Table Of ContentRECOMBINANT
TECHNOLOGY IN
HEMOSTASIS AND
THROMBOSIS
RECOMBINANT
TECHNOLOGYIN
HEMOST ASIS AND
THROMBOSIS
Edited hy
Leon W. Hoyer
and
William N. Drohan
Jerome H. Holland Laboratory
American Red Cross B100d Services
Rockville, Maryland
SPRINGER SCIENCE+BUSINESS MEDIA, LLC
Llbrary of Congress Cataloglng-ln-Publlcatlon Data
American Red Cross Scientific Symposium (21st 1990 Washington,
D.C. )
Recombinant technology in hemostasis and thrombosis I edited by
Leon W. Hoyer .nd William N. Drohan.
p. cm.
"Proceedlngs of the American Red Cross Twenty-first Annual
Scientific Symposium, impact of recombinant technalogy in hemostasis
and thrombosis, held May 15-16, 1990, in Washington, DC"--T.p.
verso.
Includes bibliographical references and index.
ISBN 978-1-4613-6644-7 ISBN 978-1-4615-3698-7 (eBook)
DOI 10.1007/978-1-4615-3698-7
i. Blooa--Coagulation, Dlsorders of--Congresses. 2. Recombinant
proteins--Congresses. 3. Blood--Coagulation--Congresses. 4. Blood
coagulatlon factors--Biotechnology--Congresses. 1. Hoyer, Leon W.
II. Drohan, Wi 11 iam. III. T1tle.
[DNLM, 1. Blood Coagulation Disorders--diagnosis--congresses.
2. Blood Coagulation Disorders--drug therapy--congresses. 3. Biood
Coagulat ion Factors--congresses. 4. Recombinant Proteins-
-congresses. 5. Thrombosis--drug therapy--congresses. WH 322
A215r 19901
RC647.C55A53 1990
616. 1 '57--dc20
DNLM/DLC
for Llbrary of Congress 91-3009
CIP
Proceedings of the American Red Cross Twenty-first AnnuaJ Scientific
Symposium: Impact of Recombinant Technology in Hemostasis and
Thrombosis, held May 15-16, 1990, in Washington, D.C.
ISBN 978-1- 46 13 -6644-7
© 1991 Springer Science+Business Media New York
OriginaJly published by Plenum Press, New York in 1991
Softcover reprint ofthe hardcover 1s t edition 1991
Ali rights reserved
No part of this book may be reproduced, stored in a retrieval system, Of transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise, without written permission from the Publisher
PREFACE
Recent progress in molecular biology has led to a rapid expansion of our
understanding of the proteins that are essential for hemostasis and
thrombosis. The goal of the XXI Annual Scientific Symposium of the American
Red Cross was to provide a forum to explore and document the impact of
recombinant DNA technology in this field. The speakers described the
essential features of the genes responsible for key plasma proteins important
in hemostasis, including procoagulant Factors VIII and IX and anticoagulant
proteins, Antithrombin III and Protein C. They emphasized the advances in
recombinant DNA technology that have led to the cloning of these genes.
Careful examination of the gene sequence has then provided a clearer
understanding of the structure of the encoded proteins, and has given
additional insight into their functional domains and their interactions in
hemostasis.
At the same time, these advances have made it possible to better
characterize hemostatic disorders. A large number of published studies have
shown that the mutations affecting biological activity are clustered in areas
that define functional domains. They have led to fundamental advances in our
understanding of specific diseases and they have made it possible to develop
more accurate and sensitive diagnostic tests for the detection of the disease
states.
v
Recombinant DNA technology is also beginning to be used to produce
coagulation proteins for clinical use. The first example of this is the
outstanding technical accomplishment of producing human coagulation Factor
VIII in mammalian tissue culture cells, and the subsequent evaluation of the
recombinant-produced protein in clinical trials. During this symposium, the
advantages and limitations of protein production by recombinant technology
were carefully examined by the participants. The rapid progress during the
past decade provides strong support for the view that even more remarkable
advances will soon be available to improve the treatment of diseases affecting
hemostasis and thrombosis.
