Handbook of

Experimental Pharmacology

Volume 146 Editorial Board

G. V.R. Born, London D. Ganten, Berlin H . Herken, Berlin K. Starke, Freiburg i . Br. P. Taylor, La Jolla, C A

Springer-Verlag Berlin Heidelberg GmbH

Fibrinolytics and Antifibrinolytics

Contributors

F. Bachmann, D.C. Berridge, C. Bode, H . Bounameaux, FJ. Castellino, XH. Chesebro, A.C. Chiu, D. Collen, G. J. del Zoppo, W. Dietrich, P. Donner, J.J. Emeis, L. Flohe, V. Fuster, R. Gallo, W.A. Günzler, V. Gurewich, E. Haber, S.B. Hawley, R. Hayes, T. Herren, J.L. Hoover-Plow, J. Horrow, T. Kooistra, E.K.O. Kruithof, H.R. Lijnen, V.J. Marder, L . A . Miles, K. Peter, E.F. Plow, V.A. Ploplis, A . Redlitz, M.S. Runge, C M . Samama, M . M . Samama, A . A . Sasahara, W.-D. Schleunig, G.V.R.K. Sharma, O. Taby, E.J. Topol, M . Verstraete, S. Xue

Editor: F. Bachmann

09 Springer

Professor emeritus F. B A C H M A N N FRAT - Höpital Nestle 2077 C H U V CH-1011 Lausanne Switzerland e-mail: Fedor.Bachmann@dmed.unil.ch

Private address: Chemin Praz Mandry 20 CH-1052 Le Mont Switzerland

With 47 Figures and 50 Tables

ISBN 978-3-642-63029-3 ISBN 978-3-642-56637-0 (eBook) DOI 10.1007/978-3-642-56637-0

Library of Congress Cataloging-in-Publication Data Fibrinolytics and antifibrinolytics / contributors, F. Bachmann . . . [et al.]; editor, Fedor Bachmann,

p. cm. - (Handbook of experimental pharmacology; v. 146) Includes bibliographical references and index.

1. Thrombolytic therapy. 2. Fibrinolytic agents. 3. Fibrinolysis. 4. Antifibrinolytic agents. I. Bachmann, Fedor. II. Series.

[DNLM: 1. Fibrinolytic Agents. 2. Antifibrinolytic Agents. QV 190 F4431 2001] QP905 .H3 vol. 146 [RC694.3] 615'.ls - dc21 [6i5'.7i8]

00-059471

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Preface

In 1978, Fritz Markwardt edited Fibrinolytics and Antifibrinolytics, vol. 46 of the Handbook of Experimental Pharmacology series. Since then tremendous strides have been made in our understanding of the pathophysiology of the fibrinolytic system, of myocardial infarction and of stroke.

r am very grateful to the contributors to this new edition of Fibrinolytics and Antifibrinolytics, all outstanding scientists and/or clinicians in their field. They have succeeded in presenting an up-to-date account of basic aspects of the fibrinolytic system, of both older and new thrombolytic agents, and have analyzed in a balanced fashion the results of first to third generation throm­bolytic drugs in clinical trials.

The book is arranged in three major sections. The first one deals with the molecular biology, biochemistry, physiology and pharmacology of the plas­minogen-plasmin enzyme system. The progress that has been made in this field since 1978 is staggering. Methods taken from molecular biology have been used successfully for cloning the zymogen plasminogen, the two princi­pal activators and three major inhibitors of the system, many receptors for plasmin( ogen) and its activators, and some modulators of this clot-dissolving system. For many compounds the crystal structure has been uncovered. Numerous regulators of the system, particularly cytokines, growth factors and hormones, have been identified and their action and signal transduction elu­cidated. This section is of particular interest to both basic scientists and phar­macologists.

The large second section on the clinical use of thrombolytic agents is sub­divided into three subsections. In the first of these subsections, a great deal of space is devoted to the use of thrombolytic agents for patients suffering from acute myocardial infarction. Analysis encompasses all major megatrials up to early 2000. Given that the formation of an occlusive thrombotic blood clot is a very dynamic process, which involves platelets and activated clotting factors (particularly thrombin), ample space is devoted to the discussion of adjuvant therapy with antiplatelet and anti thrombotic agents. This part of the book will be of great interest to cardiologists. The second subsection provides a compe­tent discussion of thrombolytic therapy of massive pulmonary embolisms, deep venous thrombosis, peripheral arterial occlusion and thromboembolic

VI Preface

cerebrovascular disease. It should make for interesting reading for internists, angiologists, vascular surgeons and neurologists. The third subsection deals mostly with thrombolytic agents that are still in development and with com­pounds capable of stimulating the endogenous production and release of tissue-type plasminogen activator.

The last major section of the volume deals with the pharmacology and clinical use of antifibrinolytic agents and will interest surgeons, internists and obstetricians. I have personally edited many of these chapters in order to prevent significant overlaps. Several chapters that had been submitted earlier by the authors have been updated to reflect the state of the art in early 2000.

This book is dedicated to my loving wife Edith who - understanding the demands on time that editing this book made - laid aside many projects for common activities in recent years. Special thanks are due to Ms. Doris Walker and her collaborators of Springer-Verlag for their uncomplicated and fruitful collaboration.

