Protein C: Biochemical and Medical Aspects. Proceedings of the International Workshop, Titisee, Federal Republic of Germany, July 9–11, 1984 9783110852707, 9783110102222

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Protein C Biochemical and Medical Aspects

Protein C Biochemical and Medical Aspects Proceedings of the International Workshop Titisee, Federal Republic of Germany July 9-11,1984

Editor Irene Witt

W DE Walter de Gruyter G Berlin • New York 1985

Editor Irene Witt, Prof. Dr. Klinisch-Chemisches Labor Biochemisches Labor Universitäts-Kinderklinik Mathildenstraße 1 D-7800 Freiburg Federal Republic of Germany

Library of Congress Cataloging in Publication Data Protein C: biochemical and medical aspects. Includes bibliographies and index. 1. Protein C-Congresses. I. Witt, Irene. [DNLM: 1. C-Reactive Protein-congresses. WH 400 P967 1984] QP93.5.P766 1985 612'.015752 85-16126 ISBN 0-89925-094-7 (U.S.)

CIP-Kurztitelaufnahme der Deutschen

Bibliothek

Protein C: biochem. and med. aspects; proceedings of the internat, workshop, Titisee, Fed. Republic of Germany, July 9-11,1984/ed. Irene Witt. Berlin; New York: de Gruyter, 1985. ISBN 3-11-010222-6 (Berlin...) ISBN 0-89925-094-7 (New York) NE: Witt, Irene [Hrsg.]

ISBN 3 11010222 6 Walter de Gruyter • Berlin • New York ISBN 0-89925-094-7 Walter de Gruyter, Inc., New York Copyright © 1985 by Walter de Gruyter & Co., Berlin 30 All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. - Binding: Dieter Mikolai, Berlin. - Printed in Germany.

PREFACE Thrombosis has been known since about 2 000 B.C. and pulmonary embolism was first described in 1676 by WISEMAN. In the 1840's Rudolf VIRCHOW recognized the connection between thrombosis and pulmonary embolism. According to VIRCHCW's triad changes of the vessel wall, blood flow and blood properties are the most important pathogenic factors for the development of thrombi with the risk of consecutive embolism. For a long time it was assumed that hypercoagulability is mainly caused by an increased intravascular thrombin generation. During the. last decade a key role in the development of hypercoagulability was attributed to proteinase inhibitors. The importance of antithrombin III became well established when it was observed at the end of the 1960's that an inherited or acquired deficiency is combined with a high risk of thromboembolism. A few years ago, it became evident that thrombin generation can be inhibited not only by proteinase inhibitors but also by the action of a specific proteinase - Protein C. The inhibition of coagulation by Protein C may be of equal importance to that of antithrombin III. The protein, which has already been known since 1960 as autoprothrombin II a, had a rennaissance when in 1981 GRIFFIN found the first family with a hereditary deficiency of this protein and its association with a high risk of thromboembolic diseases or thromboembolic complications. Since then there has been increasing interest and research regarding Protein C. It belongs to a complex system of interacting proteins, it needs vitamin K for the synthesis of its biologically active form, it needs a vitamin K dependent protein as cofactor and gains its full activity only after activation by thrombin and thrombomodulin. Moreover

a specific

inhibitor exists for Protein C. It is imposing that only a few years were necessary to clarify the complex basic biochemistry of Protein C and related proteins. New insights into the regulation of coagulation by plasmatic inhibitors arose. Many families have since been found with a congenital deficiency of this protein and many cases of spontaneous or recurrent thrombosis could be elucidated as a deficiency of Protein C or its cofactor Protein S.

VI

Despite advanced knowledge there are still many problems in determining Protein C functionally. Most of them arise from its characeristic features. The difficulties of the functional assay in routine use are presumably the reason why little information is available about the changes in Protein C activity during the course of various diseases. The aim of the workshop on Protein C, which was held form July 9th - 11th, 1984, in Titisee near Freiburg i.Br., was to sum up present knowledge in the biochemistry, physiology and pathophysiology of the inhibitor and related proteins. Important topics were the possibilities of determining Protein C concentration immunologically or the functional activity by chromogenic or clotting assays. The basic information on Protein C and connected proteins was followed by a number of clinical reports on inherited and acquired disorders. Multifarious open questions were outlined in the lectures and subsequent discussions . I hope the proceedings of the workshop which are documented in this book will reflect the current status of the complex Protein C system and spread information on this new field of cagulation. I wish to express special thanks to Dr. Ernst Zimmer, Mannheim, for his thorough help with the editing of the manuscripts. Sincere gratitude is extended to Mrs. Monika Krumnow, Mrs. Evelyne Glowka and Dr. Rudolf Weber of Walter de Gruyter Publishers, whose helpful cooperation has made the task of editing this book a pleasant one. Freiburg i. Br., July 1985

Irene Witt

ACKNOWLEDGEMENT

The workshop was made possible by the generous sponsorship of Boehringer Mannheim GmbH. Further valuable contributions came from MERZ & DADE (AHS), München, Deutsche KABI VITRUM, München, PENTAPHARM, Basel and SCHWAB, Brunn (Austria). I wish to express my sincere gratitude to all of them.

LIST OF PARTICIPANTS

Dr. J. Amiral

Diagnostica Stago 6 ter rue Denis Papin 92600 Asnieres

Prof. Dr. E. A. Beck

Häraatologisches Zentrallabor Inselspital Bern Freiburgstr. 30 CH-3013 Bern

Prof. Dr. H. Beeser

Institut f. Transfusionsmedizin der Universität Freiburg Hugstetterstr. 55 D-7800 Freiburg i.Br.

