. Jiang X, Williams KM, Liauw WS, et al. (2005). Effect of ginkgo and ginger on the pharmacokinetics and pharmacodynamics of warfarin in healthy subjects. British Journal of Clinical Pharmacology 59(4): 425–432. Mayanglambam A, Dangelmaier CA, Thomas D, Damodar Reddy C, Daniel JL, Kunapuli SP (2010). Curcumin inhibits GPVI‐mediated platelet activation by interfering with the kinase activity of Syk and the subsequent activation of PLCgamma2. Platelets 21(3): 211–220. Voelcker G, Fortmeyer HP (1979). Intravenous injections in mice by the retrobulbar plexus route. Zeitschrift Fur Versuchstierkunde 21(3): 177–181. Srivastava KC, Bordia A, Verma SK (1995). Curcumin, a major component of food spice turmeric (Curcuma longa) inhibits aggregation and alters eicosanoid metabolism in human blood platelets. Prostaglandins, Leukotrienes and Essential Fatty Acids 52(4): 223–227.

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5 Herbs and Platelet Function ­Abbreviations Used in This Chapter Cyclic GMP, cGMP; cyclic AMP, cAMP, Adenosine diphos­ phate, ADP, Glycoprotein IIb/IIIa, GPIIB/IIIa

5.1 ­Introduction Platelets play a pivotal role in both health and disease, through their central involvement in hemostasis and thrombotic events. The process of primary hemostasis is initiated through platelet adhesion/activation at sites of vascular injury. This is followed by platelet aggregation initiated by several stimuli, such as ADP, collagen, adrenaline, and TXA2. Platelets are also involved in the pathogenesis of atherosclerosis, via different mechanisms, such as shedding of platelet membrane particles, expression of adhe­ sive proteins, cytokine secretion. Platelet‐monocyte aggregates also contribute to the initiation and progression of atherosclero­ sis. There is growing interest in the possible beneficial effects of specific dietary components on cardiovascular health. A recent increase in the popularity of herbs has revived interest in tradi­ tional remedies that have been used for the treatment of CVD. Herbs are used in food for flavoring or fragrances, owing to their aromatic properties. In fact, culinary use typically distin­ guishes herbs from spices. Herbs refers to the leafy green or flowering parts of a plant, while spices are produced from other parts of the plant, including seeds, berries, bark, roots and fruits. Nutraceuticals and Human Blood Platelet Function: Applications in Cardiovascular Health, First Edition. Asim K. Duttaroy © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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Herbal medicine refers to using a plant’s seeds, berries, roots, leaves, bark, or flowers for medicinal purposes [1]. In the early 19th century, extraction and isolation of the active ingredients from plants began, and almost one‐fourth of pharmaceutical drugs are derived from plants. Recently, the World Health Organization estimated that 80% of people worldwide rely on herbal medicines for some part of their primary health care. Herbs have been used as medical treatments since the beginning of civilization, and some deriva­ tives, such as aspirin, reserpine, and digitalis have become main­ stays of human pharmacotherapy. For CVD, herbal treatments have been used in patients with congestive heart failure, systolic hypertension, angina pectoris, atherosclerosis, cerebral insuffi­ ciency, venous insufficiency, and arrhythmia [2]. However, many herbal remedies used today have not undergone careful scien­ tific assessment, and some have the potential to cause serious toxic effects and major drug‐to‐drug interactions. More research is necessary to elucidate the pharmacological activities of the many herbal remedies now being used in CVD. Herbs such as feverfew, garlic, ginger, ginseng, motherwort, St  John’s wort, and willow bark have been found to prevent platelet activation/aggregation via different mechanisms (see Table 5.1). In vitro studies showed promise in the reduction of platelet activation/aggregation for Andrographis, feverfew, garlic, ginger, ginkgo, ginseng, hawthorn, horse chestnut, and Table 5.1  Effects of spices/herbs on platelet function. Spice

Effect on platelets

Garlic

Reduces TxA2 generation; reduced AA incorporation to membrane PL

Onion

Reduces TxA2 and 12‐Lipoxygenase products

Ginger

Reduces aggregation

Cloves

Antiaggregatory; reduces cyclooxygenase and lipoxygnease products

Cumin

Inhibits AA‐induced aggregation

Turmeric

Reduces TxA2 generation; reduced AA incorporation to membrane PL

Herbs and Platelet Function

turmeric. In addition, cranberry, ginkgo, ginseng, green tea, and St John’s wort were found to have potential interactions with warfarin [3]. Furthermore, St John’s wort interacted with clopi­ dogrel and danshen with aspirin. Therefore, repeat testing of platelet function and coagulation studies, particularly for patients on warfarin therapy, may be required after exclusion of herbal medicines. Blood platelets, besides their physiological function, play a central role in the pathogenesis of some CVDs, such as arterial hypertension, atherosclerosis and subsequent ischemic events [4]. Indeed, several experimental studies have shown that plate­ lets became more sensitive to their agonists and also hyperac­ tive [5–8]. Different therapeutic approaches were developed in order to prevent this abnormal platelet hyperactivity, including the consumption of natural products. Platelet activity may play a major role in the development of several diseases, as well as in the stability of atherosclerotic plaques. Aspirin or acetylsalicylic acid is the most widely used anti‐platelet drug, and remains the gold standard of anti‐platelet therapy. Aspirin inhibits platelet‐derived TxA2 synthesis via acetylation of COX‐1. Herbal medicines may have the potential to achieve this anti‐platelet role, and the anti‐thrombotic prop­ erty of dietary nutrients and non‐nutrients of plants and vegeta­ bles origin have been documented [9]. With the high prevalence of herbal use in the world today, further research is necessary to elucidate the pharmacological activities of the many herbal rem­ edies now being used to treat CVD. Evidence is emerging on the anti‐platelet property of herbs. In vitro studies have demonstrated that certain herb extracts are effective in inhibiting platelet aggregation. Markers for platelet activation are activation‐dependent changes in the GPIIb/IIIa complex, exposure of granular membrane proteins, and binding of secreted platelet proteins and those directed against granular membrane proteins, such as P‐selectin. There are several herbs known to have anti‐platelet aggregation. These are Andrographis, cranberry, danshen, dong quai, feverfew, garlic, ginger, Ginkgo biloba, ginseng, green tea, hawthorn, horse chestnut, mother­ wort, St John’s wort, turmeric, and willow bark. A number of herbs contain potent cardioprotective active gly­ cosides, which have positive inotropic actions on the heart. The

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drug digitoxin, derived from either D. purpurea (foxglove) or D. lanata, and digoxin, derived from D. lanata alone, has been used in the treatment of congestive heart failure for many dec­ ades. Some common plant sources of cardiac glycosides include D. purpurea (foxglove), Adonis microcarpa and Adonis vernalis (adonis), Apocynum cannabinum (black Indian hemp), Asclepias curassavica (redheaded cotton bush), Asclepias fruticosa (bal­ loon cotton), Calotropis procera (king’s crown), Carissa spectabilis (wintersweet), Cerebera manghas (sea mango), Cheiranthus cheiri (wallflower), Convallaria majalis (lily of the valley, con­ vallaria), Cryptostegia grandiflora (rubber vine), Helleborus niger (black hellebore), Helleborus viridis, Nerium oleander (ole­ ander), Plumeria rubra (frangipani), Selenicereus grandiflorus (cactus grandiflorus), Strophanthus hispidusand Strophanthus kombe (strophanus), Thevetia peruviana (yellow oleander), and Urginea maritima (squill). Spices and their active principles inhibit 5‐lipoxygenase and also formation of leukotriene C4. In this study, we report the modulatory effect of spice active principles, including eugenol, capsaicin, piperine, quercetin, curcumin, cinnamaldehyde and allyl sulfide on in vitro human platelet aggregation. We have demonstrated that spice active principles inhibit platelet aggre­ gation induced by different agonists, namely ADP (50 μM), col­ lagen (500 μg/ml), arachidonic acid (AA) (1.0 mM) and calcium ionophore A‐23187 (20 μM). Spice active principles show preferential inhibition of arachi­ donic acid‐induced platelet aggregation, compared with other agonists. Among the compounds present in spices, eugenol and capsaicin are the most potent inhibitors of ARA‐induced platelet aggregation. The order of potency of spice principles in inhibiting ARA‐induced in vitro platelet aggregation is: eugenol > capsaicin >curcumin> cinnamaldehyde > piperine> allyl sulfide> quercetin [10]. Eugenol is found to be 29 times more potent than aspirin in inhibiting ARA‐induced human platelet aggregation. Eugenol and capsaicin inhibits TxB2 formation in platelets in a dose‐ dependent manner challenged with ARA, apparently by the inhibition of the COX‐1. Further, eugenol and capsaicin inhibits platelet aggregation induced by agonists collagen, ADP and cal­ cium ionophore, but to a lesser degree compared with ARA. Spices have modulating effects on human platelets.

Herbs and Platelet Function

This chapter describes mostly on the effects of several herbs on human platelet function.

5.2  ­In Vitro Platelet Aggregation Studies: Effects of Different Herb Extracts The effect of different herbs, such as alfalfa, origan, mint, thyme, sage, nettle, onions, garlic, and ginger on platelet and whole blood aggregation in vitro has been investigated [11]. Overnight fasted venous blood was collected from volunteers who had not taken any medication for at least 14 days before donation. Table 5.2 shows the inhibitory effect of aqueous extracts of sev­ eral herbs at 1 mg/ml on human platelet aggregation in platelet‐ rich plasma induced by ADP (10 μM). Twenty‐eight herbs were used as herbs/nutraceuticals on human platelet aggregation in vitro. Among these extracts used, chamomile, nettle, alfalfa, garlic, and onion were the most effec­ tive against ADP‐induced platelet aggregation, inhibiting plate­ let aggregation by more than 45% (P < 0.05). The maximum inhibitory effect (90%) was observed with garlic, followed by alfalfa, (73%), onion (71%), fresh nettle (65%), and chamomile (60%), whereas peppermint and lime flower inhibited platelet aggregation by 25% and 20%, respectively [11]. Other herbs, such as feverfew, hawthorn, dandelion leaves, devils claw, licorice root, lily of the valley, mint, marigold, mistletoe, motherwort, origan, passiflora, prickly ash, rosemary, thyme, and black tea had little or no effect [11]. The dose‐dependent inhibition of ADP‐induced platelet aggregation by aqueous extracts was observed by three herbs – alfalfa, chamomile, and nettle. Both chamomile and alfalfa inhibited collagen‐induced platelet aggregation by 80 and 60%, respectively, whereas nettle had no inhibitory effect [11]. Alfalfa, chamomile, and nettle had very little or no effect on arachidonic acid‐induced platelet aggregation. In addition, these herbs failed to inhibit thrombin‐induced aggregation of washed platelets. Nettle, alfalfa, and chamomile at 1 mg/ml inhibited colla­ gen‐induced whole blood aggregation by 60%, 50%, and 30%, respectively [11]. Both chamomile and alfalfa inhibited TxB2 synthesis in response to ADP and collagen, with a great inhibitory effect observed for collagen‐induced thromboxane

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Table 5.2  Effect of various herbs and plant extracts on human platelet aggregation induced by ADP. Adapted from [11]. Herbs