Lausanne, June 2000 Fedor Bachmann

List of Contributors

BACHMANN, E, Department of Medicine, University of Lausanne Medical Centre, FRAT - Hopital Nestle 2077, CHUV, CH-1011 Lausanne, Switzerland Private address: Chemin Praz Mandry 20, CH-1052 Le Mont, Switzerland e-mail: Fedor.Bachmann@dmed.unil.ch

BERRIDGE, D.c., Department of Vascular and Endovascular Surgery, First Floor, Block A, Lincoln Wing, St. James's University Hospital, Leeds, LS9 7TF, United Kingdom e-mail: David Berridge@GW.SJUH.NORTHY.NHS.UK

BODE, c., Medizinische Universitatsklinik Freiburg, Abteilung Innere Medizin III, Kardiologie und Angiologie, Hugstetter Strasse, D-79106 Freiburg, Germany e-mail: bode@mm31.ukI.uni-freiburg.de

BOUNAMEAUX, H., University of Geneva School of Medicine, Division of Angiology and Hemostasis, Department of Internal Medicine, University Hospital of Geneva, CH-1211 Geneva 14, Switzerland e-mail: bounamea@cmu.unige.ch

CASTELLTNO, EI, Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA e-mail: castellino.1@nd.edu

CHESEBRO, IH., Cardiovascular Institute, Mount Sinai Medical Center, Box 1030, One Gustave L. Levy Place, New York, NY 10029-6574, USA e-mail: james.chesebro@mssm.edu

CHIU, A.C., SMDC Health System Section of Cardiology 407 East Third Street Duluth, MN 55805 e-mail: achiu@smdc.org

VIII List of Contributors

Present address: Section of Cardiology, SMDC Health System, 400 East Third Street, Duluth, MN 55805, USA e-mail: achiu@smdc.org

COLLEN, D., Centre for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O&N, Herestraat 49, B-3000 Leuven, Belgium e-mail: desire.collen@med.kuleuven.ac.be

DEL Zappa, G.I, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, MEM-132, La Jolla, CA 92037, USA e-mail: grgdl.zop@hermes.scripps.edu

DIETRICH, W., Deutsches Herzzentrum, Institut flir Anaesthesiologie, Lothstrasse 11, D-80335 Mtinchen, Germany

DONNER, P., Preclinical Drug Research, Schering Research Laboratories, Mtillerstr. 178, D-13342 Berlin, Germany

EMEIS, II, Gaubius Laboratory, TNO-Prevention and Health, P.O. Box 2215, NL-2301 CE Leiden, The Netherlands e-mail: jj.emeis@pg.tno.nl

FLOHE, L., Department of Biochemistry, TU Braunschweig, Mascheroder Weg 1, D-38124 Braunschweig, Germany e-mail: lfl@gbf.de

FUSTER, Y, Cardiovascular Institute, Mount Sinai Medical Center, Box 1030, One Gustave L. Levy Place, New York, NY 10029-6574, USA

GALLO, R, Cardiovascular Institute, Mount Sinai Medical Center, Box 1030, One Gustave L. Levy Place, New York, NY 10029-6574, USA

GUNZLER, w.A., Grtinenthal GmbH, Postfach 50 04 44, D-52088 Aachen, Germany

GUREWICH, Y, Harvard Medical School, Vascular Research Laboratory, 21-27 Burlington/BIDMC, POB 15709, Boston, MA 02215, USA e-mail: vgurevic@caregroup.harvard.edu and: Institute for the Prevention of Cardiovascular Disease, Beth Israel-Deaconess Medical Center, Boston, MA, USA

HABER, E., Center for the Prevention of Cardiovascular Disease, Division of Biological Sciences, Harvard School of Public Health and Harvard Medical School, 677 Huntington Avenue, Boston, MA 02115, USA

List of Contributors

HAWLEY, S.B., Department of Vascular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA

IX

HAYES, R, Cardiovascular Institute, Mount Sinai Medical Center, Box 1030, One Gustave L. Levy Place, New York, NY 10029-6574, USA

HERREN, T., Joseph I Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic Foundation/NB50, 9500 Euclid Avenue, Cleveland, OH 44139, USA

HOOVER-PLOW, IL., Joseph I Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic Foundation/NB50, 9500 Euclid Avenue, Cleveland, OH 44139, USA

HORROW, I, IBEX Technologies Corporation, 5 Great Valley Parkway, Suite 300, Molvern, PA 19355, USA

KOOISTRA, T., Gaubius Laboratory, TNO-Prevention and Health, P.O. Box 2215, NL-2301 CE Leiden, The Netherlands e-mail: T.Kooistra@pg.tno.nl

KRUITHOF, E.K.G., Research Laboratories, Division of Angiology and Hemostatis, University Hospital, CH-1211 Geneva, Switzerland e-mail: Egbert.Kruithof@hcuge.ch

LIJNEN, H.R, Center for Molecular and Vascular Biology, Campus Gasthuisberg, O&N, Herestraat 49, B-3000 Leuven, Belgium e-mail: roger.lijnen@med.kuleuven.ac.be

MARDER, V.I, Orthopaedic Hospital/UCLA, Vascular Medicine Program, 2400 S. Flower Street, Los Angeles, CA 90007, USA e-mail: vmarder@laoh.ucla.edu