Dr. R. M. Bertina

Hemostasis and Thrombosis Research Unit Academisch Ziekenhuis Leiden Rijnsburgerweg 10 2333 AA Leiden

Dr. W. Brockhaus

Klinikum Nürnberg Flurstr. 17 D-8500 Nürnberg

Dr. A. W. Broekmans

Hemostasis and Thrombosis Research Unit Academisch Ziekenhuis Leiden Rijnsburgerweg 10 2333 AA Leiden

Dr. J. P. Cazenave

Service d'Hemostase et de Thrombose Centre le Transfusion Sanguin F-67100 Strasbourg

Dr. M. Colucci

Center for Thrombosis and Vascular Research Katholieke Universiteit Leuven Department of Medical Research B-3000 Leuven

Dr. H. Droste

Boehringer Mannheim GmbH Sandhoferstr. 116 D-6800 Mannheim

VIII Dr. J. H. Griffin

Dept. of Molecular Immunology Scripps Clinic and Research Foundation 10666 North Torrey Pines Road La Jolla, California 92037

Prof. Dr. W. Heller

Chirurgische Univ.-Klinik Calwer Straße 7 D-7400 Tübingen

Dr. P. Hellstern

Universitätskliniken Abtlg. Klin. Haemostaseologie und Transfusionsmedizin D-6650 Homburg/Saar

Dr. H. P. Kreuzer

Biochemisches Labor Universitäts-Kinderk1inik Mathildenstraße 1 D-7800 Freiburg i. Br.

Prof. Dr. K. Lechner

I. Medizinische Univ.-Klinik Lazarettgasse 14 A-1090 Wien

Dr. H. Li 11

Boehringer Mannheim GmbH Biochemica Werk Tutzing Bahnhofstr. 5 D-8132 Tutzing/Obb.

Prof. Dr. G. Müller-Berghaus

Klinische Forschungsgruppe für Blutgerinnung und Thrombose Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Gaffkystr. 11 D-6300 Gießen

Doz. Dr. G. Oehler

Klinikum der Justus-Liebig-Univ. Zentrum für Innere Medizin Klinikstr. 36 D-6300 Gießen

Prof. Dr. W. G. Owen

Cardiovascular Center, Departments of Pathology and Biochemistry University of Iowa Iowa City, Iowa 52242

Dr. I. Pabinger-Fasching

I. Medizinische Univ.-Klinik Lazarettgasse 14 A-1090 Wien

Dr. F. W. Rabe

Deutsche Kabi Vitrum GmbH Levelingstr. 18 D-8000 München 80

IX

Prof. Dr. L. Rôka

Klinikum der Justus-Liebig-Univ. Inst. f. Klinische Chemie und Pathobiochemie Klinikstr. 32 b D-6300 Gießen

Dr. S. Rosén

AB KABI Peptide Research S-43122 Mölndal

Prof. Dr. I. Scharrer

Zentrum der Inneren Medizin der Univ.-Kliniken Frankfurt Theodor-Stern-Kai 7 D-6000 Frankfurt/M.

Doz. Dr. U. Schmitz-Huebner

Medizinische Klinik u. Poliklinik der Westfälischen Wilhelms-Univ. Abtlg. Innere Medizin A Domagkstr. 3 D-4400 Münster

Prof. Dr. T. H. Schöndorf

Klinikum der Justus-Liebig-Univ. Zentrum f. Innere Medizin Klinikstr. 36 D-6300 Gießen

Dr. H. H. Seydewitz

Biochemisches Labor Universitäts-Kinderklinik Mathildenstr. 1 D-7800 Freiburg i.Br.

Dr. E. Spanuth

Boehringer Mannheim GmbH Sandhoferstr. 116 D-6800 Mannheim

Dr. T. Sugo

Department of Clinical Chemistry University of Lund Malmö General Hospital S-21401 Malmö

Prof. Dr. K. Suzuki

Dept. of Laboratory Medicine Mie University School of Medicine Tsu-City, Mie 514

Prof. Dr. T. Vukovich

Institut f. Med. Physiologie Schwarzspanierstr. 17 A-1090 Wien

Prof. Dr. F. J. Walker

Terre Haute Center for Medical Education at Indiana State Univ. 135 Holmstedt Hall Terre Haute, Indiana 47809

X Prof. Dr. H. Wehinger

Städtische Kliniken Kassel Kinderklinik Mönchebergstr. 41/43 D-3500 Kassel

Prof. Dr. E. Wenzel

Universitätskliniken Abtlg. Klin. Haemostaseologie und Transfusionsmedizin D-6650 Homburg/Saar

Prof. Dr. I. Witt

Biochemisches Labor Universitäts-Kinderklinik Mathildenstr. 1 D-7800 Freiburg i.Br.

