Handbook of Synthetic Substrates: For the Coagulation and Fibrinolytic System

181 Pages·1983·6.637 MB·English

by  H. C. Hemker (auth.)

Handbook of Synthetic Substrates
HANDBOOK OF
SYNTHETIC
SUBSTRATES
For the Coagulation and Fibrinolytic System
by
H.C.HEMKER
Department ofB iochemistry
Faculty of Medicine
University of Limburg
Maastricht, The Netherlands
with contributions by
M.C.E. VAN DAM-MIERAS
S.IWANAGA
C.M. JACKSON
J .M. NIGRETIO
K.C. ROBBIN:S
1983 MARTINUS NIJHOFF PUBLISHERS
a member of the KLUWER ACADEMIC PUBLISHERS GROUP
BOSTON / THE HAGUE / DORDRECHT / LANCASTER
Distributors
for the United States and Canada: Kluwer Boston, Inc., 190 Old Derby Street,
Hingham, MA 02043, USA
for all other countries: Kluwer Academic Publishers Group, Distribution Center,
P.O.Box 322,3300 AH Dordrecht, The Netherlands
Library of Congress Cataloging in Publication Data
Hemker, H. C.
Handbook of synthetic substrates for the coagulation
and fibrinolytic system.
Bibliography: p.
Includes index.
1. Blood--Coagulation. 2. Fibrinolysis. 3. Chromo
genic compounds--Analysis. I. Dam-Mieras, M. C. E. van.
II. Title.
QP93.5.H44 1983 612' .115 82-24678
ISBN -13: 978-94-009-6692-5 e-l SBN -13:978-94-009-6690-1
DOl: 10.1007/978-94-009-6690-1 87-28241
ISBN -13: 978-94-009-6692-5
Copyright
© 1983 by Martinus Nijhoff Publishers, Boston.
Softcover reprint of the hardcover 1st edition 1983
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, mechanical,
photocopying, recording, or otherwise, without the prior written permission of
the publishers,
Martinus Nijhoff Publishers, 190 Old Derby Street, Hingham, MA 02043, USA.
Contents
Preface IX
Foreword XI
I. Basic enzymology 1
I. I. Introduction 1
I. 2. Maximal velocity 3
I. 3. Michaelis constant 4
I. 4. Low substrate concentration;
the efficiency of an enzyme 5
I. 5. High substrate concentration;
the saturation curve 6
I. 6. The characteristic parameters of an enzymatic
reaction 7
I. 7. Enzyme inhibition 9
I. 8. Rate-constants and enzymatic mechanism 10
I. 9. The reaction mechanism of serine proteases 12
o.
1.1 Transient state kinetics 16
1.11. Data from a reaction going to completion 17
1.12. Active site titration 18
1.13. Simultaneous reactions in complicated reaction
mixtures 21
II. Measuring the conversion of a chromogenic substrate 27
II. 1. Photometric assays 27
II. 2. Absorption spectrophotometry 28
II. 3. Choice of wavelength and absorbancy range 32
II. 4. Temperature 33
II. 5. The reaction medium 35
v
vi
II. 6. Substrate solution 36
II. 7. Methodology 38
II. 8. Execution of the experiment 38
II. 9. Fluorescence 44
I1.l O. Electrochemical determinations 47
llI. Substrates S3
III.l. Specificity in serine proteases 53
III.2. Substrate selectivity and sensitivity 57
I1L3. Inhibition by artificial substrates 64
IliA. Data on commercially available substrates 65
IlLS. Fluorogenic substrates 83
I1L6. General principles of fluorogenic peptide substrates
with 7 -amino-4-methylcoumarine (MeA) as a leaving
group 83
III.7. Thioesters 88
I1L8. Substrates for electrochemical determinations 89
III.9. Luminogenic substrates 90
IV. Determinations that can be carried out with chromogenic
substrates 9S
IV.l. Thrombin and derived determinations 95
1.1. thrombin 96
1.2. prothrombin 96
1.3. antithrombin III and heparin cofactor 98
104. prothrombinase, factor V, platelet factor 3 99
1.4.1. prothrombinase 100
1.4.2. factor V 101
1.4.3. platelet factor 3 101
IV.2. Factor Xa and derived determinations 102
2.1. factor Xa 102
2.2. factor X 103
2.3. antifactor Xa (antithrombin III,
heparin cofactor) 104
2.4. heparin 105
2.5. platelet factor 4 and other antiheparins 106
2.6. factor X activating enzymes 107
vii
2.6.1. factor VIII 108
2.6.2. factor IX 108
2.6.3. platelet factor 3 108
2.6.4. factor VII and tissue thromboplastin 108
IV.3. Determination of fibrinolytic pathway components
in plasma 109
3.1. plasminogen 109
3.2. plasminogen activator 110
3.3. 'free' protease activity 110
3.4. plasmin generation rates 111
3.5. cx -p1asmin inhibitor 111
2
IV.4. The contact activation system 112
4.1. plasma prekallikrein 112
4.2. factor XII 114
4.3. activated factor XI 116
IV.5. Miscellaneous determinations 117
5 .1 . glandular kallikrein 11 7
5.2. brinase and brinase inhibitors 118
5.3. bacterial endotoxins 119
Bibliography of synthetic substrates (as available Fall 1982) 125
Index of subjects 169
Preface
The need for a handbook on the use of synthetic substrates for assay of
proteases of the coagulation and fibrinolytic systems became evident
several years ago during the activities of the Subcommittee on Synthetic
Substrates of the International Committee on Thrombosis and Haemo
stasis (lCTH). Production of such a handbook, which was recommended
during discussions of the ICTH at its meeting in London in 1979 was
made possible by the generous efforts of Professor HC Hemker with the
aid of several contributors with particular interests in the use of synthetic
substrates in coagulation and fibrinolysis. As current Chairman and
Secretary General of the ICTH we would like to express our sincere
thanks to Professor Hemker for producing this handbook and look for
ward to seeing the benefits of this tremendous effort reflected in the
advancement of our understanding of thrombosis and hemostasis and the
transfer of such knowledge into improved diagnosis and treatment of
thrombotic and hemorrhagic disorders.
Craig M Jackson
Professor of Biological Chemistry