MILES, L.A., Department of Vascular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA

PETER, K., Medizinische Universitatsklinik Freiburg, Abteilung Innere Medizin III, Kardiologie und Angiologie, Hugstetter Strasse, D-79106 Freiburg, Germany

PLOW, E.F., Joseph I Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic Foundation/NB50, 9500 Euclid Avenue, Cleveland, OH 44139, USA e-mail: plowe@ccf.org

x List of Contributors

PLOPLIS, V.A, Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA

REDLITZ, A, Joseph I Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic Foundation/FF20, 9500 Euclid Avenue, Cleveland, OH 44139, USA

RUNGE, M.S., Division of Cardiology and Sealy Center for Molecular Cardiology, University of Texas Medical Branch at Galveston, 5.106 John Sealy Hospital, 301 University Boulevard, Galveston, TX 77555-0553, USA

SAMAMA, CM., Department d'Anesthesie-Reanimation, H6pital Avicenne, 125 route de Stalingrad, F-93009 Bobigny Cedex, France e-mail: cmsamama@invivio.edu

SAMAMA, M.M., H6pital Hotel Dieu, Place du Parvis de Notre Dame, F-75004 Paris, France e-mail: mmsamama@aol.com

SASAHARA, AA., Cardiovascular Division and the Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, 1115 Beacon Street, Unit 12, Newton, MA 02461-1154, USA e-mail: arthur.a.sasahara@abbott.com

SCHLEUNIG, W.-D., Preclinical Drug Research, Schering Research Laboratories, Mtillerstr. 178, D-13342 Berlin, Germany e-mail: Wolfdieter.Schleuning@Schering.de

SHARMA, G.v.R.K., the Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, and the Cardiology Section, Veterans Affairs Medical Center, West Roxbury, MA 02132, USA

TABY, 0., H6pital Hotel Dieu, Place du Parvis de Notre Dame, F-75004 Paris, France

TOPOL, E.I, Department of Cardiology, Desk F-25, II Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA e-mail: topole@ccf.org

VERSTRAETE, M., Centre for Molecular and Vascular Biology, University of Leuven, O&N, Herestraat 49, B-3000 Leuven, Belgium e-mail: marc.verstraete@med.kuleuven.ac.be

XUE, S., Department of Vascular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA

Contents

Section I: Molecular Biology, Biochemistry, Physiology, and Pharmacology of the Plasminogen-Plasmin Enzyme System

CHAPTER 1

The Fibrinolytic System and Thrombolytic Agents F. BACHMANN. With 1 Figure .................................... 3

A. The Fibrinolytic System .................................... 3 B. Mechanisms which Lead to the Lysis of a Thrombus . . . . . . . . . . . . 5 C. Pathophysiology of the Fibrinolytic System . . . . . . . . . . . . . . . . . . . . 7

I. Decreased Fibrinolytic Activity and Deep Venous Thrombosis ............................. 7

1. Familial Thrombophilia with High PAI-1 Levels. . . . . . . 7 2. Acquired Association of Hypofibrinolysis and

Deep Venous Thrombosis. . . . . . . . . . .. . . . .. . . . . . . . .. 7 II. Impaired Fibrinolysis and Coronary Heart Disease. . . . . . . . 9

D. Thrombolytic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 I. Basic Considerations ................................. 10

1. The Proteolytic State and Bleeding Complications . . . . . 10 2. Thrombin Generation and Reocc1usion .............. 10 3. Delay to Treatment ............................... 10 4. Platelet Activation ................................ 11 5. Contraindications for Thrombolytic Therapy . . . . . . . . . . 11 6. Fibrin Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 7. Antigenicity..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 8. Treatment of Elderly Patients. . . . . . . . . . . . . . . . . . . . . . . 12

E. Milestones in Thrombolytic Therapy of Acute Myocardial Infarction ................................ 13

I. First Generation Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 II. Second Generation Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

III. Third Generation Agents .............................. 13 IV. The Future of Thrombolytic Therapy. . . . . . . . . . . . . . . . . . . . 15

XII Contents

List of Abbreviations and Acronyms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

CHAPTER 2

Plasminogen and Streptokinase EI CASTELLINO and v.A. PLOP LIS. With 3 Figures 25

A. Primary Structure of Human Plasminogen .................... 25 B. Gene Organization of Human Plasminogen. . . . . . . . . . . . . . . . . . . . 28 C. The Activation of Human Plasminogen ....................... 29

I. General Considerations ... . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 II. Activation of Human Plasminogen by Two-chain Urokinase

and Tissue-type Plasminogen Activator. . . . . . . . . . . . . . . . . . . 33 1. Mechanism of Activation of Human Plasminogen. . . . . . 33 2. Positive and Negative Activation Effectors. . . . . . . . . . . . 34

III. Activation of Human Plasminogen by Single-chain Urokinase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

IV. Activation of Human Plasminogen by Streptokinase. . . . . . . 36 1. Structural and Activation Features of Streptokinases . . . 36 2. Mechanism of Activation of Human Plasminogen

by Human Host-derived Streptokinase. . . . . . . . . . . . . . . 39 3. Functional Regions of Human Plasminogen and

Human-derived Streptokinase that Mediate Activation ............................... 41