CONTENTS

Biochemistry and Physiology of Protein C W.G. Owen

1

Mechanism of Action of Protein C H.P. Schwarz, J.H. Griffin

5

Biochemistry of Protein S and its Role in the Regulation of the Anticoagulant Activity of Activated Protein C F.J. Walker

19

Does Human Protein C Exert Profibrinolytic Activity? M. Colucci, D. Collen

29

Biochemistry and Physiology of Protein C Inhibitor K. Suzuki

43

Isolation of Protein C Using High Performance Liquid Chromatography (HPLC) - Experiences and Problems C. Miyashita, P. Hellstern, G. von Blohn, H. Hammer, E. Wenzel

59

Determination of Protein C by an Immunoenzymatic Assay J. Amirai, C. Boyer, C. Rothschild, M. Wolf

67

Functional Assays of Plasma Protein C R.M. Bertina, A.W. Broekmans

81

XII

Hereditary Protein C Deficiency A.W. Broekmans, R.M. Bertina

93

Production of an Antibody Against Human Protein C and its Use to Detect Patients with Congenital Protein C Deficiency Associated with Thrombosis J.-M. Freyssinet, L. Grunebaum, M.-L. Wiesel J.-P. Cazenave, R. Fritsche, S. Greber, A. Lampart, R. Spaethe

107

Protein C Deficiency in a Newborn Infant with Purpura fulminans H. Wehinger, I. Witt

117

Protein C in Patients under L-Asparaginase Treatment T.H. Schöndorf, I. Witt

125

Protein C in Various Liver Diseases G. Oehler, K. Matthes

135

Protein C Determination in Patients with Thrombotic Disease and Disseminated Intravascular Coagulation I. Pabinger-Fasching, U. Stoffels, K. Lechner

141

Investigations of the Incidence of Different Inherited Fibrinolysis Disorders in Juvenile Thrombosis Patients I. Scharrer

151

Protein C Concentration after Elective Hip Surgery T.H. Schöndorf, I. Witt

163

XIII

Protein C Activity and Concentration in Patients with Thromboembolic Disease and in Patients Treated with Phenprocoumon G. Müller-Berghaus, W. Thiel, K.T. Preissner, G. Oehler

169

Protein C under Long-Term Dicoumarol Treatment P. Hellstern, G. von Blohn, C. Miyashita, M. Köhler P. Scheffler, E. Wenzel

177

Protein C under Heparin Treatment U. Schmitz-Huebner

183

AUTHOR

189

SUBJECT

INDEX

INDEX

191

BIOCHEMISTRY AND PHYSIOLOGY O F PROTEIN C

W h y t e G. O w e n U n i v e r s i t y of Iowa, I o w a C i t y , I o w a 52242, U S A

Identified by

originally

treating

prothrombin

tein C w a s p u r i f i e d tion

(2) , after

component member serine

linked

cance,

of

a

single

to

4),

The

is

as

a

third,

(hence,

K-dependent and has

zymogen

of

155

and

from

the is

"C").

at

the

core

a of

first

structure of

acid

unknown

heavy

As

zymogens

amino

yet (5).

the

unknown,

comprised

260

Of

fl-hydroxylated

arginine-specific

an

purifica-

disulfide.

tetradecapeptide

protein C

of p r o t h r o m b i n

is ^ - c a r b o x y l a t e d

(3,

elicited (1) , p r o -

chromatogram

vitamin

polypeptides,

activity thrombin

identified

family.

aspartate-71

N-terminal

been

residues

by

with

a by-product

protein C

chymotrypsin

non-identical dues,

as

family

proteinases,

the

anticoagulant

ion-exchange

the

eleven glutamate of

an

preparations

having

in an

of

as

resi-

signifi-

Removal chain

two

of

an

activates

endopeptidase,

with

the

c a t a l y t i c c e n t e r c o n f i n e d to the h e a v y c h a i n . The

only

natural

identified In

a

are

reaction

factors

Va

and

concentrations specific appears

substrates

the a c t i v e Villa

inactivated

of

are

activated bonds. for

v a t e d p r o t e i n C, w h i c h b l o o d or p l a s m a

(9,

vated

is

to

identification

volume,

as

an

factors

on

account

protein C

activated of

dependent

peptide to

of

forms

phospholipid

of

protein

obligate

anticoagulant S,

a for

activity that

elsewhere

activated

(11-13) .

Protein C - Biochemical and Medical Aspects © 1985 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

catalytic

cleavage

of

cofactors of

acti-

and _in v i t r o

finding

discussed

cofactor

by

activity

is c o m p a r a b l e Jji v i v o

10). T h e

to

far

(6-8) .

platelets),

procoagulant

anticoagulant

species-specific, of

owing

so

VIII

(or

rapidly

p r o t e i n C,

Cleavage the

protein C V and

of

acti-

has in

in led

this

protein C

2 A

fibrinolytic

served

action

in d o g s

elicits

a

of

(9, 10,

arises

from The

but

circulation response

as d i s c u s s e d of

human

Intravascular

of

a

or

endogenous

the r e s p o n s e

tissue-type

protein C

activation pendent the

of

the

zymogens

cofactor

detergent

preparations

extracts

is a single contains

of

about

10

protein

high affinity The

bound

(19, change

is a

thrombin

other

heparin

of

The

38

thrombomodulin, and

cultured

purified purified

weight

half-cystines.

Seemingly

is a n

integral

II

of

p r o t e i n C,

(procoagulant) and

reversible

upon

Studies

of

tivation

(21, 22) .

From

viewpoint

the

of

in

components

thrombin-catalzyed

biochemistry,

is

This

dissociation of 2+ the Ca -depend-

derivatives

participate

but

c£, 351 (1983). 7. H o r e l l o u , M . H . , C o n a r d , J., B e r t i n a , R . M . , S a m a m a , M. : Br. M e d . J. 289, 1 2 8 5 - 1 2 8 7 (1984). 8. T h a l e r , E., L e c h n e r , K. : C l i n . H a e m a t o l . 10, (1981).