The success of the symposium was due to the efforts of the Program
Committee that planned and chaired the scientific sessions: Morris A.
Blajchman, M.D., William N. Drohan, Ph.D., Leon W. Hoyer, M.D., Kenneth G.
Mann, Ph.D., and Frederick J. Walker, Ph.D. The rapid publication of these
proceedings has been facilitated by the excellent editorial assistance of
Debbie Wilder.
Leon W. Hoyer, M.D.
William N. Drohan, Ph.D.
vi
CONTENTS
CHARACTERIZATION OF GENE AND PROTEIN STRUCTURE
Biosynthesis and Assembly of the Factor VIII-von Willebrand
Factor Complex................................... . . . . . . . . . . . . . . 3
Jan A. van Mourik, Anja Leyte, Harm B. van Schijndel,
Martin Ph. Verbeet, Jan Voorberg, Ruud D. Fonteijn,
Hans Pannekoek, and Koen Mertens
Factor IX: Gene Structure and Protein Synthesis..................... 13
D.B.C. Ritchie, D.L. Robertson, and R.T.A. MacGillivray
Antithrombin III Genetics, Structure and Function....... ......... ... 25
Susan Clark Bock
Interactions Between the Functional Domains of
Antithrombin III................................. . . . . . . . . . . . . . . 47
Paula R. Boerger, Robert M. Wolcott, Morgan Lorio,
and Michael N. Blackburn
Protein C: Gene Structure and Protein Synthesis...................... 65
George L. Long
Structural and Functional Properties of Protein C....... .......... ... 79
Frederick J. Walker
MOLECULAR DEFECTS AFFECTING HEMOSTASIS
Molecular Defects in Hemophilia A .................................... 101
Leon W. Hoyer
Molecular Defects in Hemophilia B. ................................... 115
Arthur R. Thompson
Molecular Defects in Human Antithrombin III
Deficiency .................................................... 133
W.P. Sheffield, F. Fernandez-Rachubinski, R.C. Austin,
and M.A. Blajchman
The Biologic Impact of Hereditary Defects that Cause
Thrombosis..................................................... 147
Kenneth A. Bauer
PROTEIN PRODUCTION BY RECOMBINANT TECHNOLOGY
Factors Limiting Expression of Secreted Proteins in Mammalian
Cells. . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Randal J. Kaufman, Robert J. Wise, Louise C. Wasley,
and Andrew J. Dorner
Synthesis of Biologically Active Vitamin K-Dependent
Coagulation Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Barbara C. Furie and Bruce Furie
The Expression of Therapeutic Proteins in Transgenic
Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 197
Rekha Pa1eyanda, Janet Young, William Ve1ander,
and William Drohan
CLINICAL USE OF PROTEINS PRODUCED BY RECOMBINANT TECHNOLOGY
The Use of Animal Models to Evaluate Proteins Produced by
Recombinant Technology ......................................... 213
Alan R. Giles
Experiences with Recombinant Factor VIla in
Hemophiliacs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 223
Ulla Hedner
Clinical Trials of Factor VIII Produced by Recombinant
Technology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 229
Richard S. Schwartz
Clinical Trials of Recombinant Factor VIII ........................... 235
Gilbert C. White, II, and Clinical Study Group
CONCLUDING OVERVIEW
The Impact of Recombinant Technologies in Understanding
Plasma Proteins Important for Hemostasis and
Thrombosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 245
Kenneth G. Mann
Contributors ........................................................ 251
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 253
viii
CHARACTERIZATION OF GENE AND PROTEIN STRUCTURE
BIOSYNTHESIS AND ASSEMBLY OF THE FACTOR VIII-VON WILLEBRAND FACTOR
COMPLEX
Jan A. van Mourik, Anja Leyte, Harm B. van Schijndel,
Martin Ph. Verbeet, Jan Voorberg, Ruud D. Fonteijn,
Hans Pannekoek, Koen Mertens
Central Laboratory of the Netherlands Red Cross
Blood Transfusion Service
P.O. Box 9190, 1006 AD Amsterdam
The Netherlands
INTRODUCTION
Factor VIII and the von Willebrand factor (vWF) are plasma
proteins that serve an essential role in the hemostatic proces;
factor VIII functions as a cofactor in the intrinsic coagulation
pathway (1,2) whereas VWF is hemostatically important in the mediation