V. Activation of Human Plasminogen by Staphylokinase ..... 42 D. Spontaneous and Induced Phenotypes of

Plasminogen Deficiency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 List of Abbreviations .......................................... 44 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

CHAPTER 3

Tissue-type Plasminogen Activator (tPA) F. BACHMANN. With 2 Figures ................................... 57

A. Milestones in Tissue-type Plasminogen Activator (tPA) Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

B. Sources of tPA ............................................ 58 C. Metabolism of tPA ........................................ 59 D. Structure and Biochemistry of tPA ........................... 60

I. Gene Structure of tPA ................................ 60 1. Translated Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 2. 5' and 3' Flanking Regions ......................... 60 3. Polymorphisms ................................... 62

II. Protein Structure of tPA .............................. 62 1. Primary Amino Acid Sequence ..................... 62

Contents XIII

a) The Finger Domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 b) The Epidermal Growth Factor Domain ........... 63 c) The Kringle Domains .......................... 64 d) The Protease (Catalytic) Domain. . . . . .. ......... 65

2. Crystal Structure of the Protease Domain ............ 65 3. Glycosylation of tPA .............................. 65

III. Enzymatic Properties of tPA ........................... 66 1. Enzyme Kinetics in the Absence and Presence

of Fibrin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 2. Differences Between sc-tPA and tc-tPA .............. 67 3. Localization of the Epitopes in Fibrin that Enhance

the Rate of Plasminogen Activation by tPA .......... 68 IV. Interactions of tPA with Serpins . . . . . . . . . . . . . . . . . . . . . . . . 68

E. The tPA Receptors ........................................ 70 I. Activation Receptors ................................. 70

II. Clearance Receptors ................................. 71 1. The Mannose Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 2. The LDL Receptor-Related Protein

(LRP, arMacroglobulin Receptor) .................. 72 F. Pathophysiology of tPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

I. Intravascular Function of tPA .......................... 72 II. Other Functions of tPA ............................... 74

List of Symbols and Abbreviations .............................. 75 References ................................ . . . . . . . . . . . . . . . . . . . 76

CHAPTER 4

Urinary-type Plasminogen Activator (uPA) w.A. GUNZLER and L. FLOHE. With 2 Figures. . . . . . . . . . . . . . . . . . . . . . 91

A. Milestones in uPA Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 B. Nomenclature of uPA Forms. . . .. . .. . . . .. . . . . . . .. . . . . . . .. . . . 92 C. Sources of uPA ............................................ 93 D. Assay of uPA ............................................ 93 E. Biochemistry of uPA ....................................... 94

I. The uPA Gene. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . .. . . . 94 II. The uPA Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

III. Non-protein Components of uPA . . . . . . . . . . . . . . . . . . . . . . . 97 IV. Proteolytic Conversions of uPA ........................ 98 V. uPA Activity ........................................ 99

F. Physiology of uPA ......................................... 100 I. Intravascular and Intratubular Functions of uPA. . . . . . . . . . . 102

II. Role of uPA in Tissue Remodeling and Cell Migration .... 102 List of Abbreviations .......................................... 103 References .................................................. 103

XIV Contents

CHAPTERS

The Inhibitors of the Fibrinolytic System E.K.o. KRUITHOF. With 1 Figure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

A. Introduction............................................... 111 B. arAntiplasmin ............................................ 111

I. Gene and Protein Structure ........................... 111 II. Target Enzyme Specificity ............................. 113

III. arAntiplasmin Deficiencies. . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 C. Plasminogen Activator Inhibitor Type 1 ....................... 114

I. Gene Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 II. Protein Structure ..................................... 115

III. Target Enzyme Specificity of PAI-1 .................... 116 IV. Deficiencies, Knockout, and Overexpression in Mice ...... 116 V. Role of PAI-1 in Cell Migration. . . . . . . . . . . . . . . . . . . . . . . . 116

VI. Distribution of PAl-lIn Vivo.. . . .. . . .. . . . . ... . . . . ... . . 117 VII. Gene Regulation of PAl-I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

VIII. PAI-1 as a Marker for Vascular and Arterial Disease ...... 118 IX. PAI-1 and the Acute Phase Response. . . .. . . . . . . . . . . . . .. . 118 X. PAI-1 and Septicemia ................................ 119

D. Plasminogen Activator Inhibitor Type 2 . . . . . . . . . . . . . . . . . . . . . . . 119 1. Gene and Protein Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

II. Target Enzyme Specificity ............................. 120 III. Distribution of PAI-2 In Vivo .......................... 120 IV. Gene Regulation of PAI-2 ............................. 121 V. The Physiological Role of PAI-2 ....................... 121

E. Plasminogen Activator Inhibitors in Pregnancy. . . . . . . . . . . . . . . . . 121 F. Therapeutic Applications of PAI-1 and PAI-2 . . . . . . . . . . . . . . . . . . 122

I. PAI-1 ............................................... 123 II. PAI-2 ............................................... 123

G. Other Protease Inhibitors of the Fibrinolytic System. . . . . . . . . . . . 123 H. Inhibitors of Plasminogen Binding to Fibrin or Cell Surfaces. . . . . 124

I. Histidine-rich Glycoprotein ............................ 124 II. Lipoprotein(a) ....................................... 125

III. Carboxypeptidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 I. Conclusions............................................... 126 List of Abbreviations .......................................... 127 References .................................................. 127

CHAPTER 6

Assembly of the Plasminogen System on Cell Surfaces E.F. PLOW, A. REDLITZ, S.B. HAWLEY, S. XUE, T. HERREN, IL. HOOVER-PLOW, and L.A. MILES. With 3 Figures. . . . . . . . . . . . . . . . . 141

A. Introduction .............................................. 141 B. Plasminogen Receptors .................................... 142

Contents XV

I. General Characteristics ............................... 142 II. Nature of Plasminogen Receptors ...................... 143

III. Regulation of Plasminogen Receptors. . . . . . . . . . . . . . . . . . . 145 C. tPA Receptors ............................................ 148

I. General Characteristics ............................... 148 II. Recognition of tPA ................................... 148

III. Cellular Distribution of tPA Receptors. . . . . . . . . . . . . . . . . . 148 1. Endothelial Cells ................................. 148 2. Circulating Blood Cells ............................ 149 3. Cells of Neuronal Origin. . . . . . . . . . . . . . . . . . . . . . . . . . . 149 4. Vascular Smooth Muscle Cells. . . . . . . . . . . . . . . . . . . . . . 149 5. Tumor Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

D. Urokinase Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 I. General Characteristics ............................... 150

II. The Urokinase Receptor, uPAR ....................... 150 1. Cellular Distribution of uPAR ..................... 150 2. Recognition Specificity of uPAR .................... 150 3. The uPAR Gene ................................. 151 4. The uPAR Protein ................................ 151 5. Regulation of uPAR Expression .................... 153 6. Non-proteolytic Functions of uPAR ................. 153

III. Other uPA Receptors ................................ 154 E. Functional Consequences of Receptor Occupancy . . . . . . . . . . . . . . 155

I. Kinetic Consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 1. Plasminogen Receptors ............................ 155

a) Enhanced Plasminogen Activation ............... 156 b) Amplification Loops ........................... 156 c) Protection of Plasmin Activity ................... 156 d) Enhancement of Plasmin Activity ................ 156

2. uPA Receptors ................................... 156 3. tPA Receptors ................................... 157

II. Functional Consequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 List of Abbreviations .......................................... 158 References .................................................. 158

Section II: Clinical Use of Thrombolytic Agents In Acute Myocardial Infarction

CHAPTER 7

Streptokinase and Anisoylated Lys-Plasminogen Streptokinase Activator Complex Y.I MARDER and F. BACHMANN. With 2 Figures .................... 173

A. Introduction............................................... 173 I. Pharmacology........................................ 173

1. Streptokinase (SK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

XVI Contents

2. Anisoylated Lys-Plasmin( ogen) Streptokinase Activator Complex (APSAC, anistreplase) .................... 174

II. The Proteolytic State ................................. 174 III. Factors Influencing the Lysis of Thrombi and Emboli . . . . . . 176

B. Therapeutic Results in Acute Myocardial Infarction ............ 177 I. Streptokinase ........................................ 177

1. Early Studies with Intravenous Streptokinase ......... 177 2. Intracoronary Administration. . . . . . . . . . . . . . . . . . . . . . . 178 3. High-Dose Intravenous Administration

of Streptokinase .................................. 179 a) Dose-Ranging Studies and Anti-SK Antibodies .... 179 b) Patency vs Recanalization Rate .................. 180 c) Mortality Reduction ........................... 182 d) Delay to Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 e) Bleeding Complication ......................... 185 f) Patient Selection, Contraindications .............. 187 g) Other Side Effects ............................. 188 h) Laboratory Monitoring ......................... 188 i) Rethrombosis ................................. 188

II. Anisoylated Lys-Plasmin( ogen) Streptokinase Activator Complex (APSAC, Anistreplase) ....................... 189

1. Randomized Clinical trials ......................... 189 2. Pre-hospital Treatment ............................ 189

List of Abbreviations .......................................... 193 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

CHAPTER 8

Tissue-Type Plasminogen Activator (tPA) D. COLLEN. With 2 Figures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

A. Introduction .............................................. 209 B. Development of tPA as a Thrombolytic Agent ................. 212

I. Isolation and Purification of tPA ....................... 212 II. Cloning and Expression of the tPA cDNA . . . . . . . .. ...... 214

III. Experimental Coronary Thrombosis Models for AMI. . . . . . 215 IV. First Administration of tPA to Patients with AMI . . . . . . . . . 215

C. Clinical Trials with tPA in AMI .............................. 216 I. Early Clinical Studies in AMI .......................... 216

II. tPA Trials, 1986-1989. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 III. tPA Trials, 1990-1995 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 IV. Further Attempts to Improve the Efficacy of Thrombolysis

and to Reduce the Incidence of Reocclusion ............. 221 List of Abbreviations .......................................... 222 References .................................................. 223

Contents XVII

CHAPTER 9

Urokinase and Single-Chain Urokinase-Type Plasminogen Activator (Pro-urokinase) V. GUREWICH ................................................. 231

A. History .................................................. 231 B. Nomenclature............................................. 232 C. Biochemistry of Pro-UK/UK (sc-uPA/tc-uPA) ................. 232