369-390

9. B e r t i n a , R . M . , B r o e k m a n s A . W . , K r o m m e n h o e k - v a n E s , C . , V a n W i j n g a a r d e n , A . : T h r o m b . H a e m o s t a s . _51, 1 - 5 (1984). 10. M a n n u c c i , P.M., V i g a n o , S . : L a n c e t j^i, 463-466 11. G r i f f i n , J . H . , M o s h e r , D . F . , Z i m m e r m a n , T . S . , A . J . : B l o o d 60, 261-264 (1982).

(1982). Kleiss,

12. V i g a n o , S., M a n n u c c i , P . M . , S o l i n a s , S., B o t t a s s o , B . , M a r i a n i , G . : Br. J. H a e m a t o l . 57, 213-220 (1984). 13. M a r c i n i a k , E . , W i l s o n , H . D . , M a r l a r , R . A . : B l o o d suppl. 303 a (1983).

62,

14. S a m a m a , M . , H o r e l l o u , M . H . , S o r i a , J., C o n a r d , J . , N i c o l a s , G . : T h r o m b . H a e m o s t a s . 51, 1 3 2 - 1 3 3 (1984). 15. Comp, P.C., N i x o n , R . R . , E s m o n , C . T . : B l o o d 63^, 15-21 (1984). 16. B e r c o f f , E., M o r c a m p , D., B o u r r e i l l e , J . , B o r g , J . Y . , P i g u e t , H., S o r i a , J . , S o r i a , C., D o r n e r , M . : P r e s s e M e d . 13, 49 (1984).

105

17. Wintzen, A.R., Broekmans, A.W., Bertina, R.M., Briet, E., Briet, P.E., Zecha, A., Vielvoye, G.J., Bots, G.Th.A.M.: Br. Med. J., accepted for publication. 18. Broekmans, A.W., Bertina, R.M., Loeliger, E.A., Hofmann, 244 (1983). V., Klingemann, H.G.: Thromb. Haemostas. 19. McGehee, W.G., Klotz, T.A., Epstein, D.J., Rapaport, S.I.: Ann. Intern. Med. 100, 59-60 (1984). 20. Loeliger, E.A., Broekmans, A.W. In: Dukes MNG, ed. Meyler's side effects of drugs. Amsterdam, Oxford, Princeton: Excerpta Medica, 10th ed. 648-693 (1984). 21. Hofmann, V., Frick, P.G.: Thromb. Haemostas. 48, 245-246 (1982). 22. Branson, H.E., Katz, J., Marble, R., Griffin, J.H.: Lancet ii, 1165-1168 (1983). 23. Marlar, R.A., Sills, R.H., Montgomery, R.R.: Blood 62, suppl. 303 a (1983). 24. Jespersen, J., Bertina, R.M. Unpublished observations (1983). 25. Broekmans, A.W., Van der Linden, I.K., Veitkamp, J.J., Bertina, R.M.: Thromb. Haemostas. 50^ 350 (1983). 26. Kluft, C., Bertina, R.M., Preston, F.E., Malia, R.G., Blarney, S.L., Lowe, G.D.O., Forbes, C.D.: Thromb. Res. 32, 297-304 (1984). 27. Jarrett, P.E.M., Morland, M., Browse, N.L. In: Davidson J.F., ed. Progress in chemical fibrinolysis and thrombolysis. Edinburgh: Churchill Livingstone 317-321 (1979).

PRODUCTION OF AN ANTIBODY AGAINST HUMAN PROTEIN C AND

ITS

TO D E T E C T P A T I E N T S W I T H C O N G E N I T A L P R O T E I N C D E F I C I E N C Y CIATED WITH

USE

ASSO-

THROMBOSIS

Jean-Marie Freyssinet, Lelia Grunebaum, Marie-Louise Jean-Pierre Cazenave

Wiesel,

S e r v i c e d ' H é m o s t a s e et de T h r o m b o s e , C e n t r e R e g i o n a l de T r a n s f u s i o n S a n g u i n e , 10 rue S p i e l m a n n , 67085 S t r a s b o u r g , France Rodolphe Fritsché, Susanne Greber, Alfred M e r z and Dade A G , 3186 D ü d i n g e n , Reiner

Lampart

Switzerland

Spaethe

AHS Deutschland, Department Merz and Dade, München,

Human

protein C

molecular

(PC)

weight

of

is

a vitamin K-dependent

62,000

which

c o n c e n t r a t i o n of a b o u t 3 >jg/ml logical the

activator

presence

of

of PC

surface

protein

PC.

physiological

At

is

either (3) or

circulates

alpha-thrombin.

factor

gulant Villa

in

Va

(4)

concentration,

as

(6, 7)

by

proteolytic

cofactor

calcium

in the p r e s e n c e

cleavage

a

at

a

physiorequires

endothelial is

to an

of

cell

activate inhibitor

form P C a by

(5). PCa is a serine p r o t e a s e w h i c h

activity

with

plasma known

Thrombin

an

of the c o n v e r s i o n of P C into its a c t i v a t e d b i n alone

zymogen

(1, 2). The o n l y

thrombomodulin,

FRG

exerts

throm-

anticoa-

factors

Va

and

of a c o f a c t o r , p r o t e i n S, also

a

vitamin K-dependent protein

(8). P C a is i n a c t i v a t e d by a n a t u -

ral

present

and

specific

inhibitor

Several

clinical

studies

of

plained, recurrent thrombosis

in c i r c u l a t i n g

families have

with

recently

blood

histories

of

suggested

i n h e r i t e d d e f i c i e n c y of PC m i g h t p r e d i s p o s e p a t i e n t s to

Protein C - Biochemical and Medical Aspects © 1985 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

(9). unex-

that

an

throm-

108 boembolic

disease

of the PC s y s t e m of c l i n i c a l In

the

of

human

(10).