% Inhibition

Alfalfa

73 ± 12

Black tea

5±2

Chamomile

61 ± 8

Dandelion leaves

0

Devil’s claw

7±2

Feverfew

5±2

Garlic

91 ± 12

Ginseng

1±2

Ginger

6±3

Hawthorn berry

7±1

Horse chestnut

4±2

Lemon grass

3±1

Licorice root

5±3

Lily of the valley

6±2

Lime flower

20 ± 5

Marigold

2±1

Mint

0

Mistletoe

0

Mother wort

3±2

Nettle

65 ± 9

Onion

71 ± 12

Origan

0

Passiflora

1±2

Prickly ash

0

Rosemary

0

Thyme

0

Yarrow

12 ± 5

Herbs and Platelet Function

synthesis (48–53%) compared with ADP (30–41%). ADP‐ induced TxB2 synthesis was inhibited by 41% and 30% by alfalfa (17 ng/ml) and chamomile (20 ng/ml), respectively, compared with control (28ng/ml) [11]. Collagen‐induced TxB2 synthesis (79 ng/ml) was also inhibited by 53% and 48% by alfalfa (37 ng/ml) and chamomile (41 ng/ml), respectively. However, nettle did not inhibit thromboxane synthesis, either induced by ADP or collagen. cGMP levels in platelets cGMP, by 50% and 32%, significantly compared with the control, whereas chamomile had no inhibitory effect. Caulis Spatholobi, Flos Carthami and Rhizoma Curcumae have potent anti‐platelet properties, and their inhibitory actions are mediated via different mechanisms. Caulis Spatholobi inhib­ ited ADP‐induced platelet aggregation but not thrombin‐ induced aggregation, whereas the effect of Caulis Spatholobi and Flos Carthami was associated with suppressing the expres­ sion of P‐selectin [12]. The crude aqueous extract of parsley was evaluated for its anti‐platelet activity in experimental animals on platelet aggre­ gation in vitro and ex vivo. Crude aqueous extract inhibited dose dependently platelet aggregation in vitro induced by thrombin, ADP, collagen and epinephrine [13]. The oral administration of crude aqueous extract (3 g/kg) significantly inhibited platelet aggregation ex vivo, without changes in the platelet counts. The prolongation of bleeding time by crude aqueous extract may be attributed to the observed inhibition of platelet aggregation. These effects could be related, in part, to the polyphenolic com­ pounds present in the crude aqueous extract. These results sup­ port the hypothesis that the dietary intake of parsley may normalize platelet hyperactivity, and are potentially interesting in the development of new prevention strategies of CVD. 5.2.1  Andrographis (Andrographis Paniculata) Andrographis, commonly known as the “king of bitters,” is an herbaceous plant that has been used as a traditional medicine in India, China, Thailand, and Scandinavia. Andrographis contains several active constituents, namely: diterpene lactones, includ­ ing andrographolide (AP1); 14‐deoxy‐11,12‐didehydroandro­ grapholide (AP3); and neoandrographolide (AP4) [14]. In vitro

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studies demonstrate that Andrographis reduces platelet aggre­ gation. One study found that an extract of Andrographis and its active diterpenoids, AP1 and AP3, inhibited thrombin‐induced platelet aggregation at concentrations of 10–100 µg/mL [14]. Andrographis and andrographolide inhibited platelet‐activating factor (PAF)‐induced human blood platelet aggregation in a dose‐ dependent manner. Andrographolide (75 μM) inhibited collagen‐ induced platelet aggregation and relative Ca2+ mobilization. In vitro studies show the potential of Andrographis in reducing platelet aggregation. Future research is suggested to further explore these effects ex vivo. 5.2.2  Cranberry (Vaccinium Macrocarpon) Cranberries (Vaccinium macrocarpon) are primarily used as a juice, or in herbal supplement form for the prevention and treatment of urinary tract infections. Cranberries are a particu­ larly rich source of phenolic phytochemicals, including phe­ nolic acids (benzoic, hydroxycinnamic, and ellagic acids) and fla­ vonoids (proanthocyanidins, anthocyanins, flavonols, and flavan‐­3‐ols) [15, 16]. Studies are suggesting that polyphenols, including those found in cranberries, may contribute to reduc­ ing CVD risk by increasing the resistance of LDL to oxidation, reducing blood pressure, and inhibiting platelet aggregation, via antithrombotic, antioxidant, and anti‐inflammatory mecha­ nisms [16]. There are several case reports of interactions between cran­ berry and warfarin. Although several of the active constituents of cranberry have been found to inhibit platelet aggregation [16], there appears to be no clinical data on the effect of cran­ berry on altering platelet aggregation. Further clinical research is suggested to investigate the effects of cranberries on platelet function. 5.2.3  Feverfew (Tanacetum Parthenium) Feverfew (Tanacetum parthenium) has numerous active con­ stituents, including sesquiterpene lactones (the principal one being parthenolide), flavonoids (including quercetin, apigenin, luteolin), volatile oils, and the coumarin isofraxidin [17]. Platelet

Herbs and Platelet Function

aggregation studies were investigated on 10 patients who had taken feverfew for 3.5–8 years. The platelets of all patients on feverfew characteristically aggregated to ADP and thrombin, and these responses were similar to the control patients who had not used feverfew for at least six months. The threshold for initiating platelet aggregation in response to U46619, a prosta­ glandin endoperoxide, was increased in patients continuously taking feverfew daily for more than four years. In addition, platelet aggregation in response to serotonin was greatly decreased in patients using high doses of feverfew [18]. In vitro studies found that feverfew extract and parthenolide reduced platelet aggregation induced by ADP, adrenaline, ARA, collagen, U46619, and the calcium ionophore, A23187. In addition, feverfew extract and parthenolide reduced platelet serotonin secretion [19]. In vitro studies also found that par­ thenolide enhanced platelet production from megakaryoblas­ tic cell lines, and enhanced platelet production from both human and mouse megakaryocytes. Parthenolide decreased the expression of P‐selectin in platelets, following activation by collagen. There was also a decrease in soluble CD40L release when platelets were pre‐incubated with parthenolide before collagen or thrombin activation [19, 20]. 5.2.4  Green Tea (Camellia Sinensis) Green tea can be consumed as a tea or in supplement form, and has many biologically active constituents, including flavonols and polyphenols. The flavonols include the catechins, which are divided into several components, the epigallocatechin gallates (EGCG) (contributing more than 50% of polyphenols), epigal­ locatechins, epicatechin (EC), and gallates [21]. An inverse rela­ tionship has been found between the consumption of green tea and cardiovascular mortality. Observational studies have shown that the consumption of green tea significantly reduced levels of cholesterol and triglycerides [22]. Green tea has several properties, including antioxidant, anti‐ inflammatory [22], hypoglycemic, anti‐viral, antihypertensive activities, increased insulin sensitivity, improved endothelial function, reduced LDL oxidation [22], and hypocholesterolemic properties [21]. Oral green tea catechins significantly inhibited

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ARA‐induced platelet aggregation at doses of 25 and 50 mg/kg body weight in Sprague‐Dawley rats [23]. In vitro studies found that EGCG isolated from tea reduced ADP‐induced platelet aggregation in a dose‐dependent manner. EGCG also reduced the ADP‐induced phosphorylation of p38 mitogen‐activated protein kinase and heat shock protein. In addition, EGCG inhibited the ADP‐stimulated release of plate­ let‐derived growth factor‐AB and soluble CD40 ligand. Green tea catechin inhibited in vitro platelet aggregation‐induced by ARA, collagen, and U46619. In addition, it also inhibited ATP release from dense granules and ARA release stimulated by col­ lagen in platelets [23]. Green tea may alter the anti‐coagulant effect of warfarin. It is suggested that patients on warfarin who already consume green tea should maintain their current dosage of green tea and moni­ tor their international normalized ratio (INR). 5.2.5  Hawthorn (Crataegus Oxyacantha) Hawthorn (Crataegus oxyacantha) is used as folk medicine for the treatment of diarrhea, gall bladder disease, insomnia, and as an antispasmodic agent in the treatment of asthma. Hawthorn has several active constituents, including flavonoids (e.g., rutin, hyper­ oside, vitexin, and vitexin‐2″‐O‐α‐L‐­rhamnoside), catechin/­EC‐ derived oligomeric procyanidins, triterpenic acids (ursolic, oleanolic, and crataegolic acids) and phenol carboxylic acids (chlorogenic and caffeic acids) [24]. In vitro studies found that hawthorn extract inhibited ADP‐ induced human platelet aggregation and platelet serotonin release [25]. An extract of the flowery heads of hawthorn inhib­ ited synthesis of TxA2. Vitexin‐2″‐O‐rhamnoside, hyperoside, catechin, and EC showed significant inhibition of in vitro bio­ synthesis of TxA2 [24]. Hawthorn shows promise in reducing platelet aggregation. Further clinical trials are suggested to investigate the impact of hawthorn on platelet aggregation and function. 5.2.6  Horse Chestnut (Aesculus Hippocastanum) Horse chestnut (Aesculus hippocastanum) is used as an herbal medicine for chronic venous insufficiency. The main constituents

Herbs and Platelet Function

of horse chestnut are triterpene saponins (escin), flavonoids (e.g., quercetin, kaempferol, EC, proanthocyanidin A2, anthocyanins), and coumarins (e.g., esculin, esculetin) [26]. An in vitro study found that horse chestnut significantly decreased ADP‐induced human platelet aggregation [26]. Clinical trials are required to establish its use on modifying platelet function, and whether or not the coumarin present in  horse chestnut may play a therapeutic role in reducing coagulation. 5.2.7  Motherwort (Leonurus Japonicus) The aerial parts of motherwort (Leonurus japonicus or Leonurus cardiaca) are often used to treat various diseases, such as blood stasis, menstrual disturbance, dysmenorrhea, and amenorrhea. Motherwort has antioxidant, anti‐inflammatory, and analgesic properties. Studies are showing beneficial effects of motherwort on the cardiovascular system. Motherwort contains several active constituents, including alkaloids, diterpenes, flavones, phenylethanoid glycosides, essen­­ tial oils, and spirolabdane diterpenoids. Although not typically used intravenously, a study found a decrease in platelet aggrega­ tion after 15 days of daily intravenous infusions of motherwort (10 mL (5 g/mL) in 250 mL of 5% glucose) [24]. The in vitro effects of the active constituents of motherwort were investigated on ADP‐induced platelet aggregation. Bis‐ spirolabdane diterpenoids 1, 2, and the positive control drug (clopidogrel hydrogen sulfate), at a concentration of 0.1 mM, significantly reduced ADP‐induced platelet aggregation significantly [27]. Compound 3 ((2S,5S)‐2‐hydroxy‐2,6,10,10‐­ tetramethyl‐1‐oxaspiro[4.5]dec‐6‐en‐8‐one), at a concentration of 10 μM, inhibited platelet aggregation induced by ADP [28]. Motherwort shows potential as an anti‐platelet herbal medi­ cine, and clinical trials are suggested to test this possibility. 5.2.8  St John’s Wort (Hypericum Perforatum) One of the oldest used and most widely investigated herbal medicines, St John’s wort, or Hypericum, is used in the treat­ ment of mild‐to‐moderate depression, seasonal affective disor­ der, sleep difficulties, and anxiety. The leaves and flowering tops