I. The Intrinsic Activity of Pro-UK (sc-uPA) . . . .. .......... 233 II. Fibrinolysis by Pro-UK/UK (sc-uPA/tc-uPA) ............. 234

III. Contrasting Properties of Pro-UK and tPA .............. 235 IV. Pro-UK/UK and Platelets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

D. Pharmacokinetics .......................................... 238 E. Clinical Studies in Acute Myocardial Infarction ................ 239

I. Urokinase ........................................... 239 1. Controlled Trials of UK vs Controls . . . . . . . . .. ....... 239 2. Prehospital vs In-hospital Thrombolysis with UK . . . . . . 240 3. Comparative Trials of UK vs SK or tPA . . . . . . . . . . . . . . 240

II. Pro-urokinase...................... . . . . . . . . . . . . . . . . . . 241 1. Early Dose-finding Studies ......................... 241 2. Randomized Trials of Pro-UK vs SK . . . . . . . . . . . . . . . . . 243 3. Randomized Trial of Pro-UK vs tPA . . . . . . . . . . . . . . . . . 245

III. Non-specific Plasminogen Activation . . . . . . . . . . . . . . . . . . . . 245 IV. Combinations of Pro-UK and UK or tPA . . . . . . . . . . . . . . . . 246

1. Early Dose-finding Studies of Pro-UK and UK. . . . . . . . 246 2. Early Dose-finding Studies of Pro-UK and tPA ....... 247 3. Larger Clinical Trial with Pro-UK and tPA ........... 247

V. Bolus administration of Pro-UK. . . . . . . . . . . . . . . . . . . . . . . . . 248 VI. Heparin and Pro-UK ................................. 248

F. Second Generation Pro-UK ................................ 248 G. Conclusions ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 List of Abbreviations .......................................... 251 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

CHAPTER 10

Comparative Evaluations of tPA vs SK with Particular Reference to the GUSTO-I Trial A.c. CHIU and E.J. TOPOL. With 1 Figure ......................... 261

A. Introduction .............................................. 261 B. Evolution of Thrombolytic Therapies ......................... 261

I. Rationale for Thrombolysis ............................ 261 II. Early Angiographic Patency Trials, tPA vs SK ............ 261

III. Adjunctive Therapies; Heparin and Aspirin. . . . . . . . . . . . . . . 263 IV. Major Comparative Thrombolytic Trials. . . . . . . . .. ....... 264

XVIII Contents

C. The GUSTO-I Trial 268 268 271 274 278 278 281 281 282

I. Design of the Trial ................................... . II. Primary Results of GUSTO-I ......................... .

III. The GUSTO-I Angiographic Substudy ................. . IV. Further Analysis of GUSTO-I Results .................. . v. Other GUSTO-I Sub studies .......................... .

VI. Epilogue ........................................... . List of Abbreviations and Acronyms ............................ . References .................................................. .

CHAPTER 11

Conjunctive Therapy to Reduce the Occurrence of Coronary Reocclusion After Thrombolytic Treatment of AMI R. HAYES, R. GALLO, V. FUSTER, and 1. CHESEBRO. With 8 Figures. . . . . 287

A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 B. The Role of Thrombin and Platelets in Thrombus Formation .... 287 C. Evidence for the Efficacy of Thrombolytic Therapy . . . . . . . . . . . . . 289

I. Importance of Achieving Patency of the Infarct Related Artery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

II. Limitations of Thrombolytic Therapy . . . . . . . . . . . . . . . . . . . . 292 III. The Role of Platelets and Thrombin in Resistance. . . . . . . . . 292

to Therapy .......................................... 292 1. Thrombin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 2. Platelets .......................................... 295

D. Antithrombotic Therapy ................................... 296 I. Thrombin Inhibition .................................. 296

1. Heparin . . . . . .. ................................... 296 2. Low-Molecular-Weight Heparin ..................... 300 3. Hirudin .......................................... 301 4. Hirulog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 5. Argatroban ....................................... 304 6. Efegatran ......................................... 305

II. Antiplatelet Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 1. Aspirin ....... ................................... 305 2. Glycoprotein lIb/IlIa Antagonists ................... 306 3. Other Drugs Inhibiting Platelet Function ............. 308 4. Physical Coujunctive Therapy. . . . . . . . . . . . . . . . . . . . . . . . 309

E. Outlook ................ ................................. 309 List of Abbreviations ......................................... 309 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Contents XIX

Thrombolytic Treatment of Other Clinical Thromboembolic Conditions

CHAPTER 12

New Concepts in Thrombolysis of Pulmonary Embolism A.A. SASAHARA and G.v.R.K. SHARMA. With 5 Figures. . . . . . . . . . . . . . 325

A. Introduction .............................................. 325 B. Treatment with Thrombolytic Therapy ........................ 327 C. Established Clinical Effect of Thrombolytic Therapy. .. ......... 328

I. Thrombolytic Agents Hasten Thromboembolic Resolution .......................................... 328

II. Results of Pulmonary Angiography ..................... 328 III. Results of Perfusion Lung Scan ........................ 328 IV. Hemodynamic Observations ........................... 329 V. Thrombolytic Therapy Removes Pulmonary

Thromboemboli More Completely than Heparin Therapy ..................................... 330