Washington University School of Medicine, St. Louis, MO
Chairman, ICTH
Harold R Roberts
Professor of Internal Medicine
University of North Carolina, Chapel Hill, NC
Secretary General, ICTH
ix
Foreword
The advent of synthetic substrates for the study of blood coagulation
and fibrinolysis was a significant step forward in the investigation of
these systems. Both basic research and clinical laboratory investigations
can profit from these advanced tools.
The International Committee on Thrombosis and Haemostasis, through
its subcommittee on chromogenic substrates, rapidly became aware that
the progress possible in this field could be improved by the presentation
in a concise form of the relevant information available.
The necessary information appeared to come in four kinds:
1) Basic knowledge on enzyme kinetics.
This is mandatory for understanding the use of chromogenic sub
strates, but it is not standard knowledge of the coagulation patho
physiologist.
2) The scientific basis of the practise of the measuring techniques.
This contains the necessary background information for the new
types of benchwork introduced in the coagulation laboratory.
3) The product information, including the enzyme kinetic data of the
various substrates. As yet these were available in many scattered
publications only.
4) An overview of the coagulation and fibrinolysis methods in which
synthetic substrates have been employed.
These four kinds of information are given in the four chapters of this
book. A substantial part of the contents of this book is based on para
graphs contributed by scientists known to be experts in different aspects
of the use of synthetic substrates. These paragraphs, listed below in
alphabetical order, were especially written for this book.
1) S Iwanaga, H Kato, T Morita, T Sugo, Y Ohno, M Ohki, K. Takada,
S Sakakibara (Faculty of Sciences, Kyushu University, Fukuoka,
Japan),
Fluorogenic substrate assay methods for proteinases in blood coagu
lation, kallikrein-kinin and fibrinolysis systems.
2) CM Jackson (Washington University School of Medicine, St Louis,
MO, U.S.A.),
xi
xii
Methods for increasing the specificity of chromogenic and fluoro
genic substrate assays of plasma proteases.
3) 1M Nigretto, M 10zefowicz (Universite de Paris Nord, Villetaneuse,
France),
Electrochemical activity determination of trypsin-like enzymes in
whole blood using electrogenic substrates.
4) KC Robbins, RC Wohl (Michael Reese Research Foundation,
Chicago, Ill, U.S.A.),
Determination of fibrinolytic pathway components in plasma.
I edited these contributions in order to obtain a certain unification of
presentation and to prevent duplications. Except for these minor adap
tations the manuscripts have been incorporated in the text unaltered.
A first draft of the text was circulated among the thirty main
researchers active in the field of synthetic substrates. I would like to
thank them for their suggestions, corrections and remarks, all of which
have had their impact on the final version.
For reasons beyond my control the preparation of the final version
has taken much more time than foreseen. Dr van Dam-Mieras (Department
of Biochemistry, Faculty of Medicine, University of Limburg, Maastricht)
has rewritten some parts of the text, contributed other parts and has
seen to it that the references have been kept up to date. The Bibliography
added at the end of the book also has been prepared by her.
I hope that the book will serve its purpose.
HC Hemker
Maastricht, Spring 1983
I. Basic enzymology
Summary
In the first paragraphs of this chapter (1-6) it is attempted to give an
explanation of enzyme kinetics in such a form that those without previous
knowledge of this matter can use it as an introduction. Basic information
of this kind is necessary for the understanding of the use and applications
of chromogenic substrates.
The further paragraphs are not compulsory reading for the under
standing of the rest of the chapters but treat subjects that may become
important under circumstances. Enzyme inhibition is treated in para
graph 7 a.o. because unwanted inhibition by competition between arti
ficial and natural substrates can cause confusion in practise.
Paragraphs 8, 9 and 10 link the formalism of kinetics with the chemical
events taking place in the reaction medium. Paragraph 11 discusses the
information that can be obtained from a reaction going to completion.
Active site titration, which is the cornerstone of standardisation in this
field is discussed in paragraph 12 whereas the subject of simultaneous
reactions, as they often occur in plasma, is treated in the last paragraph.
1.1. Introduction
An enzyme is a protein that due to its specific action can convert mol
ecules, called the substrate(s) into others, called the product (s). As all
catalysts an enzyme accelerates a reaction without being converted
itself and without influencing the chemical equilibrium. This text con
cerns the assessment of the enzymes active in blood coagulation and
fibrinolysis. We, therefore, will need a discussion of the basic concepts in
enzymology. Blood coagulation and fibrinolysis factors are examples
of those (pro) enzymes known as the serine proteases, therefore, we will
treat general enzymology only insofar as it pertains to that class of
enzymes. Also we cover the theory of enzyme action only insofar as
it is necessary for the practice of using chromogenic substrates. For a