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Nutraceuticals and Human Blood Platelet Function

of St John’s wort contain hypericin, hyperforin, flavonoids, naphthodianthrones, and xanthones [29]. St John’s wort can inhibit or induce various CYP isozymes and/or P‐glycoprotein [29]. Numerous drugs are substrates for CYP isozymes and/or P‐glycoprotein, and this may be the main origin of interac­ tions between herbal medicines and drugs [29]. St. John’s wort enhances the platelet inhibitory activity of clopidogrel by increasing CYP3A4 metabolic activity. Healthy volunteers were tested for St John’s wort responsive­ ness after consuming 300 mg of clopidogrel. After a washout period, the mild‐responders (n = 10) received 14 days of St John’s wort (300 mg, three times a day) followed by a second 300 mg clopidogrel dose. St John’s wort significantly decreased ADP‐ induced platelet aggregation, and increased CYP3A4 activity significantly [30]. The combined use of anti‐platelet or anticoagulant drugs, especially those primarily cleared via CYP isoenzymes, with St John’s wort should be done with caution until clinical trials determine a safe dosage range of St John’s wort with these drugs. However, this does open the door for potential positive or addi­ tive effects by St John’s wort, thus leading to a possible reduction in the dosage of anti‐platelet medications. Further research is recommended on for deeper understanding of this interaction. 5.2.9  Willow Bark (Salix Alba) Willow bark is commonly regarded as one of the first examples of modern drug development from a herb. This can, historically, be traced back to ancient Egypt, where extracts of willow bark were used to treat inflammation. Willow bark contains several active constituents, most notably, salicin. The oxidation of sali­ cin results in a new substance, termed “salicylic acid”, and the acetylated derivative of this substance (acetylsalicylic acid) was manufactured into aspirin. The origins for the development of acetylsalicylic acid were from the historical uses and therapeutic actions of willow bark that included anti‐inflammatory, analge­ sic, anti‐rheumatic, and anti‐pyretic actions [31]. In a double‐blind, randomized controlled trial, 35 patients suffering from acute exacerbations of chronic low back pain randomly received either willow bark extract (240 mg salicin)

Herbs and Platelet Function

per day (n = 19) or placebo (n = 16). In addition, 16 patients with stable chronic ischemic heart disease were given 100 mg acetyl­ salicylate per day. The mean maximal ARA‐induced platelet aggregation was 78%, 61%, and 13% in the placebo, willow bark extract, and acetylsalicylate groups, respectively. Acetylsalicylate had a significant inhibitory effect on platelet aggregation, compared with the willow bark extract. In addition, there was a significant difference between the placebo and the willow bark‐­ treated groups in the maximal platelet aggregation induced by ARA and ADP. No statistical difference was found between the groups for collagen‐induced platelet aggregation. This study showed that daily consumption of willow bark extract with 240 mg salicin per day affected platelet aggregation to a lesser magnitude than acetylsalicylate [32].

5.3 ­Effects of Herbs on Signaling Molecules in Human Platelets The TxA2 pathway is the most important pathway involved in CVD events in cardiovascular patients (Figure 5.1). Aspirin has been used for decades, but other TxA2 pathway inhibitors have been investigated, and are potentially more potent at inhibiting TxA2‐mediated events that lead to a pro­ gression of atherosclerosis and thrombus formation. To deter­ mine whether the inhibitory effect herbal extracts on platelet aggregation was due to the decreased synthesis of TxA2, levels of TxB2, the stable breakdown product of TxA2, were measured in post‐aggregated plasma. TxB2 was measured in the plasma after platelet aggregation induced by ADP or collagen in the presence and absence of these herbal extracts. Both chamomile and alfalfa inhibited TxB2 synthesis in response to ADP and collagen, with a great inhibitory effect observed for collagen‐induced TxA2 synthesis (48–53%), com­ pared with ADP (30–41%). ADP‐induced TxB2 synthesis was inhibited by 41% and 30% by alfalfa (16 ng/ml) and chamomile (19 ng/ml), respectively, compared with control (28 ng/ml). The effect was observed by all of these herbs, suggesting that platelet COX‐1 per se was not inhibited by these extracts. Inhibition at upstream of COX‐1 at the release of ARA from membrane

95

Phospholipases Herbs/spices

Arachidonic acid, 20:4 n-6

Herbs/spices

Isoprostanes HETEs

Prostaglandin G2

Cyclooxygenase

Prostaglandin H2

Thromboxane receptors Platelet

Thromboxane synthase Thromboxane A2

Platelet Activation/Aggregation

Figure 5.1  The thromboxane pathway in platelets.

Herbs and Platelet Function

phospholipids is, therefore, a more likely possibility. However, further investigations are required for better understanding the mode of actions of these extracts. Both nettle and alfalfa appear to inhibit platelets via the mod­ ulation of cyclic nucleotides. Both of these herbs increased cGMP levels in platelets. cGMP is known to inhibit platelet aggregation. It is important to note that the aggregation of plate­ lets was performed in vitro, in a different environment than the blood stream. Studies on human volunteers would be required to ascertain their effects in vivo. Nevertheless, this study showed that consuming these herbs may be beneficial in CVD. Chamomile, nettle and alfalfa are equally effective at inhibit­ ing platelet aggregation in vitro. These data suggest that three herbs may have potential for inhibiting platelet activation and platelet‐mediated events in CVD. Further investigation of the effects of individual herbs on ex vivo platelet function is required, in order to elucidate their role in the relationship between herbs, platelet aggregation response and the risk reduction for CVD.

5.4 ­Conclusions CVD is a multifactorial disease, where platelet hyperactivity plays a very important role, that affects a large proportion of the population. Herbal medicines have been used in the manage­ ment of CVD, either alone or in conjunction with standard medical therapy, via anti‐platelet, anti‐inflammatory, and anti­ oxidant actions. Herbal medicines, such as feverfew, garlic, gin­ ger, ginseng, motherwort, St John’s wort, and willow bark, Andrographis, feverfew, garlic, ginger, ginkgo, ginseng, haw­ thorn, horse chestnut, and turmeric have been shown to reduce platelet aggregation in vitro. Herbal products that modify platelet function have the poten­ tial to be used in the management of CVD. Since herbs can interact with so many other anti‐platelet and anti‐coagulant drugs, proper guidelines should be followed, or more informa­ tion may be required on herbs‐drugs interactions.

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thromboxane A2 biosynthesis in vitro by the main components of Crataegus oxyacantha (Hawthorn) flower heads. Prostaglandins, Leukotrienes and Essential Fatty Acids 50(4): 173–175. Grice ID, Rogers KL, Griffiths LR (2011). Isolation of Bioactive Compounds That Relate to the Anti‐Platelet Activity of Cymbopogon ambiguus. Evidence‐Based Complementary and Alternative Medicine 2011: 467134. Felixsson E, Persson IA, Eriksson AC, Persson K (2010). Horse chestnut extract contracts bovine vessels and affects human platelet aggregation through 5‐HT(2A) receptors: an in vitro study. Phytotherapy Research 24(9): 1297–1301. Xiong L, Zhou QM, Peng C, et al. (2015). Bis‐spirolabdane diterpenoids from Leonurus japonicus and their anti‐platelet aggregative activity. Fitoterapia 100: 1–6. Xiong L, Zhou QM, Peng C, et al. (2013). Sesquiterpenoids from the herb of Leonurus japonicus. Molecules 18(5): 5051–5058. Russo E, Scicchitano F, Whalley BJ, et al. (2014). Hypericum perforatum: pharmacokinetic, mechanism of action, tolerability, and clinical drug‐drug interactions. Phytotherapy Research 28(5): 643–655. Lau WC, Welch TD, Shields T, Rubenfire M, Tantry US, Gurbel PA (2011). The effect of St John’s Wort on the pharmacodynamic response of clopidogrel in hyporesponsive volunteers and patients: increased platelet inhibition by enhancement of CYP3A4 metabolic activity. Journal of Cardiovascular Pharmacology 57(1): 86–93. Schmid B, Ludtke R, Selbmann HK, et al. (2001). Efficacy and tolerability of a standardized willow bark extract in patients with osteoarthritis: randomized placebo‐controlled, double blind clinical trial. Phytotherapy Research 15(4): 344–350. Krivoy N, Pavlotzky E, Chrubasik S, Eisenberg E, Brook G (2001). Effect of salicis cortex extract on human platelet aggregation. Planta Medica 67(3): 209–212.

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6 Tomato Extract and Human Platelet Functions ­Abbreviations Used in This Chapter CVD, Cardiovascular disease; tAF, Total active fraction; ACE, Angiotensin‐converting enzyme; EPA, Eicosapentaenoic acid, 20:5n‐3; TRAP, thrombin receptor‐activating peptide; DHA, docosahexaenoic, 22:6n‐3; WSTC, Water‐soluble tomato concentrate; EFSA, European Food Safety Authority; PDI, Protein disulphide isomerase; TF; Tissue factor, PRP, platelet rich plasma; VSMC, vascular smooth muscle cells; TxA2, thromboxane A2

6.1 ­Introduction During the last 50 years, tomato (Lycopersicon esculentum) has become a highly consumed healthy food [1]. Tomato contains several components that are beneficial to overall health, including vitamin E, flavonoids, phytosterols, carotenoids, and several water‐soluble vitamins and minerals [2–4]. Since oxidative stress triggers inflammatory disorders, the basis for the development of several diseases, such as immune disorders, atherosclerotic lesions and rupture of plaque [5], the antioxidants present in tomato are, therefore, believed to slow the progression of many diseases, including cardiovascular disease (CVD) [6]. Platelets play an important role in CVD, both in the pathogenesis of atherosclerosis and in the development of acute thrombotic events (Figure 6.1). Nutraceuticals and Human Blood Platelet Function: Applications in Cardiovascular Health, First Edition. Asim K. Duttaroy © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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Nutraceuticals and Human Blood Platelet Function

Pathological disorders (Insulin resistance, diabetes, hyperlipideamia, ageing, smoking, sedentary life style, high fat diets etc.)

Platelet hyperactivity

Atherosclerosis

CVD events Coronary heart disease angina, myocardial infarction cerebrovascular disease ischaemic stroke

Figure 6.1  Hyperactivity of platelets and their roles in the development of CVD. Platelets play an important role in CVD, both in the pathogenesis of atherosclerosis and in the development of acute thrombotic events. Hyperactive platelets are involved in the development of atherosclerosis by different mechanisms, such as membrane shedding, growth factor secretion, and expression of several adhesive factors. In addition, hyperactive platelets are involved in the well‐known penultimate thrombotic events. Adapted from [6].

Their importance in CVD is indirectly confirmed by the benefit of anti‐platelet agents such as aspirin, clopidogrel, and glycoprotein IIb/IIIa inhibitors abciximab/eptifibatide [7]. In fact, intravascular thrombosis is a factor in the generation of a wide variety of CVDs. Platelets in individuals with diabetes, sedentary life style, obesity, and insulin resistance show increased activity at baseline and in response to agonists, ultimately leading to increased aggregation and plaque development [8–10].

Tomato Extract and Human Platelet Functions

Aspirin remains a cornerstone of anti‐platelet therapy, but does not benefit all patients equally, as evidenced by the phenomenon of aspirin resistance [7]. Aspirin therapy is also responsible for a number of serious side‐effects, rendering it unsuitable for use in primary prevention of CVD [11, 12]. However, very few new anti‐thrombotics are currently progressing beyond Phase II trials, and those that have been developed are similarly unsuitable for use in primary prevention [12]. There is an interest in naturally occurring compounds which might lack the side‐effects currently so prevalent. We therefore systematically investigated the effects of bioactive compounds in fruits and vegetables on human blood platelet aggregation, and utilized the findings to characterize the mechanisms involved in this process, in the hope of identifying potential dietary anti‐platelet components. In a variety of studies, it was demonstrated that water‐soluble components of tomatoes are capable of inhibiting platelet aggregation both in vitro and in vivo [13–16]. These water‐­ soluble tomato components were also found to inhibit angiotensin‐converting enzyme (ACE), and to relax the vascular endothelium, the other important limbs of the cardiovascular system [17, 18]. A water‐soluble tomato extract containing all the bioactive components was developed, and later given the trade name Fruitflow®. Fruitflow® is now an established naturally derived functional food ingredient. Since its discovery in 1999, several mechanistic studies and human trials with Fruitflow® have been carried out. Studies included localization of the anti‐platelet activity within the tomato fruit, its modes of action, its stability under various conditions, and identification of the compounds with anti‐platelet activity. The presence of a range of compounds suggested that all have anti‐platelet activity, but act on different parts of the platelet activation/aggregation pathway. The chemical properties of the active compounds indicated their potential suitability as therapeutic agents or as functional food ingredients. There are several excellent reviews available on overall health benefits of tomatoes [1, 2, 4, 5, 19, 20]. This chapter will discuss the anti‐platelet factors present in tomato, as well as mechanisms of action and efficacy established in human trials.