VI. Thrombolytic Agents Hasten Dissolution of Thrombi in the Venous System of the Legs . . . . . . . . . . . . . . . . . . . . . . . 332

VII. Thrombolytic Therapy Can Be Administered Safely .. . . . . . 332 D. Possible Unestablished Clinical Effects. . . . . . . . . . . . . . . . . . . . . . . . 334

I. Thrombolytic Agents May Decrease Mortality from Pulmonary Embolism ................................ 334

II. Thrombolytic Therapy Improves Long-term Hemodynamic Status ................................. 335

E. New Thrombolytic Regimens .............. . . . . . . . . . . . . . . . . . . 336 F. Surgical Therapy .......................................... 341 G. Adjunctive Therapy ........................................ 343 List of Abbreviations .......................................... 343 References .................................................. 344

CHAPTER 13

Deep Venous Thrombosis H. BOUNAMEAUX .............................................. 349

A. Introduction............................................... 349 B. Treatment Regimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 C. Immediate Effects of Thrombolytic Therapy of DVT:

The Definite Risk ......................................... 350 D. Late Effects of Thrombolytic Therapy of DVT:

The Potential Benefit ...................................... 352 E. The Benefit-to-Risk Balance ................................ 353 F. How to Reduce the Risk and Improve the Benefit? . . . . . . . . . . . . . 354

I. Reducing Hemorrhagic Risk ........................... 354 II. Improving Thrombolytic Efficacy ....................... 355

G. Miscellaneous Venous Indications ........................... 356

xx Contents

I. Phlegmasia Caerulea Dolens .......................... 356 II. Superior or Inferior Vena Cava Syndrome ............... 356

III. Deep Venous Thrombosis of the Upper Limbs. . . . . . . . . . . . 356 IV. Venous Thrombosis of Unusual Localization ............. 356 V. Thrombosis of Central Venous Catheters ................ 357

H. Laboratory Monitoring ..................................... 357 I. Treatment of Bleeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358 1. Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358 List of Abbreviations and Acronyms ............................ 359 References .................................................. 359

CHAPTER 14

Peripheral Arterial Occlusions D.C. BERRIDGE ................................................ 363

A. Introduction............................................... 363 B. Current Indications ........................................ 364 C. Contra-Indications ......................................... 366 D. Drugs Available ........................................... 367

I. Streptokinase ........................................ 367 II. APSAC, Anistreplase (Acylated-Plasminogen-

Streptokinase-Activator Complex) . . . . . . . . . . . . . . . . . . . . . . 367 III. Urokinase and Pro-Urokinase. . . . . . . . . . . . . . . . . . . . . . . . . . 368 IV. Recombinant Tissue Plasminogen Activator (tPA) ........ 368 V. Staphylokinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368

E. Techniques Used .......................................... 368 I. Systemic Administration .............................. 368

II. Local Low-dose Intra-thrombotic Administration ......... 369 III. High-dose Bolus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 IV. Pulse-Spray Administration ............................ 370 V. Enclosed Thrombolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

VI. Intra-operative Lysis ................... . . . . . . . . . . . . . . . 372 F. Comparative Results ....................................... 373

I. Streptokinase vs Recombinant Tissue Plasminogen Activator ................................ 373

II. UK vs Recombinant Tissue Plasminogen Activator. . . . . . . . 374 III. Pro-UK vs. UK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 IV. Surgery vs Thrombolysis .............................. 374

G. Long-Term Results ........................................ 378 H. Adjuvant Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 I. Complications............................................. 379

I. Major Hemorrhage ................................... 379 II. Minor Hemorrhage ........................ . . . . . . . . . . . 379

III. Cerebrovascular Accident. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 IV. Distal Embolization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 V. Pericatheter Thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

Contents XXI

VI. Allergic Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 VII. Other Complications ................................. 380

1. Summary................................................. 381 List of Abbreviations and Acronyms ............................ 381 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

CHAPTER 15

Thrombotic Cerebrovascular Disease G.1. DEL Zoppo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387

A. Introduction .............................................. 387 B. Focal Cerebral Ischemia .................................... 388

I. Considerations for Tissue Salvage. . . . . . . . . . . . . . . . . . . . . . . 388 II. Hemorrhagic Transformation .......................... 389

III. Neurological Outcome ................................ 390 C. Plasminogen Activators in Focal Cerebrovascular Ischemia ...... 392

1. Completed Stroke .................................... 393 II. Acute Stroke ........................................ 394

1. Carotid Territory Ischemia ......................... 394 a) Angiography-Controlled Trials .................. 394

a) Intra-Arterial Local Infusion ................. 394 f3) Intravenous (Systemic) Infusion .............. 397 y) Single Photon-Emission Computed

Tomography (SPECT) ....................... 400 b) Sympton-Based (Clinical Outcome) Trials . . . . . . . 400

2. Vertebrobasilar Territory Ischemia. . . . . . . . . . . . . . . . . . . 408 a) Angiography-Controlled Trials .................. 408

a) Intra-Arterial (Local) Infusion. . . . . . . . . . . . . . . . 408 f3) Intravenous (Systemic) Infusion .............. 409

III. Other Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409 1. Retinal Vascular Occlusion ........................ 409 2. Dural Sinus Thrombosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . 410