It

is

possible

that other

(protein S, t h r o m b o m o d u l i n )

components

will prove

present PC

study,

we

from h u m a n

describe

the

large

cryosupernatant

in

scale

order

P u r i f i c a t i o n of h u m a n PC from p l a s m a was

routinely

following Kisiel

a

time

isolated

procedure

from

liters

from

precipitate.

After

several

fractionated

s u c c e s s i v e l y by 35 % and 65

solutions last

the 6.0

in the p r e s e n c e

in

the

presence

factor X

could

be

eluted

peak. PC w a s

sulfate-Sepharose preparation acrylamide

was

barium

gel

decyl sulfate m o s t of our

proteins

protein

to

two

the

7.5

by

clotting

this

assays.

described

p u r i f i e d on

PC

%

in

pure, the

as

(11),

X eluted (Fig. of

(molecular

of

PC

Purified

a

small

weight

contained

than

PC

Poly-

sodium

38 ,000, 1 %

in a

1) . by

proportion

about

less

PC

dextran

(Fig. 2). As p o i n t e d o u t by other a u t h o r s contained

fac-

affinity

in

judged

presence

only

and

4B

at run

stage,

the same

chromatography 95

g r a d e d PC h e a v y c h ^ p 41,000) .

collected

DEAE-Sephacel

sulfate-sepharose

further than

were

ammonium

s e c o n d one w a s

(6). At

conditions

electrophoresis

preparations

its

citrate

% saturated the

pH

4B a f f i n i t y greater

of

The f i r s t one was p e r f o r m e d

by d e x t r a n

Following

on

f i r s t in a s y m m e t r i c a l p e a k , then factor

symmetrical

(11).

thromboplastin

eluted

subjected

EDTA, while at

detected

separated

chromatography.

of

the

dialysis,

was

Ca2+

of

by

electroimmunoassay,

adsorbed

washes,

chromatographies.

pH

tor X w e r e

were

(1). After

precipitate

ion e x c h a n g e

published

the a b i l i t y

or by r o c k e t

proteins

in

cryosupernatant

available.

Vitamin K-dependent

sulfate

of

methods

activated partial

(APTT) of n o r m a l p l a s m a

w h e n the a n t i s e r u m b e c a m e

a

cryosupernatant

detected by

to p r o l o n g

obtain

purposes.

(6) a n d F r e y s s i n e t e t al.

of PC w a s e i t h e r

form, P C a ,

25

derived

(1), Suzuki et al.

The p r e s e n c e active

be

isolation

to

s p e c i f i c a n t i s e r u m u s e f u l for c l i n i c a l or r e s e a r c h

PC

to

importance.

do-

(1, 6) of

de-

instead

factor

X,

109 OH

210 tin. Ratlin C

X

IX

e.s o50

75

FRACTION

100

nuueen

B

piotlin C.

0-d at 710 n*. 0.5 0

St

I

r

75

too

FRACTION MUMSER

F i g . l . A. D e x t r a n s u l f a t e - S e p h a r o s e 4B a f f i n i t y c h r o m a t o g r a p h y of the pool e n r i c h e d in PC from D E A E - S e p h a c e l c h r o m a t o g r a p h i e s . Buffer is 5 m M M O P S c o n t a i n i n g 8 m M t r i s o d i u m c i t r a t e and 0.01 % N a N 3 , pH 7.5. G r a d i e n t , G , is from 0 to 1 M N a C l (2x400 m l ) . V o l u m e of the c o l u m n is 40 m l . V o l u m e of a f r a c t i o n is 7 m l . B. R e c h r o m a t o g r a p h y of the PC pool o b t a i n e d in A on d e x t r a n s u l f a t e - S e p h a r o s e 4B. C o n d i t i o n s are i d e n t i c a l to those of A e x c e p t v o l u m e of c o l u m n is 10 m l and v o l ume of g r a d i e n t is 2x100 ml. V o l u m e of a f r a c t i o n is 10 m l and v o l u m e of g r a d i e n t is 2x100 m l . V o l u m e of a f r a c t i o n is 1.8 ml. based

on c l o t t i n g

assays.

The

isolation method

of P C

is

sum-

m a r i z e d in T a b . 1. P r e p a r a t i o n of a n t i - p r o t e i n C a n t i s e r u m Rabbit

anti-human

rabbits with in F r e u n d ' s

PC

antiserum

four w e e k l y (in)complete

was

prepared

by

immunizing

i n j e c t i o n s of 30 - 50 jig p u r i f i e d adjuvant.