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6.2 ­Epidemiology of Tomato Consumption and Cardiovascular Disease Risk Reduction Epidemiologic studies focused on tomato and tomato products  have associated their intake with a reduced risk of CVD [19, 21, 22]. However, tomatoes and tomato‐based products are important dietary sources of lycopene in observational studies, and most human lycopene trials are performed using tomato‐ based interventions. Studies showed that increased plasma lycopene levels were associated with reductions in CVD risk factors [23–25]. The strongest population‐based evidence for the beneficial effects of tomato lycopene came from a multi‐ centre case‐control study [26]. However, a subsequent dietary intervention study observed that consumption of a carotenoid‐ rich diet did not have an effect on plasma antioxidant status or markers of oxidative stress [27]. Several [25, 28], but not all [28–32] prospective studies relating circulating lycopene concentrations and CVD risk have reported inverse associations, while studies based on dietary intake did not find any such significant associations [32–36]. Also, the focus on lycopene in tomatoes cannot explain the fact that, taking tomato consumption into account, individuals in the Mediterranean area have a lower risk of CVD, when compared with their North American and other European counterparts [2, 37, 38]. Thus, it is difficult to separate out the potential lycopene contribution to cardiovascular health from the overall contribution from tomato products and other components present in tomatoes. The lack of coherence between available epidemiological data and dietary intervention data underscores the requirements for further studies to unravel other non‐lycopene components in tomato, and their roles in CVD risk reduction. Tomatoes contain several known and unknown compounds that might affect platelet function, lipid metabolism, blood pressure and endothelial function – important determinants of CVD [5, 39]. Lycopene or other antioxidants act through inhibition of LDL oxidation alone, and so may only partly contribute to the CVD risk reduction. Recent studies showed that tomato also contains gamma‐ aminobutyric acid, 13‐oxo‐9,11‐octadecadienoic acid, and

Tomato Extract and Human Platelet Functions

esculeoside A, which may provide heart and psychological health benefits [40]. 13‐oxo‐ODA is, however, found only in tomato juice [41]. Esculeoside A reduces plasma lipids and may, therefore, ameliorate atherosclerotic lesions in ApoE‐deficient mice [42]. This indicates that other unidentified compounds (non‐lycopene compounds) may also contribute to the cardio‐ protective effects of tomatoes, as observed in epidemiological and interventional studies.

6.3 ­ In Vitro Studies with Water‐Soluble Tomato Extract on Human Blood Platelet Aggregation The anti‐aggregatory effects of different aqueous fruit extracts on human platelets in vitro have been published previously [13]. The maximum inhibitory effect (70–75%) was found to be with tomato and kiwi fruit extracts, whereas apple and pear had very little activity (2–5%). Grapefruit, melon and strawberry had intermediate activities on platelet aggregation (33–44%). The anti‐platelet potential of the fruits tested appeared to have no relationship with their antioxidant activity [13]. The anti‐platelet compounds in tomato had a molecular mass less than 1000 Da, were highly water soluble and stable to boiling. The compounds of interest were concentrated into an aqueous extract produced by homogenising fresh tomatoes, removing particulate matter. The aqueous extract was then further fractioned by gel filtration, using a Biogel P2 column [13]. Adenosine, a known anti‐platelet factor, was identified in one fraction, but its removal from the whole extract did not substantially decrease the anti‐platelet activity, indicating the presence of additional, different anti‐platelet agents. Further work showed that the aqueous tomato extract consisted largely of soluble sugars (85–90% of dry matter), which showed no in vitro anti‐platelet activity [15]. The non‐sugar material that was ­isolated (tomato total active fraction, tAF) accounted for 4% of the aqueous tomato extract dry matter, and showed strong ­inhibition of platelet aggregation in vitro (Figure 6.2). Isolation of many individual components from tAF followed,  and it was found that most fell into one of three

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Nutraceuticals and Human Blood Platelet Function Tomato extract preparation protocol Ripe tomatoes

Homogenisation and centrifugation at 3000xg at 25 C for 15 min

Ultrafiltration with membrane MW cut-off 1000 da

Aqueous extract (soluble sugars 85–90%) Inactive (soluble sugars 85–90%) are removed by solid-phase extraction with styrene divinylbenzene cartridge at pH 2.5

Total active fraction (tAF) is 4% of the aqueous extract dry matter Figure 6.2  Outline of tomato extract preparation. Tomatoes were homogenized and the juice was centrifuged at 3000 g for 25 min. The supernatant was then filtrated, using a filter with molecular cut‐off less than 1000 Da. The filtrated fraction was de‐sugarized by solid‐phase extraction with a styrene divinylbenzene cartridge at pH 2.5. Total active fraction (tAF) is 4% of the aqueous extract dry matter.

categories – nucleosides, simple phenolic derivatives and flavonoid derivatives (Table 6.1). All showed anti‐platelet activities consistent with their compound categories. Proteomic experiments carried out to examine effects of tAF on platelet‐signaling pathways showed that tAF components altered a range of platelet functions. One of the most strongly affected proteins was protein disulphide isomerase (PDI), an oxidoreductase which catalyses the formation and the isomerization of disulfide bonds. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding [43, 44]. Other investigators [45] have reported similar functional effects after blockage of cell‐surface thiol isomerases. Glycosides related to quercetin, of which several are present in tAF, have

Tomato Extract and Human Platelet Functions

Table 6.1  Composition of active components. Components

Mg per gm of soluble solids

Adenosine 5’ monophosphate

4063

Cytidine

1247

Uridine

1020

2‐deoxycytidine

94

Adenosine

919

Guanosine

530

Deoxyadenosine

5

Deoxyguanosine

38

Hydroxymethylfurfural

16

Furaneol

105

3‐0‐methyl adenosine

480

Caffeoyl‐3‐0‐glucoside

162

Feruloyl derivative

239

Chlorogenic acid

227

Coumaric acid

0.1

Benzoic acid

115

Rutin

1.3

Myricetin

68

Querecetin

10

Luteolin

70

been shown to interact with PDI in this way [46, 47]. Interaction of polyphenols with PDI suggested a possible mechanism by which tomato extract components could inhibit different pathways of platelet aggregation. Figure 6.3 describes the functional effects of tAF. tAF components were therefore examined in a series of experiments. tAF and its sub‐fractions F1, F2 and F3, prepared by semi‐preparative reversed‐phase HPLC, as described by O’Kennedy et al. (2006) [48], were observed to prevent activation of integrin αIIbß3 (i.e., GPIIb/IIIa). Inhibition of the GPIIb/IIIa activation step  –  which is common to multiple

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AF1 1

3000

4 6

2000 mA

7

AF3 1000

AF2 2

3

5

0 0.0

2.5

5.0

7.5

10.0

12.5 Minute

15.0

17.5

20.0

22.5

25.0

Tomato Extract and Human Platelet Functions

aggregation pathways – could underlie the wide‐ranging effects of tAF [15]. This is consistent with the observation that basal platelet cyclic AMP concentrations (controlled by phospholipase C enzyme family‐mediated cascade reactions) are unaltered by tomato extract active components in vitro. In addition, tAF reduced the expression of P‐selectin (CD62P) on the platelet surface in response to ADP‐induced platelet activation in whole blood [15]. In resting platelets, P‐selectin is localized in the membranes of platelet α‐granules. On platelet activation, it is redistributed to the platelet surface, where it initiates adhesion to leukocytes. Under conditions of blood flow and shear stress, this glycoprotein promotes platelet cohesion and stabilizes newly formed aggregates. Thus, tAF components can potentially affect the size and longevity of platelet aggregates. tAF components were also found to affect the binding of tissue factor (TF) to activated platelets, at least in part, due to effects on P‐selectin. These results, demonstrating the actions of tAF on different platelet functions, were all consistent with potential effects mediated partly through polyphenols and PDI, and partly through nucleosides elevating cAMP and cGMP levels in platelets [13, 48]. Effects on TF binding suggested that tAF components could have a larger effect on some aspects of the coagulatory response, such as thrombin generation, than previously imagined. Figure 6.4 summarizes the actions of different ingredients of Fruitflow® on the platelet activation pathway.

Figure 6.3  HPLC chromatography of total active fraction (tAF) isolated from tomato. A: The HPLC chromatogram shows the three sub‐fractions (AF1, AF2, and AF3), which are monitored by ultraviolet absorbance at 254 nm. Components within these sub‐fractions that have anti‐platelet activity in vitro (data not presented here) are numbered on the chromatogram. B: HPLC chromatogram of AF2, which was obtained under altered chromatographic conditions suitable for the separation of these components. C: HPLC chromatogram of AF3, which was obtained under altered chromatographic conditions to obtain increased component separation. Adapted from Am. J. Clin. Nutrition 2006, 84, 570–579. American Society for Nutrition.

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tAF

Thrombin

VWF

Collagen

Lowers expression of active form of GIIb/IIIa GP1b/IX/V

GPVI

−

α11bβ3

α2β 1

Inside-out Gαq signaling α G 12 α G j PAR-1 Gαq Gα12 Gαi Gαq PAR-4 Thrombin

Fibrinogen VWF

+

P2Y1

ADP

P2Y12

Outside-in signaling

Gαz

Gαq

Gα12

TP α

2A

TxA2

Epinephrine

in

pir

As

COX

ARA Irreversible inhibition of COX

Tomato Extract and Human Platelet Functions

6.4 Fruitflow®: Compositional and Structural Aspects Fruitflow® is now made in two ingredient formats: Fruitflow® 1 and Fruitflow® 2. The raw material for both formats is high grade, minimally processed tomato commodity products. Fruitflow® 1 is a syrup, of which more than 50% w/w comprises tomato‐derived carbohydrates, and ≈ 3% w/w comprises known bioactive compounds with measured anti‐platelet activity. This ingredient format is especially suitable for use in drinks and foods with high water content. Fruitflow® 2 is a low carbohydrate powder, of which more than 55% w/w comprises bioactive compounds, dried to produce a tablet‐grade powder. Fruitflow® 2 can be compressed into tablets, and has flow properties which render it suitable for capsule formation or for use in dry‐blend food processes. Both ingredient formats are lycopene‐ and fat‐free, low in inorganic salts and low in organic acids. The individual bioactivity profiles of compounds occurring in Fruitflow® are three representative components, with one from each of the three broad fractions F1, F2 and F3 [15] having been selected. These three compounds were: adenosine, which represents a group of nucleosides/nucleotides found in F1; chlorogenic acid, which represents a group of phenolic derivatives found in F2; and rutin, which represents a group of flavonoid derivatives found in F3. Figure 6.4  Platelet activation via multiple pathways and sites of action of Fruitflow® ingredients. Key agonists, their receptors and triggering signaling pathways involved in platelet activation and subsequent aggregation. In addition, the mechanisms by which Fruitflow® ingredients anti‐platelet effects are detailed. VWF: Von Willebrand factor; TxA2: thromboxane‐A2; TP: thromboxane receptor; ADP: adenosine diphosphate; PAR: proteinase activated receptor. The activation of platelets is accompanied by a conformational change in glycoprotein (GP) IIb‐IIIa exposes a binding site for fibrinogen and the release of aggregating agents such as TxA2 and ADP. Adenosine nucleotides signal through P2 purinergic receptors (P2Y) on the platelet membrane. Activation of these receptors initiates a complex signaling cascade that ultimately results in platelet activation, aggregation and thrombus formation. Thrombin acts via cell surface Protease Activated Receptors (PARs). Both stimulate PLC, giving rise to PIP2 hydrolysis and consequent activation of PI3K. GPIV: collagen receptor. Adapted from the DSM publication.