of platelet-vessel wall interactions at sites of vascular injury
(3,4). In blood, factor VIII and VWF are not present as distinct proteins
but rather circulate as a linked complex. Several lines of evidence
indicate that this phenomenon is of physiological significance. For
instance, it now seems clear that VWF functions as a carrier protein
and as such has a stabilizing effect on factor VIII. This view stems
from the observation that reduced or absent synthesis of VWF (as
seen in von Willebrand's disease) is associated with markedly reduced
concentrations, or absence, of plasma factor VIII (5). Similarly, a rise
in VWF concentration, as observed in disorders associated with acute-phase
reactions, is accompanied with concommitant rises in plasma factor VIII
concentrations (6). Taken into account that the half life of factor VIII
infused in animals is determined by the presence of endogenous vWF (7),
and vWF also stabilizes factor VIII in vitro (8), these observations
clearly illustrate that vWF not only binds to factor VIII but also
confers stability to factor VIII. As VWF protects factor VIII from proteo
lytic attack by proteases including thrombin and activated factor X (9,10),
it seems likely that limited proteolysis is a factor that determines
the stability of factor VIII in vitro and in vivo. The importance of the
apparent stabilizing effect of vWF on factor VIII is underscored by recent
observations which show that an aberrant interaction between factor VIII
and vWF predisposes to a bleeding diathesis (11,12) Taken together,
these observations clearly document the physiological importance of
the factor VIII-VWF complex formation.
The cloning of the factor VIII- and VWF gene and the expression of
recombinant proteins and mutants thereof, together with immunochemical
Recombinant Technology in Hemostasis and Thrombosis
Edited by L.W. Hoyer and W.N. Drohan, Plenum Press, New York, 1991 3
studies, have provided the basis for significant advancements in the
understanding of the structure-function relationship of factor VIII and
vWF and of the nature of the interaction between these molecules.
BIOSYNTHESIS AND ASSEMBLY OF VON WILLEBRAND FACTOR
In the early 1970's, when antibodies to the factor VIII-vWF complex
became available, immunofluorescence studies revealed that vascular
endothelial cells of a variety of human tissues contain immuno-
reactive material (13,14). Soon it became clear that most normal
endothelial cells synthesize and secrete vWF, including endothelial
cells isolated from large and smaller veins, capillaries, aorta, and
arteries (15,16). Besides megakaryocytes (17,18), the endothelial cell
is the only cell type that synthesizes vWF.
vWF gena,chromosome '2, -'50kb
NUCLEUS
-9000nl
cotT'lj)lelely c:l •• WflII
larat j'ftuHi"".u
Fig. 1. Schematic representation of the processing steps involved
in the biosynthesis of vWF. The bars on the right represent the
vWF protein precursor and the products derived from it. The hatched
area represents the propeptide (vWAg II) of vWF and the dark area
mature vWF. WB = Weibel-Palade body; SV = secretory vesicles; * =
=
noncovalent interaction; S disulfide bonds.
Endothelial cells or megakaryocytes do not synthesize factor VIII (see
below). VWF distinguishes itself from many other endothelial proteins
in that it can be secreted by the cell by more than one pathway.
VWF is either released directly by the constitutive pathway or is released
into the medium upon treatment with endothelial cell agonists that trigger
release of previously synthesized vWF from storage vesicles (regulated
pathway). The constitutive secretory nature of the endothelium is reflected
by the observation that soon after synthesis vWF, and other proteins such
as fibronectin or thrombospondin, accumulate extracellularly in the absence
of a stimulus (15,19). No external stimulus or trigger is required for
this type of secretion. On the other hand, if endothelial cells are
exposed to stimuli such as thrombin, the Ca-ionophore A23187 or phorbol
esters, vWF rapidly (within minutes) accumulates outside the cell (19,
4