PC

110

Immune s e r a w e r e a d s o r b e d f i r s t w i t h B a S O ^

(100 m g / m l ) ,

plemented

adsorbed

with (w/v)

during

1/10

30 m i n

(v/v)

Al(OH)^

of

as

at

human

antiserum

and

plasma

described

d o u b l e d i f f u s i o n a n d by this

56°C in

then

previously

(12).

adsorbed

Analysis

Immunoelectrophoresis

contains

specific

a n t i b o d i e s a g a i n s t h u m a n PC

(Fig.

and

decom-

once

by

on

5 %

Ouchterlony

have

strongly

more

shown

that

precipitating

3).

T a b l e 1. P u r i f i c a t i o n of h u m a n PC f r o m 25 l i t e r s o f cryosupernatant

Volume (%)

Step

25 x 1 0 3

Cryosupernatant Barium citrate

Total

DEAE-Sephace1-EDTA DEAE-Sephacel-Ca

1.5 x

200*

210

80*

65

2+

105

3.5 x 1 0 3

250

eluate

Recovery (%)

protein (mg)

1st d e x t r a n

sulfate-Sepharose

60*

10

2nd d e x t r a n

sulfate-Sepharose

25*

7

10

*Values b e f o r e c o n c e n t r a t i o n o n A m i c o n PM 10 m e m b r a n e .

M e a s u r e m e n t of PC a n t i g e n by r o c k e t The e l e c t r o p h o r e s i s

electroimmunoassay

buffer w a s T r i c i n e

cine

(Biorad), 38.2 g T r i s , 1 m M E D T A

rose

(Standard L o w - m r ,

tion

of

1 %

dilution

of

(w/v) 1/75.

Biorad)

with The

at pH 8.6: 17.2 g

Tri-

in 4 l i t e r s of H 2 0 .

Aga-

w a s used at a f i n a l

anti-PC

antiserum

electrophoresis

was

present

at

carried

out

h o u r s at 10 m A / p l a t e . T h e g e l s w e r e t h e n w a s h e d solution 24 h o u r s . and

(0.9 % The

destained

(w/v)

gels in

NaCl

were 10 %

and

0.15 %

(v/v)

dried,

stained

with

acetic

acid

concentra-

solution.

in

a

final for

18

TWEEN-NaCl

T W E E N 20) Coomassie Fig. 4

for Blue shows

111

§

u-1 m U-l Q O 0.5

0.5

\

lA_ y v JL X j. 66K ASK 21K 14K molecular weight (daltons) F i g . 2.

S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of h u m a n PC. A. 8 p g of u n r e d u c e d P C B. 8 ¿ig of r e d u c e d PC (5 %

fl-mercaptoethanol)

The a r r o w s i n d i c a t e the d e g r a d e d form of the P C heavy chain.

112

F i g . 3.

F i g . 4.

Ouchterlony double immunodiffusion analysis of the r a b b i t a n t i h u m a n PC a n t i s e r u m . A. 1 3

a n t i - h u m a n PC a b s o r b e d ; 2 fresh normal human plasma.

B. 1 3

anti-hPC absorbed; 2 fresh normal plasma; p u r i f i e d PC; 4 PC d e f i c i e n t p l a s m a .

Rocket immunoelectrophoresis 1-3) 4-5)

purified

of h u m a n

PC;

PC.

P u r i f i e d PC d i l u t e d 1/4 (1), 1/2 (2), 1/1 (3). R e f e r e n c e p l a s m a (Coag-cal®) d i l u t e d 1/4 (4), 1/2 (5), 1/1 (6). 7-10) F r e s h n o r m a l p l a s m a p o o l d i l u t e d 1/8 (7), 1/4 (8), 1/2 (9), 1/1 (10).

113 different

samples

using

above

the

containing

antiserum.

PC

The

analyzed limit

by

of

this

technique

detection

of

PC

in

p l a s m a was a b o u t 0.08 U / m l . M e a s u r e m e n t of PC In

order

similar

to

activity

measure

activity,

we

have

used

to that d e s c r i b e d by B e r t i n a et al.

vitamin K-dependent instead about

PC

of

Al(OH)3.

80 %. A s

were

proteins The

average

pointed

out

by

recoveries

of

prothrombin

temperature

of

the

barium

recovery

were

et

on of

al.

only

procedure

(13) , e x c e p t

adsorbed

Sala

a

barium

that

citrate

prothrombin (2),

obtained

citrate

adsorption

carried

out

was

reproducible when

the

was

well

step

controlled. The

activation

thrombin plete,

in

of

the

tivity

with

was

of

EDTA.

inhibited

by

When

an

chromogenic

substrate

the p r o t e o l y t i c

this p u r p o s e ,

we

have

of c o n g e n i t a l

familial recurrent The

etiology

problem

of

for

association

disease,

the

recurrent

biologist

plasminogen

of of

PC

known

activator

been

described

and

Gen-

(Kabi).

due

to

ac-

For

all

possible

ac-

than PC w a s

subtracted.

plasma

prepared

in p a t i e n t s

and

causes XII,

thrombosis the

is a

with

clinician.

of

and

congenital

inherited

antithrombin

plasminogen. over

Until

Since

then,

the

world

and

PC

the

(12)

of

thrombotic

thrombosis III,

and h o m o z y g o u s c o n g e n i t a l all

difficult

(14) a n d B e r t i n a et al.

deficiency

factor

c a s e s of h e t e r o z y g o u s have

com-

thrombosis

other

abnormalities

was

hirudin.

its a m i d o l y t i c

S-2238

background

alpha

antibodies.