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6.5 ­Human Trials After standardization of the ingredients of Fruitflow®, a unified set of studies was carried out to establish the efficacy of these ingredients ex vivo. Studies which established the onset time of an acute anti‐platelet effect after oral ingestion of a dose of Fruitflow® 1 have been published elsewhere [15]. These studies showed that, in all subjects, an acute lowering of platelet aggregability to ADP and collagen was observed at a time three hours after consuming Fruitflow®. The range of onset times was from 1.5 hours to three hours after consumption (Figure 6.5). In contrast, the normal diurnal increases in platelet aggregability were illustrated in subjects consuming the control supplement over the time course measured. The persistence of this acute effect varied between individuals but, in all cases, platelet aggregability returned to baseline 18 hours after consumption of a single dose of Fruitflow® [16]. On average, these studies have shown an inhibition of the platelet response to ADP agonist of approximately 17–25%, and an inhibition of the response to collagen of approximately 10–18%. Platelet aggregation induced by arachidonic acid and thrombin receptor‐activating peptide (TRAP) have also been shown to fall after Fruitflow® administration. A study in which Fruitflow® 1 was administered to 93 healthy men and women [16] showed that some variability in response may occur, with men responding more than women, and subjects with higher risk factors for CVD responding more highly than others. A dose response was established in studies administering different amounts of Fruitflow® 1. This dose‐response work established that a dose of Fruitflow® equivalent to 65mg tAF, or approximately three average bowls of tinned tomato soup, already caused close to the maximum level of platelet inhibition achievable by this extract, and that no significant gain would be obtained in an acute setting from increasing the dose. The anti‐platelet effects observed in all matrices were similar to those seen in previous studies. Studies examining the effects of daily consumption of Fruitflow® showed that the size of the anti‐platelet effect observed after consuming a single dose of Fruitflow® daily for two or for

Tomato Extract and Human Platelet Functions

% Change from baseline (t0) aggregation % Change from baseline ADP-mediated aggregation

10.0 5.0

t 1.5 hr

t 3 hr

t 6 hr

n = 23

Control

0.0 −5.0 −10.0 −15.0 −20.0 −25.0 −30.0

Tomato extract ***

***

O’Kennedy et al. 2006a, AJCN

Figure 6.5  The effect of supplementation with control or tomato extract treatment on ex vivo platelet aggregation, induced by ADP agonist over a seven‐hour period.ADP‐induced platelet aggregation was significantly lower than baseline values after supplementation with tomato extract, but not after supplementation with the control drink. The overall differences between the tomato extract and control drinks were significant at both optimal (overall mean Δ% in tomato extract group: −1.58 ± 0.71%; overall mean Δ% in control group: 2.10 ± 1.15%; P = 0.03) and suboptimal (overall mean Δ% in tomato extract group: −15.23 ± 2.19%; overall mean Δ% in control group: 1.86 ± 3.56%; P < 0.001) ADP concentrations.Changes from baseline aggregation were observed at each time point for the tomato extract group. When platelets were stimulated with suboptimal concentrations of ADP, significant differences from the control group were observed for the tomato extract group at three and six hours. When optimal ADP concentrations were used, the time × treatment interaction was not significant at any time point. No significant differences arising from the different carrier volumes were observed in either the control or tomato extract group at any time point.Adapted from Am. J. Clin. Nutrition 2006, 84, 570–579. American Society for Nutrition.

four weeks was not significantly different from the size of effect observed after a single dose – that is, the observed effects were not cumulative. Suppression of platelet function achieved through chronic consumption was continuous – measurements of platelet function were made in fasted subjects in the morning, approximately 24 hours after consumption of their last Fruitflow® dose, and suppression of original baseline platelet function was observed after two and four weeks. Compounds found in Fruitflow® have been shown to affect many aspects of platelet function, including (via effects on TF

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immobilization and signaling) thrombin generation. However, in all intervention studies undertaken, clotting time measurements showed no significant increases from baseline levels. Fruitflow® does not directly affect blood coagulation at any dose tested. Even without affecting blood coagulation directly, many anti‐platelet drugs, taken on a chronic basis, give rise to excessive platelet inhibition, and are associated with internal bleeding. These potentially serious side‐effects mean that anti‐ platelet therapy, a fundamental aspect of CVD secondary prevention, is contraindicated for primary prevention, as the benefit conferred (lowering risk of a first CVD event in relatively low risk groups) is outweighed by the increased risk of gastric or intracranial bleeding [49]. This judgment was recently revisited by the US FDA, in the context of increasing obesity and type 2 diabetes mellitus levels in relatively young populations, but was upheld [50]. The known side effects of existing anti‐platelet drugs related to internal bleeding were clearly pertinent for consideration during Fruitflow® development. However, Fruitflow® differs fundamentally from anti‐platelet drugs in the reversibility of its action. The widely used anti‐platelet drugs have irreversible mechanisms of action. Over the course of ten days, approximately 90% of the circulating platelet population can be irreversibly affected for the lifetime of those platelets. This level of platelet inhibition is then maintained by daily drug treatment. Conversely, the anti‐platelet effects of Fruitflow® are not irreversible or cumulative, and can be overcome by increased agonist concentrations. This very significant difference in mode of action renders Fruitflow® suitable for use by the general population as a dietary functional ingredient, while anti‐platelet drugs cannot be used. As Fruitflow® is designed as a food ingredient, with potential for incorporation into a variety of food products, a specific study was undertaken to examine the likely effects of overconsumption. As the amount of Fruitflow® in any one food product serving is low, equivalent to approximately three bowls of tinned tomato soup, and as dose‐response studies had shown that increasing the dose significantly would not result in a much bigger acute effect on platelets, no significant dangers were anticipated.

Tomato Extract and Human Platelet Functions

6.6 ­Comparing the Dietary Anti‐Platelet Fruitflow® with the Anti‐Platelet Drug Aspirin A study comparing a single dose of Fruitflow® with 75 mg aspirin, either as a single dose or taken continuously for one week, was undertaken in order to benchmark the effects of a dietary anti‐platelet [51]. A comparison of the effects of Fruitflow® and aspirin on the platelet proteome was first carried out, to examine  similarities and differences in mechanisms of action. This comparison showed that aspirin and Fruitflow® affect broadly similar proteomic pathways, with aspirin affecting the signaling pathways more strongly than Fruitflow® – assuming full metabolism of the entire ingested dose. Proteins affected by Fruitflow® and aspirin are associated with platelet structure, platelet coagulation, platelet membrane trafficking and platelet secretion  –  actin‐binding proteins, fibrinogen beta chain 5, Ras‐related proteins, redox system proteins and HSP70s. Of the 26 proteins with altered expression after treatment, 11 were affected by both Fruitflow® and aspirin, 14 by aspirin alone and one by Fruitflow® alone. The single protein affected only by Fruitflow® was identified as PDI, known to disrupt inside‐out signaling, as described previously. The proteomic experiments suggested that, apart from disruption of disulfide bonds, broadly similar alterations in the platelet might be expected after Fruitflow® and aspirin treatment. The intervention study which followed in 47 healthy subjects showed that the effects of a single dose of Fruitflow® were similar, in terms of anti‐platelet action, effects on thromboxane  synthesis, and time to form a primary haemostatic clot (PFA‐100 closure time), to those of a single 75 mg dose of aspirin. When aspirin was taken daily for seven days, the associated increase in PFA‐100 closure time was three times higher than that associated with a single aspirin dose [51]. The cumulative anti‐platelet effect of aspirin, when taken daily, is well known, and reflects its irreversible disabling of platelet COX‐1 and associated signaling. Fruitflow®’s effects are not cumulative in this way, as its effects do not irreversibly disable platelet signaling pathways.

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Thus, taking the results for the study population as a whole, daily aspirin supplementation may be viewed as approximately three times as efficacious as daily Fruitflow® supplementation, due to the irreversibility of its action. This overall result seemed to echo the proteomic data, but further examination showed that it masked some interesting behavior in study subgroups. The anti‐platelet effects of aspirin in healthy subjects are extremely heterogeneous, with some subjects experiencing a very large increase in time to form a primary haemostatic clot, while others respond poorly. Approximately 50% of aspirin responders had a response to the drug which was lower than the average response for the treatment group, in terms of time to form a primary haemostatic clot. This group of subjects had a residually strong response to collagen after seven days of aspirin treatment, and over one‐third of the group responded better to Fruitflow® supplementation than to seven‐day aspirin supplementation. At the other end of the spectrum, for 18% of the study population, taking aspirin for seven days more than trebled the time to clot [51]. This underlines the reasons behind the known internal bleeding risks associated with aspirin, and its unsuitability for use in primary prevention. While the response to Fruitflow®, in terms of time to clot data, was also heterogeneous, it was markedly less so than the response to aspirin. The majority of the subject group experienced increases in time to form a primary haemostatic clot of up to two‐fold, with fewer subjects at either extreme. It would appear that the proteomic predictions of stronger aspirin‐led effects on platelet signaling may not be observed ex vivo, possibly due to wide variability in the extent of aspirin metabolism, but also possibly due to differences in the relative importance of platelet collagen signaling pathways between individuals. Fruitflow®, with its wider range of anti‐platelet compounds, may have a less variable metabolism and, thus, may achieve its more moderate effects more widely. These more moderate effects, which can be related to the reversi­bility of the anti‐platelet action of Fruitflow®, rather than its mode of action per se, render it a possible option for use in primary prevention of CVD, in contrast to aspirin at any dosage. However outcomes‐based studies on dietary supplements such as this are needed before their true potential can be properly assessed.

Tomato Extract and Human Platelet Functions

6.8 ­EFSA Approval of Fruitflow® In 2006, the European Union (EU) adopted a regulation on the use of nutrition and health claims for foods which lays down harmonized EU‐wide rules for the use of health or nutritional claims on foodstuffs (Regulation (EC) No 1924/2006). One of the key objectives of this regulation is to ensure that any claim made on a food label in the EU is clear, and is substantiated by scientific evidence. Different categories of claim are defined. Health claims are defined as pertaining to relationships between food and health, either with regard to a function of the body (Article 13 claims), or with regard to reducing a risk factor for a disease (Article 14a claims), or with regard to children’s development (Article 14b claims). Nutrient claims are defined as pertaining to foods with particular nutritional properties with regard to either the energy, or the nutrients, that they provide. Allowed nutrient claims are clearly defined within the Regulation. However, companies wishing to associate their food or ingredient with a health claim must submit a dossier in support of the desired claim, which is then assessed by the EFSA Panel on Dietetic Products, Nutrition and Allergies. Key to the dossier is the inclusion of human intervention studies showing evidence of the health benefit claimed, for the food/ingredient of interest. More than 2200 unique claims have been submitted for assessment to date, of which over 95% have been Article 13 claims, with an overall approval rate of close to 10%. The first Article 13 claim based on newly developed evidence or proprietary data (a special category under Article 13(5)) to be achieved, in December 2009, was for Fruitflow®, when the EU Commission authorized the health claim “water‐soluble tomato concentrate (WSTC) I and II helps maintain normal platelet aggregation, which contributes to healthy blood flow”. The authorized claim was based on the eight human studies (seven proprietary), and seven non‐human studies (three proprietary) conducted with Fruitflow. Thus, Fruitflow® is now authorized by EFSA for daily consumption.

