RECOMBINANT 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