d e s c r i p t i o n by G r i f f i n et al. an

of

protein C deficiency

familial

the

human

activation

used a PC d e p l e t e d

by i m m u n o a d s o r p t i o n w i t h P C Detection

pure

excess

t i v a t i o n by t h r o m b i n of p r o t e i n other For

by

then e s t i m a t e d by m e a s u r i n g

the

measurements,

was

presence

thrombin

erated PCa was

PC

were

fibrinogen, many

more

deficiency

associated

with

114

various

clinical

manifestations

thrombotic

events,

episodes,

arterial

necrosis

to

purpura

frequent

PC d e f i c i e n c y deficiency.

recurrent

thrombosis,

massive

fulminans

thrombosis

(A.W.

ranging

venous

of

the

Broekmans,

prevalence

of

few

or

PC

skin

microcirculation

this

no

thrombo-embolic

antivitamin K-induced

s e e m s to be m o r e f r e q u e n t

The

from

volume).

with

Congenital

than a n t i t h r o m b i n

deficiency

has

been

III

esti-

m a t e d to be one in 1 6 , 0 0 0 . In

a one

year

isolated

PC

thrombosis tion

and

clinical

and

activities,

unknown

of

of

PC

and PC

with

s u l t s are p r e s e n t e d t a t i o n s . A l l these normal

Tab.

the

antigenic

measured we

have

by

Since PC

and

are

probably

developed.

then,

we

ImmunoThe

re-

manifes-

about

heterozygous dominant.

ven-

deficiency.

rocket

2 w i t h the m a i n c l i n i c a l

t r a i t , w h i c h is i n h e r i t e d as a u t o s o m a l

the with

biological

recurrent

14 p a t i e n t s have m e a n PC l e v e l s

value

coagula-

family

and

with

D...).

for

venous

reported

French

with congenital

were

antibody

in Tab.

briefly

large

Family

screening

recurrent

platelets,

associated

2,

levels

of

have a

(both

4 new families

families,

electrophoresis

the

We

study

been

with

abnormalities

type I d e f i c i e n c y ) (15

has

patients

systems.

deficiency

have discovered

of

laboratory

in

biological

thrombosis

In these

our

fibrinolytic and

congenital ous

period, deficiency

for

50 % the

115

T a b l e 2. P a t i e n t s w i t h f a m i l i a l d e f i c i e n c y of

Patients Normal population (range) Family II II

Protein C antigen (U/ml) 0.66-1.2

Thrombotic

PC.

manifestations

none

D...

2 6

0. 54 0.49

II 8 II 15 10 n o r m a l (range)

0.31 0.45

members

0.87-1.32

varicose recurrent thrombosi recurrent recurrent

veins superficial s deep venous deep venous

venous thrombosis thrombosis

none

Family W... III. III IV IV IV IV

2 4 1 2 3 4

0.35 0.42 0. 44 0.33 0. 52 0.33

recurrent deep venous recurrent deep venous none none none none

0. 59 0.40

none none

1

0.46

recurrent venous

R O H . F.. .

0.19

recurrent deep venous under A V K (PT 44 %)

Family II II

thrombosis thrombosis

J...

1 2

Family H... II

thrombosis

The l e v e l of PC a n t i g e n w a s m e a s u r e d in p l a s m a by Immunoelectrophoresis.

thrombosis

rocket

116 References 1. K i s i e l , W. : J . C l i n . I n v e s t . 64, 7 6 1 - 7 6 9

(1979).

2. S a l a , N., O w e n , W . G . , C o l l e n , D. : B l o o d 63, (1984).

671-675

3. Esraon, C . T . , E s m o n , N . L . : S e m i n . T h r o m b . H e m o s t a s . 10^, 1 2 2 - 1 3 0 (1984). 4. S a l e m , H . H . , B r o z e , G . J . , M i l e t i c h , J . P . , M a j e r u s , Proc. Natl. A c a d . Sei. USA 80, 1 5 8 4 - 1 5 8 8 (1983). 5. A m p h l e t t , G . W . , K i s i e l , W . , C a s t e l l i n o , F . J . : 20, 2 1 5 6 - 2 1 6 1 (1981).

P.W.:

Biochemistry

6. S u z u k i , K., S t e n f l o , J., D a h l b ä c k , B . , T e o d o r s s o n , B. : J. B i o l . Chem. 258, 1 9 1 4 - 1 9 2 0 (1983). 7. V e h a r , G . A . , D a v i e , E.W. : B i o c h e m i s t r y

19, 4 0 1 - 4 0 9

(1980).

8. W a l k e r , F . J . : S e m i n . T h r o m b . H e m o s t a s . 1J), 1 3 1 - 1 3 8 (1984). 9. S u z u k i , K. : S e m i n . T h r o m b . H e m o s t a s .

10, 1 5 4 - 1 6 1

(1984).

10. G r i f f i n , J.H. : S e m i n . Thromb. H e m o s t a s . 1_0, 1 6 2 - 1 6 6 (1984). 11. F r e y s s i n e t , J . - M . , T h e v e n o n , D . , S o u q u e , A . , M . : T h r o m b . H a e m o s t a s . £ 8 , 120-124 (1982).

Suscillon,

12. B e r t i n a , R . M . , B r o e k m a n s , A.W. , V a n der L i n d e n , M o r t e n s , K. : T h r o m b . H a e m o s t a s . 48 1 - 5 (1982).