6.8 ­Conclusions Normal platelet activity is the key for the maintenance of hemostasis and normal blood flow. Hyperactive platelets interact with

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vessel walls by shedding macro particles, secreting several adhesive growth factors, and inflammatory agents interrupt the blood flow and produce a pro‐thrombotic state in people with obesity, diabetes, a sedentary life style, hypertension, or in people who smoke. In general, the molecular events underpinning these processes are broadly similar. It has long been known that disturbances in blood flow, changes in platelet reactivity and enhanced coagulation reactions facilitate pathological thrombus formation, and the maintenance of normal platelet activity is critical to overall hemostasis. Fruitflow®, developed from tomato and containing bioavailable cardioprotective compounds, can be of benefit to the people who are vulnerable to develop CVD. The outlined data suggests that Fruitflow® may be useful in the primary prevention of CVD. An array of extensive basic, mechanistic, compositional, and several human trials are testimony to its cardio‐protective benefits.

­References 1 Canene‐Adams K, Campbell JK, Zaripheh S, Jeffery EH,

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3

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6

Erdman JW, Jr. (2005). The tomato as a functional food. Journal of Nutrition 135(5): 1226–1230. Agarwal S, Rao AV (2000). Tomato lycopene and its role in human health and chronic diseases. CMAJ : Canadian Medical Association Journal 163(6): 739–744. Jacques PF, Lyass A, Massaro JM, Vasan RS, D’Agostino Sr RB (2013). Relationship of lycopene intake and consumption of tomato products to incident CVD. British Journal of Nutrition 2013: 1–7. Weisburger JH (2002). Lycopene and tomato products in health promotion. Experimental Biology and Medicine 227(10): 924–927. Willcox JK, Catignani GL, Lazarus S (2003). Tomatoes and cardiovascular health. Critical Reviews in Food Science and Nutrition 43(1): 1–18. O’Kennedy N, Raederstorff D, Duttaroy AK, (2016). Fruitflow®: the first European Food Safety Authority-approved natural cardio-protective functional ingredient. European Journal of Nutrition 56, 461–482.

Tomato Extract and Human Platelet Functions

7 Muller I, Massberg S, Zierhut W, et al. (2002). Effects of aspirin

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14

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cardiovascular disease in men. The American Journal of Clinical Nutrition 81(5): 990–997. 29 Hak AE, Ma J, Powell CB, et al. (2004). Prospective study of plasma carotenoids and tocopherols in relation to risk of ischemic stroke. Stroke 35(7): 1584–1588. 30 Ito Y, Kurata M, Suzuki K, Hamajima N, Hishida H, Aoki K (2006). Cardiovascular disease mortality and serum carotenoid levels: a Japanese population‐based follow‐up study. Journal of Epidemiology 16(4): 154–160. 31 Street DA, Comstock GW, Salkeld RM, Schuep W, Klag MJ (1994). Serum antioxidants and myocardial infarction. Are low levels of carotenoids and alpha‐tocopherol risk factors for myocardial infarction? Circulation 90(3): 1154–1161. 32 Tavani A, Gallus S, Negri E, Parpinel M, La Vecchia C (2006). Dietary intake of carotenoids and retinol and the risk of acute myocardial infarction in Italy. Free Radical Research 40(6): 659–664. 33 Sesso HD, Liu S, Gaziano JM, Buring JE (2003). Dietary lycopene, tomato‐based food products and cardiovascular disease in women. Journal of Nutrition 133(7): 2336–2341. 34 Osganian SK, Stampfer MJ, Rimm E, Spiegelman D, Manson JE, Willett WC (2003). Dietary carotenoids and risk of coronary artery disease in women. The American Journal of Clinical Nutrition 77(6): 1390–1399. 35 Ascherio A, Rimm EB, Hernan MA, et al. (1999). Relation of consumption of vitamin E, vitamin C, and carotenoids to risk for stroke among men in the United States. Annals of Internal Medicine 130(12): 963–970. 36 Hirvonen T, Virtamo J, Korhonen P, Albanes D, Pietinen P (2000). Intake of flavonoids, carotenoids, vitamins C and E, and risk of stroke in male smokers. Stroke 31(10): 2301–2306. 37 Agarwal S, Rao AV (1998). Tomato lycopene and low density lipoprotein oxidation: a human dietary intervention study. Lipids 33(10): 981–984. 38 Rissanen T, Voutilainen S, Nyyssonen K, Salonen JT (2002). Lycopene, atherosclerosis, and coronary heart disease. Experimental Biology and Medicine 227(10): 900–907. 39 Ribeiro AB, Chiste RC, Lima JL, Fernandes E (2016). Solanum diploconos fruits: profile of bioactive compounds and in vitro

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50 George MM, Copeland KC (2013). Current treatment options

for type 2 diabetes mellitus in youth: today’s realities and lessons from the TODAY study. Current Diabetes Reports 13(1): 72–80. 51 O’Kennedy N, Crosbie L, Song HJ, Zhang X, Horgan G, Duttaroy AK. (2017). A randomised controlled trial comparing a dietary antiplatelet, the water-soluble tomato extract Fruitflow, with 75 mg aspirin in healthy subjects.  European Journal of Clinical Nutrition 71, 723–730.

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7 Dietary Nitrates and Their Anti‐Platelet Effects ­Abbreviations Used in This Chapter Inorganic nitrate, NO3−; nitric oxide, NO; nitric oxide synthase, eNOS); glycoprotein VI, GPVI; integrin, α2β1

7.1 ­Introduction The cardio‐protective effects of fruits and vegetables have been attributed to their having constituents such as vitamins, minerals, fiber, and antioxidants [1]. Prospective clinical studies using dietary antioxidants in protecting against cardiovascular disease (CVD) have, however, failed to reproduce the same beneficial effects [2–4]. Such observations have stimulated the search for other possible candidates that may be responsible for the cardiovascular health benefits of fruit‐ and vegetable‐rich diets. Prospective epidemiologic studies have suggested that green leafy vegetables are inversely associated with CVD [5]. This search has suggested that dietary inorganic nitrate present in green leafy vegetables may be responsible for the observed beneficial effects of fruits and vegetable on cardiovascular health [6–8]. In fact, the blood pressure‐lowering effects of vegetables is attributed mainly to their inorganic nitrate (NO3−) content [9, 10]. It was demonstrated that that inorganic nitrates in certain fruits and vegetables can provide a physiologic substrate for reduction to nitrite (NO2) and, subsequently, to different metabolites, including nitric oxide (NO) (Figure 7.1). Nutraceuticals and Human Blood Platelet Function: Applications in Cardiovascular Health, First Edition. Asim K. Duttaroy © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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Diet Nitrate reductases (bacteria, mammallian sources) NO2− Deoxyhemoglobin/myglobins), xanthine oxidoreductase, vitamin C polyphenols

NO3− Oxidation NO2−

Oxyhemoglobin

Ceruloplasmin, oxygen NO (−)

O

(−)O

N+

O(−)

Nitrate

(−)O

N

Nitrite

O(−)

NO(−) Nitric oxide

Figure 7.1  Reduction of inorganic nitrates to different metabolites. Inorganic nitrates present in fruits and vegetables are converted to nitrite (NO2) and, subsequently, to different metabolites, including nitric oxide (NO).

Organic nitrates have been used for over a century in CVD therapies. Nitrates, together with sodium nitroprusside, exert their biologic effects via the release of NO. Dietary nitrate is absorbed and reduced to nitrite mainly by bacterial nitrate reductases in the oral cavity, and nitrite can be further reduced to NO in blood and tissues by several pathways, including deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, and non‐ enzymatic reduction. These reductive pathways are known to be greatly enhanced under hypoxic conditions, while oxygen‐ dependent endogenous NOS does not work efficiently under hypoxic situations. NO‐induced arterial vasodilation results in an improvement of arterial blood supply and/or reduction of cardiac workload in CVD. NO is a gaseous signaling molecule with a very short half‐ life (milliseconds), and it plays an essential role in regulating systemic physiological function. Therefore, NO homeostasis is important for optimal health. NO can be sequentially oxidized to nitrite (NO2−) and nitrate (NO3−), molecules, with much longer half‐lives (minutes to hours), and can be measured in the plasma. NO is produced

Dietary Nitrates and Their Anti‐Platelet Effects

from l‐arginine by isoforms of the enzyme NO synthase (L‐­arginine‐NO pathway). The generation of up to ≈ 70% of systemic nitric oxide is accomplished by endothelial nitric oxide synthase (eNOS). In healthy young adults, constitutive NO production by eNOS is sufficient for normal physiological function in most cases. However, NOS is not functional in chronic NO insufficiency. Under NO insufficiency, activation of NOS often results in increased formation of superoxide instead of NO, due to inadequate availability of tetrahydrobiopterin (BH4) or folic acid, further exacerbating oxidative stress. Platelet‐derived NO is synthesized by membrane‐bound endothelial‐type nitric oxide synthase (eNOS). A common signaling mechanism is shared by many eNOS agonists that  bind to membrane receptors, followed by activation of phosphatidylinositol‐3‐kinase (PI3K), which results in phosphorylation of eNOS at serine 1179, through Akt [11]. The activated eNOS converts L‐arginine to L‐citrulline, and produces NO as a result. NO is a water‐soluble free radical that can bind to the heme‐ soluble site of guanylate cyclase in platelets and smooth muscle cells, which increases synthesis of cGMP. cGMP can bind to phosphodiesterase III, which reduces metabolism of cAMP. Elevated levels of cGMP and cAMP can result in increased activity of protein kinase G and protein kinase A, which inhibit protein kinase C activation and intracellular Ca2+ mobili­zation. The consequence of this signal transduction cascade is  the inhibition of platelet activation and the relaxation of vascular smooth muscle, resulting in blood vessel dilation (Figure 7.2). As a small, hydrophilic free radical, NO is highly diffusible in the aqueous environment of the blood. However, it is also highly reactive, with a very short half‐life, estimated to be only on the order of a few seconds in the blood. Previous studies have demonstrated that platelets can produce NO in the absence of erythrocytes. Recently, it has been recognized that nitro‐vasodilators have anti‐platelet effects both in vitro and in vivo. Nitrovasodilators non‐selectively inhibit platelet aggregation induced by different aggregating agents, such as ADP, collagen, and so on. Their aggregation and adherence to the endothelium is inhibited by a number of endogenous anti‐platelet agents, including NO [12].

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Nutraceuticals and Human Blood Platelet Function Collagen

Thrombin

NO donors NOS

NO sGC cGMP PKG

PDE3

cAMP PKA

Inhibition of platelet aggregation

Figure 7.2  Nitric oxide synthase inhibits platelet aggregation. Collagen or thrombin‐activated eNOS converts L‐arginine to L‐citrulline and produces NO. NO regulates cGMP via activation of soluble guanylate cyclase (sGC) and Ca2+. cGMP can bind to phosphodiesterase 3 (PDE3), which reduces metabolism of cAMP. Elevated levels of cGMP and cAMP can result in increased activity of protein kinase G (PKG) and protein kinase A, which inhibit protein kinase C activation and intracellular Ca2+ mobilization. The consequence of this signal transduction cascade is the inhibition of platelet activation, and the relaxation of vascular smooth muscle, resulting in blood vessel dilation.

To date, four publications have reported the acute effect (2.5–3 hours, n = 5) [13, 14] of nitrate intake (dose: 31–1054 mg) and the chronic effect (42 days, n = 1) [14] of nitrate intake (dose: 375 mg/d) on platelet function. All studies observed a significant reduction in platelet aggregation and reactivity. The beneficial vascular effects of dietary nitrate highlight the strong potential for nitrate‐rich vegetables to be used as a therapeutic and preventive strategy to improve cardiovascular health ­worldwide. A meta‐analysis of 12 clinical trials, conducted in 2014, found a significant inverse association between dietary nitrate supplementation (1.5 hours to 28 days) and endothelial function. However, clinical trials with long‐term nitrate supplementation yielded conflicting results.

Dietary Nitrates and Their Anti‐Platelet Effects

The beneficial effects of nitrates are often compromised by the generation of deleterious oxidative stress, causing endothelial dysfunction by continuous nitrate administration, as NOS often results in increased formation of superoxide instead of NO, due to inadequate availability of BH4, which further exacerbates oxidative stress. However, combination therapies with nitrovasodilators and antioxidants have shown some promise in  reducing or reversing tolerance, potentiating anti‐platelet effects, and improving clinical outcome, such as lowering blood pressure. The circulatory levels of inorganic nitrates are determined by endogenous NOS and dietary sources of NO. It has been estimated that up to 50% of human plasma nitrate is derived from dietary sources [15], with green leafy vegetables and certain types of lettuce having the highest levels of nitrate. Inorganic nitrate is a key nutritional constituent of many vegetables. All vegetables take up nitrate from the soil, since it is critical in providing a supply of nitrogen. There is convincing evidence from both acute and chronic human studies demonstrating the blood pressure‐lowering effects of dietary nitrates. This chapter describes the potential inhibitory effects of dietary nitrite and nitrate and their mechanisms of action in human blood platelets.

7.2 ­Nitrate and Cardiovascular Health Atherosclerosis is involved with endothelial dysfunction, which is a central feature in the early development of CVD. Impairment of endothelium‐dependent vasodilation precedes structural atherosclerotic lesions. LDL oxidation is recognized as an early stage in the development of atherosclerosis, leading to CVD. Plant foods contain many components that could contribute to significant protection of endothelium structure and function by a wide variety of phenolic phytochemicals. In addition to their antioxidant properties, polyphenolic compounds increase eNOS activity, as shown in cellular and animal studies. The degree of eNOS expression is a robust biomarker for vascular tone and blood pressure regulation. One of the mechanisms by which eNOS expression is regulated takes place via epigenetic

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mechanisms. For instance, oxidized LDL increases Histone 3 Lysine 9 (H3K9) methylation of eNOS promoter in endothelial cells [16]. It also concurrently decreases acetylation of H3 and H4, as well as methylation of H3K4 in the proximity of eNOS promoter. Together, these findings are indicative of downregulation of eNOS expression and, thus, a decrease in the bioavailability of NO. Diets deficient in methyl donors, like betaine, choline, folate, and methionine are a pointer to dysregulation of metabolic and cardiovascular function. This is mainly caused by disturbances in DNA hypomethylation, plasma homocysteinemia, increases in S‐adenosylhomocysteine (SAH), reduction in S‐adenosylmethionine (methyl donor), and an unfavorable plasma lipid composition. This milieu can be considered as a contributory risk factor for the pathogenesis of cardiovascular diseases. Notably, ingestion of phytochemicals could be very beneficial in adjusting the level of hypomethylation [17]. Diets deficient in methyl donors, like betaine, choline, folate, and methionine are a pointer to dysregulation of metabolic and cardiovascular function [17]. This is mainly caused by disturbances in DNA hypomethylation, plasma homocysteinemia, increases in S‐adenosylhomocysteine (SAH), reduction in S‐adenosylmethionine (methyl donor), and an unfavorable plasma lipid composition [17]. This milieu can be considered as a contributory risk factor for the pathogenesis of CVDs [18]. Notably, ingestion of phytochemicals could be very beneficial in adjusting the level of hypomethylation [17]. Folate acts as a methyl donor, and is important for synthesis and regulation of DNA. Indeed, a diet containing low folate content prior to pregnancy has a bearing on the future offspring and its health into adult life, causing an obese phenotype and increasing the risk of hypertension [19]. Several studies have reported that the phytochemicals present in fruits and vegetables retard the susceptibility of LDL oxidation, owing to their antioxidants activity. Recently, however, there has been growing support for the view that the inorganic nitrate present in vegetables may also play a role in cardiovascular health protection. Ingestion of vegetables rich in inorganic nitrate has emerged as an effective method, via the formation of a nitrite intermediate, for acutely elevating vascular NO levels.

Dietary Nitrates and Their Anti‐Platelet Effects

Dietary nitrates may have a role in increasing peripheral vasodilation and therefore lower blood pressure. Dietary nitrates lower blood pressure and vascular reactivity via a pathway that is independent of the endothelium, and relies initially on the reduction of nitrate to nitrite by oral bacteria and further to NO in blood vessels. However, long‐term and well‐ designed human intervention studies are required in order to establish the roles of the dietary nitrates on CVD.

7.3 ­Effects of Nitrates on Human Blood Platelet Function In Vitro Platelet activation can occur by various stimuli released from the endothelium and different blood cells. Numerous activating agonists transduce activation signals through their respective platelet membrane receptors. For example, both ADP and thrombin act via G‐protein coupled receptors, such as P2Y and protease‐activated receptors (PARs), respectively. In addition, collagen, one of the major components of the vessel wall, acts as a signal on platelets when exposed at the site of injury. Collagen activates platelets through their glycoprotein VI (GPVI) and integrin (α2β1) receptors, while prostacyclin and NO increase cAMP and cGMP levels, respectively, in platelets, through their own receptors. Elevation of these second messengers reduces platelet activation processes. Therefore, a stringent balance between pro‐ and anti‐platelet activation signals within the circulation is necessary to precisely regulate platelet function and keep normal vascular blood flow. NO is one of the potent inhibitors of platelet function. NO is generated from the amino acid L‐arginine and molecular oxygen in the endothelium by eNOS, and diffuses into the lumen of the vessel and enter blood cells, as well as diffusing to the smooth muscle cells of the arteries and arteriolar vessels. The ability of NO to regulate cGMP via activation of soluble guanylate cyclase (sGC) and Ca2+ mobilization is the main mechanism by which NO inhibits platelet activity [20]. While the effect of endothelium‐derived NO on platelet function is investigated [12, 20–23], more data are now emerging on the effects of NO generated from dietary nitrite and nitrate.

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Nitrite ions can inhibit activation/aggregation of human platelets in the presence of erythrocytes through its reduction to NO.  This platelet inhibitory effect of nitrite can be enhanced by  deoxygenation, since deoxyhemoglobin efficiently reduces nitrite to NO [24]. This suggests that nitrite might play a critical role in regulating platelet reactivity, especially in small vessels under hypoxic conditions. The effect of nitrite on different platelet activation pathways was investigated by monitoring the activation of membrane markers, P‐selectin and fibrinogen receptor (glycoprotein IIb/ IIIa (GPIIb/IIIa) [25]. P‐selectin (CD62L) is secreted from the alpha granules of platelets, is translocated to the membrane upon activation, and mediates stable adhesions between interacting cells. The fibrinogen receptor, GPIIb/IIIa, undergoes conformational changes upon platelet activation, which allows fibrinogen to bind to GPIIb/IIIa, resulting in platelet‐platelet aggregation. Nitrite inhibits P‐selectin expression via the GPIIb/IIIa expression in response to ADP, collagen and thrombin in the presence of erythrocytes, and this inhibition is enhanced by increasing hematocrit and deoxygenation of erythrocytes. At 0.1–1.0 μM concentrations, Nitrite significantly decreases the expression of these platelet membrane receptors. The inhibitory effect of nitrite is augmented by increasing hematocrit values and decreasing oxygen saturation. The important roles of hemoglobin in the production of NO from nitrite was demonstrated in the mammalian vasculature [26]. Platelets are known to be more involved in arterial thrombosis rather than venous thrombus formation, in which trapped erythrocytes greatly contribute due to the low pressure in veins. Inhibition of platelet activity by nitrite in venous thrombosis is more effective, as deoxygenated erythrocytes converts nitrite to NO. This is a plausible mechanism that explains how normal physiology of circulation regulates blood flow through the interaction between erythrocytes and platelets in vein. Another in vitro study was designed to demonstrate the effect of nitrite on platelet functions [27]. The effect of nitrite on agonist‐induced platelet aggregation depends on the ­concentration of nitrite ions.

Dietary Nitrates and Their Anti‐Platelet Effects

7.4 ­Clinical Studies with Dietary Nitrate: Effects on Ex Vivo Platelet Function Dietary nitrate may be one of the factors that underlie some beneficial effects of consuming fruits and vegetables. To this end, animal and human studies were carried out using nitrates on CVD risk factors, including blood pressure and platelet function. Small‐scale clinical studies demonstrated that orally ingested inorganic nitrate is sequentially activated to inorganic nitrite (NO2–). Some of the nitrite appears in the circulation, where it is converted to NO by a number of enzyme‐­dependent and independent nitrite reductase pathways. The resultant NO exerts a number of beneficial effects, as discussed elsewhere. Considering the potential effect of blood flow and tissue nitrite and nitrate levels after their conversion to NO, it is important to study how those anions affects blood platelet function. Emerging evidence suggests that dietary nitrate, via nitrite as an intermediate, can inhibit platelet reactivity. Consumption of dietary nitrate attenuated ex vivo stimulation of platelet aggregation, and the effect was lost if the conversion of nitrate to nitrite was somehow inhibited [28]. The effects of acute consumption of nitrate‐containing ­beetroot juice or nitrate capsules were investigated in randomized crossover trials, using 24 healthy subjects. Inorganic nitrate, ingested either from a dietary source or via supplementation, raised circulating nitrate and nitrite levels in both sexes, and attenuated ex vivo platelet aggregation responses to ADP in males only, but not in females. These inhibitory effects were associated with a reduced platelet membrane P‐selectin expression and elevated platelet cGMP levels in platelets. The effects of nitrate and nitrite on platelet aggregation and ATP release from dense granules were demonstrated in mice. An inverse correlation between platelet activity and NO metabolite levels in whole blood was observed. In healthy young adult men, oral L‐arginine inhibited platelet aggregation by way of the nitric oxide pathway. However, it had no effect on systemic hemodynamic variables, plasma nitrosylated protein levels, or endothelium‐dependent dilation. Therefore, at certain doses, oral L‐arginine may result in

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a relatively platelet‐specific increase in nitric oxide production [29]. Normal functioning of human vasculature requires the presence of both nitrite and NO, along with the necessity to respond to these important signaling molecules. These enzymes synthesize nitric oxide from L‐arginine, using molecular oxygen to accomplish vasodilation, blood pressure regulation, inhibition of endothelial inflammatory cell recruitment, and platelet aggregation. As a result, the normal production of nitric oxide and nitrite, and the ability of the endothelium to respond to these species, may prevent various types of CVD,  including hypertension, atherosclerosis, cardiac events, and stroke. In cholesterol‐fed rabbits, oral L‐arginine correlated with the plasma level of L‐arginine, and it could be completely or partially reversed by ex vivo incubation with N‐monomethyl‐ L‐arginine, a specific nitric oxide synthase inhibitor. Platelet cGMP levels were increased after consuming L‐arginine. The risk of primary cardiac arrest is transiently increased during vigorous exercise, whereas habitual physical exercise decreases the risk of primary cardiac arrest [30, 31]. This is due to the fact that the acute strenuous exercise increases platelet adhesiveness and aggregability, whereas acute moderate exercise and exercise training suppress sudden platelet hyperactivity parameters [32, 33]. The modest anti‐platelet activity rendered by dietary intervention using nitrate and L‐arginine may provide a better option via providing an important, but less dramatic, anti‐platelet effect, than aspirin does.