I.K.,

13. B e r t i n a , R . M . , B r o e k m a n s , A . W . , K r o m m e n h o e k - v a n , E., W i j n g a r d e n , A . : T h r o m b . H a e m o s t a s . SI, 1-5 (1984).

van

14. G r i f f i n , J . H . , E v a t t , B., Z i m m e r m a n , T . S . , K l e i s s , A . J . , W i d e m a n , C. : J. Clin. Invest. £ 8 , 1 3 7 0 - 1 3 7 3 (1981) . 15. C a z e n a v e , J . - P . , W i e s e l , M . - L . , G r u n e b a u m , L . , F r e y s s i n e t , J . - M . , D o r n e r , M . , F r i t s c h e , R., G r e b e r , S., S p a e t h e , R . , L a m p a r t , A. : V l l l t h I n t e r n a t i o n a l C o n g r e s s o n T h r o m b o s i s , I s t a n b u l , June 4-7 (1984) (Abstract).

PROTEIN C DEFICIENCY

IN A N E W B O R N I N F A N T W I T H

PURPURA

FULMINANS

H e l m u t W e h i n g e r and Irene W i t t K i n d e r k l i n i k der S t ä d t i s c h e n K l i n i k e n D-3500 Kassel B i o c h e m i s c h e s Labor der U n i v e r s i t ä t s - K i n d e r k l i n i k D-7800 Freiburg

Purpura

fulminans

mortality.

The

is

a

disease

The p a t h o g e n e s i s h a s ly

protein

C

with purpura

rare may

The

in

newborn

deficiency fulminans

was

diagnosed

(3). W e

report

was

pregnancy years

born

weighing

old;

one

baby

infants

in

a

a

high

(1,

definitely.

on

newborn

similar

2).

Recentinfant

case

and

phenprocoumon.

both

episode

presented

on

2920 g.

were of with

17th A u g u s t 1983 The

father

healthy.

The

vital

in

ation

of

testes.

A

scrotum,

diagnosis

and

of

it

sorbed

after

stead. O n the the

right

a

few

days

sixth day

and of

other

life

appeared,

on

uneventful

the

mother

grandmother leg.

Apgar to

the

hematomas

d a y 11

a

has

was

10

discolor-

palpate

the

torsion

was

hematoma

a bluish-black

21

newborn

score

testicular

when

The

and bluish

impossible

false,

an

was

re-

developed

in-

discoloration

necrotizing

hemor-

rhage of the lower a b d o m e n , and o n d a y 14 an e x t e n s i v e

bleed-

ing i n v o l v i n g

elbow

was

intrauterine

made. This diagnosis proved

her

signs;

five m i n u t e s . T h e r e w a s a s w e l l i n g the

22,

maternal

thrombophlebitis normal

after

was

after

of

with

history child

had

occur

disorder

not been established

successful treatment with

Case

hemorrhagic

the r i g h t s c a l p . T h e r e w e r e m o r e r e l a p s e s

Protein C - Biochemical and Medical Aspects © 1985 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

and

a

Fig. 1.

total them

L a r g e h e m a t o m a on the r i g h t side of the and on the lower a b d o m e n

of

nine

occurring

hemorrhagic on

the

delineated,

started

then

to

turned

superficial

episodes

scalp.

with

All

red

bullae

were

these

to

bluish-black,

were

domen. there

On

sonography

was

dilatation

persisted,

There the of

With

but d i d not p r o g r e s s

were

healing

lower

was

renal

later). N o

Examination

of

the

eyes

showed

on

urinary

sonography the

in

right

the

normal,

Cerebral

found.

necrotic

pelvis

ipheral

and

6 to d a y

mass

and no f r a g m e n t o c y t e s

were

sharply

Sometimes

the

From day

no rise in s e r u m c r e a t i n i n e blood

of

discoloration,

parenchyma

left

most

gangrene.

w a s no p a l p a b l e

renal the

lesions

purple

s k i n was r e j e c t e d a n d a scar w a s formed. hematuria was present.

observed,

indicating

formed.

scalp

10 abbut

(which

infection, in the

was

side

a

per-

normal. central

119 corneal the

scar,

synechisis

vitreous.

On

the

of

the

left

iris,

side

and

there

old

was

hemorrhages

corneal

h y p e r e m i a of the iris, and some s t r i p e - l i k e v i t r e o u s Using

routine

bleeding

clotting

episode

was

tests

it

accompanied

became

and - less s t r i k i n g - t h r o m b o c y t o p e n i a mers were present,

evident

by s e v e r e

in

clouding, bleeding. that

each

hypofibrinogenemia

(Tab.

1). F i b r i n

but there w a s no e v i d e n c e of s e v e r e

monohyper-

fibrinolysis. O n day

34

revealed

a biopsy extensive

from

an

involved

hemorrhage

t h r o m b i w i t h i n the c a p i l l a r y c u l i t i s a n d no other

19.8.

THERAPY HEPARIN U/24 H

22.8. 1200

FIBRINOGEN MG THROMBOZYTES//UL

24.8.

2400

52.000

was

corium

was

25.8. 2400

26.8.

27,8.

2400

28.8.

2400

3000

29.8.

vas-

Investigation

3000

300 66,000

143,000 83,000

31 8.

30.8.

3000

NORHAL

300 122,000 122,000 51,000

RANGE 102,000

100,000 - 300,000

FIBRINOGEN HG/DL

83

40

320

QUICK I

66

48

>100 •

80

> 70

&

PIT sec

35

42

25

35