7.5 ­Conclusions There has been growing interest in the possibility that dietary  nitrate may provide some beneficial effects in humans. Inorganic nitrate is found in vegetables, and is especially abundant in green‐leafy vegetables and beetroot (Beta vulgaris). However, it has to be remembered that iNOS can generate high concentrations of nitric oxide that promote carcinogenesis by inhibiting apoptosis, enhance prostaglandin formation, and promote angiogenesis in the early stage of carcinogenesis. In atherosclerosis, hypoxic conditions, combined with an oxidative environment, can limit eNOS‐derived nitric

Dietary Nitrates and Their Anti‐Platelet Effects

oxide production, and nitrite can directly induce vasodilation in hypoxic endothelium. New discoveries in the field of nitrate and nitrite biology have provided mechanistic insights into the potential new physiologic roles of dietary nitrate and nitrite, and their potential health benefits. There is a consensus that dietary nitrates are essentially inert, and acquire biological activity only after reduction to nitrite. As such, nitrate serves as a source, via successive reduction, for the production of nitrite and nitric oxide, as well as other metabolic products. Recent evidence is mainly focused on the acute effects of dietary nitrate supplementation, and there is a lack of data looking at the chronic effects of high nitrate consumption in humans. Nevertheless, due to potential health benefits, some are recommending that nitrate should be considered as a nutrient necessary for health. The acceptable daily intake (ADI) set by the European Food Safety Authority for nitrate is 3.7 mg/kg (0.06  mmol/kg). This equates to 260 mg/day for a 70 kg adult (4.2 mmol). The WHO first set an upper limit for nitrate in food in 1962, which was based on studies showing that daily doses of 500 mg nitrate per kg body weight had no adverse effects on rats and dogs. This value was divided by 100 to obtain an ADI for humans of 5 mg sodium nitrate, or 3.7 mg nitrate per kg body weight. However, food choices within a dietary pattern, such as the Dietary Approaches to Stop Hypertension (DASH) diet, can yield differences from 2,8–19.7 mmol nitrate, exceeding the ADI by 550% for a 60 kg adult. Therefore, the variability in nitrate content between vegetables and within species has a large influence on meeting the ADI for nitrate.

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improves vascular function in patients with hypercholesterolemia: a randomized, double‐blind, placebo‐controlled study. American Journal of Clinical Nutrition 103(1): 25–38. Hord NG, Tang Y, Bryan NS (2009). Food sources of nitrates and nitrites: the physiologic context for potential health benefits. American Journal of Clinical Nutrition 90(1): 1–10. Fang F, Yang Y, Yuan Z, et al. (2011). Myocardin‐related transcription factor A mediates OxLDL‐induced endothelial injury. Circulation Research 108(7): 797–807. Stefanska B, Karlic H, Varga F, Fabianowska‐Majewska K, Haslberger A (2012). Epigenetic mechanisms in anti‐cancer actions of bioactive food components – the implications in cancer prevention. British Journal of Pharmacology 167(2): 279–297. Zawada AM, Rogacev KS, Hummel B, et al. (2014). S‐adenosylhomocysteine is associated with subclinical atherosclerosis and renal function in a cardiovascular low‐risk population. Atherosclerosis 234(1): 17–22. Sinclair KD, Allegrucci C, Singh R, et al. (2007). DNA methylation, insulin resistance, and blood pressure in offspring determined by maternal periconceptional B vitamin and methionine status. Proceedings of the National Academy of Sciences of the United States of America 104(49): 19351–19356. Zhang G, Xiang B, Dong A, et al. (2011). Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation. Blood 118(13): 3670–3679. Azuma H, Ishikawa M, Sekizaki S (1986). Endothelium‐ dependent inhibition of platelet aggregation. British Journal of Pharmacology 88(2): 411–415. Radomski MW, Palmer RM, Moncada S (1987). Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 2(8567): 1057–1058. Bult H, Fret HR, Van den Bossche RM, Herman AG (1988). Platelet inhibition by endothelium‐derived relaxing factor from the rabbit perfused aorta. British Journal of Pharmacology 95(4): 1308–1314. Srihirun S, Sriwantana T, Unchern S, et al. (2012). Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation. PLoS One 7(1): e30380.

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8 Kiwifruit and Human Platelet Function ­Abbreviations Used in This Chapter Kiwi fruit extract, KFE

8.1 ­Introduction Kiwifruit is best known crop in the genus Actinidia. There is a large and diverse range of species and cultivars of Actinidia, with different characteristics and attributes, of which Actinidia deliciosa “Hayward” (green kiwifruit) and Actinidia chinensis “Hort 16A”, ZESPRI® (gold kiwifruit) are the most popular commercially available cultivars [1]. The cultivar “Hayward” is the mainstay of the kiwifruits, and is the one most commonly consumed. Today, kiwifruit is grown in many countries, notably Italy, China, Chile, France, Greece, Japan, and the United States. Consumption of fruits and vegetables protects against the devel­ opment of cardiovascular disease (CVD) [2–4]. The bioactive compounds present in fruits and vegetables, although not yet well characterized, individually or in concert may protect the cardiovascular system, by favorably modulating oxidative stress, plasma lipid levels, hypertension, platelet hyperactivity and other CVD risk factors [5, 6]. Hyperlipidemia, hypertension, and hyperactivity of blood platelets are the critical contributors to pathogenesis of CVD [7]. Kiwifruit contains very significant amounts of vitamin C, Nutraceuticals and Human Blood Platelet Function: Applications in Cardiovascular Health, First Edition. Asim K. Duttaroy © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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vitamin E, folic acid, and various phytochemicals, such as anthocyanidins and flavonols. The common green kiwifruit, “Hayward”, has been used in several human trials to examine effects on biomarkers relevant to both cancer and CVD [5, 8–10]. Human blood platelets are not only involved in thrombotic events, but are also involved in the initiation and progression of atherosclerotic plaque. Consequently, platelets act as a bridge between the processes characteristic of atherosclerosis and thrombosis [11, 12]. Hyperactive platelets, as observed in diabe­ tes mellitus, insulin resistance, obesity, sedentary life, and smok­ ing, contribute to the development of CVD [13–18]. Flavonoids are also inhibitors of cyclic nucleotide phos­ phodiesterase and TxA2 synthesis, two of the main mecha­ nisms  responsible for the inhibition of platelet aggregation. Consequently, these bioactive components in fruits may reduce more than one CVD risk factors, such as platelet hyperactivity [19, 20]. The common green kiwifruit, Actinidia deliciosa, has been used in several trials to examine effects on biomarkers relevant to CVD [5, 8–10]. Daily consumption of two or three kiwifruits reduced platelet aggregation response, blood pres­ sure and plasma lipids [5, 8–10]. The presence of these diverse activities, such as anti‐platelet and anti‐angiotensin converting enzyme (ACE), was also demonstrated in aqueous extract of kiwifruits [5, 21].

8.2 ­Kiwifruit and Its Bioactive Phytochemicals Kiwifruit are some of the most nutrient‐dense fruit, and are par­ ticularly high in vitamins C, E, and K, folate, carotenoids, potas­ sium, fiber, and contain a range of phytochemicals [1]. Kiwifruit contains very significant amounts of various phytochemicals, such as anthocyanidins and flavonols [22]. Apart from oranges, both green and gold kiwifruit are better than other fruit as sources of carotenoids, including beta‐carotene, lutein, and zeaxanthin. The carotenoids contribute to the color of the kiwi­ fruit, but the unique green color of green kiwifruit is attributed to the retention of chlorophyll during ripening (1 mg of chloro­ phyll/100 g), which masks the yellow color of the carotenoids

Kiwifruit and Human Platelet Function

[23]. Kiwifruit also contain a range of other phytochemicals/ polyphenols, although many of the phenolics and flavonoids in kiwifruit are yet to be identified. Kiwifruit is best known crop in the genus Actinidia. It con­ tains very significant amounts of various phytochemicals, such as anthocyanidins and flavonols [22]. The common green kiwifruit has been used in several trials to examine effects on biomarkers relevant to CVD [5, 8–10].

8.3 ­Kiwifruits and Human Blood Platelet Function The anti‐platelet activity of several fruits showed that kiwifruits and tomato extract (see Chapter 6) had maximum anti‐platelet activity, and that this activity was not related to the antioxidant potential of fruits [6]. Among all fruits tested for their in vitro anti‐platelet activity, tomato and kiwifruit had the highest activ­ ity, followed by grapefruit, melon, and strawberry, while pear and apple had little or no activity (see Table 8.1). Table 8.1  Effect of different fruit extracts on platelet aggregation induced by ADP. Fruit

% Inhibition of ADP‐induced aggregation

Kiwi

89

Tomato

90

Grapefruit

44

Melon

42

Strawberry

33

Orange

18

Grape

16

Plum

16

Cranberry

9

Apple

5

Pear

2

See references [5, 6].

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Nutraceuticals and Human Blood Platelet Function

In vitro studies using extracts of different fruits showed anti‐ aggregation activity mainly in kiwifruit and tomato. Among all fruits tested for their in vitro anti‐platelet activity, tomato and kiwifruit had the highest activity, followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. One hundred percent kiwifruit extract (KFE) (w/v) was used to study its inhibitory effects on platelet aggregation [5]. ADP‐ induced aggregation was inhibited by 11% with 5 mg KFE, 54% with 10 mg KFE, and 96% with 20 mg KFE. KFE also inhibited collagen‐induced platelet aggregation, although the level of inhibition was lower with the 5 and 10 mg KFE, compared with those observed with ADP‐induced platelet aggregation. Inhibition of ARA‐induced platelet aggregation exhibited a very different profile, with only 32% inhibition at the highest KFE level (20 mg) tested, and nothing at all at the lower concentrations of KFE. The KFE extract inhibited both collagen‐ and ADP‐induced platelet aggregation to a greater extent, whereas it had very lit­ tle inhibitory effects on ARA‐induced aggregation, indicating that the inhibition of platelet aggregation by kiwifruit extract may not involve the thromboxane pathway. This is quite differ­ ent from aspirin’s mode of action in platelets. Aspirin’s anti‐ platelet action involves inhibition of the COX‐1 in platelets, leading to a decreased formation of PGG2, a precursor of TxA2. Decreased formation of TxA2 would result in low platelet aggregation. The kiwifruit extract may contain a wide variety of different types of compounds that have anti‐platelet activity, and which affect different mechanisms of activation and aggregation. The compounds responsible for the observed anti‐platelet activity in kiwifruit extract have been isolated. These com­ pounds are water‐soluble and heat‐stable, and their molecular mass is less than 1000 Da [5]. The sugar‐free kiwifruit extract (KFE) extract with anti‐platelet activity was isolated as out­ lined in Figure 8.1. Delipidation, followed by ultrafiltration of the KFE, indicated that the active factors in KFE were water‐soluble, heat‐stable and the molecular mass was > 1000 Da. KFE had glucose (8.9 ± 0.4 mg/ml), fructose (9.9 ± 0.5 mg/ml) and sucrose (2.3 ± 0.2 mg/ml). Soluble sugars were removed by using SPE

Kiwifruit and Human Platelet Function

Kiwifruit Homogenisation, centrifugation at 9000xg for 15 min at 4C Boiled at 90C for 20 min and centrifuged at 10,000xg for 20 min, Supernatant prepared Delipidation of the supernantant by Lipidex-1000 column

Molecular weight cut-off (