D. Ongoing Stroke Trials with Thrombolytic Agents. . . . . . . . . . . . . . . 410 E. Conclusion ............................................... 411 List of Abbreviations and Acronyms ............................ 411 References .................................................. 412

Thrombolytic Agents in Development, Biochemistry, Pharmacology -Efficacy in Animal Experiments and First Clinical Trials

CHAPTER 16

Staphylokinase H.R. LIJNEN and D. COLLEN. With 3 Figures 425

A. Introduction............................................... 425 B. Gene and Protein Structure of Staphylokinase ................. 426

XXII Contents

I. Production of Staphylokinase .......................... 426 II. Gene Structure of Staphylokinase ...................... 427

III. Protein Structure of Staphylokinase . . . . . . . . . . . . . . . . . . . . . 428 C. Plasminogen Activation by Staphylokinase .................... 429

I. Interaction with Plasmin( ogen) . . . . . . . . . . . . . . . . . . . . . . . . . 429 II. Kinetics of Plasminogen Activation ..................... 431

III. Mechanism of Plasminogen Activation .................. 431 D. Fibrin-Specificity of Staphylokinase .......................... 432

I. Inhibition of Plasmin·Staphylokinase Complex by arAntiplasmin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432

II. Effect of Fibrin on Plasminogen Activation by Staphylokinase .................................... 433

III. Molecular Mechanism of Fibrin-specificity in Plasma ...... 433 E. Fibrinolytic Properties of Staphylokinase in Plasma In Vitro ..... 435

I. Fibrinolytic Potency and Fibrin-specificity . . . . . . . . . . . . . . . . 435 II. Species-specificity . . . . . . . . . . . . . .. ..................... 437

F. Thrombolytic Properties of Staphylokinase in Animal Models. . . . 437 I. Hamsters with Pulmonary Embolism . . . . . . . . . . . . . . . . . . . . 437

II. Rabbits with Jugular Vein Thrombosis. . . . . . . . . . . . . . . . . . . . 438 III. Rabbits with Arterial Thrombosis. . . . . . . . . . . . . . . . . . . . . . . 438 IV. Rabbit Embolic Stroke Model ......................... 438 V. Baboons with Venous and Arterial Thrombosis. . . . . . . . . . . . 438

G. Thrombolytic Properties of Staphylokinase in Patients .......... 439 I. Acute Myocardial Infarction ........................... 439

II. Peripheral Arterial Occlusion .......................... 440 H. Pharmacokinetic Properties of Staphylokinase ................. 441

I. In Animal Models .................................... 441 II. In Patients .......................................... 441

I. Immunogenicity of Staphylokinase ........................... 442 I. In Animal Models .................................... 442

II. In Patients .......................................... 442 III. Attempts to Reduce Immunogenicity . . . . . . . . . . . . . . . . . . . . 443

J. Conclusions............................................... 443 List of Abbreviations and Acronyms ............................ 444 References .................................................. 444

CHAPTER 17

Desmodus rotundus (Common Vampire Bat) Salivary Plasminogen Activator W.-D. SCHLEUNING and P. DONNER. With 5 Figures ..................... 451

A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 B. Natural History of Vampire Bats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453 C. Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453

I. Purification ......................................... 453

Contents XXIII

II. Cloning and Expression ............................... 454 III. DSPA Gene Expression in Hetereologous Host Cells ..... 455 IV. Three-dimensional Structure of the Protease Domain. . . . . . 456 V. Enzymology ......................................... 456

D. Pharmacology ............................................. 459 I. In Vitro Clot Lysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

II. Carotid and Femoral Artery Thrombosis in Rabbits ....... 461 III. Myocardial Infarction in Dogs. . . . . . . . . . . . . . . . . . . . . . . . . . 461 IV. Experimental Pulmonary Embolisms in Rats ............. 462 V. Bleeding Models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

E. Pharmacokinetics .......................................... 464 F. Toxicology................................................ 466 G. Phase I Clinical Studies .................................... 466 H. Phase Ib Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467 I. Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467 List of Abbreviations and Acronyms ............................ 468 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

CHAPTER 18

Thrombus-Targeting of Plasminogen Activators C. BODE, K. PETER, M.S. RUNGE, and E. HABER .................... 473

A. Introduction............................................... 473 B. The Principle of Antibody Targeting. . . . . . . . . . . . . . . . . . . . . . . . . . 474

I. Antifibrin Antibodies ................................. 474 II. Antiplatelet Antibodies ............................... 474

III. Chemical Conjugates or Recombinant Fusion Proteins .... 475 C. Synthesis, Purification, and Characterization of Chemical

Antibody-Plasminogen Activator Conjugates .................. 475 I. Synthesis ............................................ 475

II. Purification .......................................... 476 III. Characterization ..................................... 476

1. Antifibrin-UK Conjugates ......................... 477 2. Antifibrin-tPA Conjugates ......................... 477 3. Antifibrin-scuPA Conjugates ....................... 478 4. Antiplatelet-UK Conjugates. . . . . . . . . . . . . . . . . . . . . . . . 478 5. Antiplatelet-scuPA Conjugates ..................... 478

D. Targeting with Bifunctional Antibodies ....................... 479 I. Principal Considerations .............................. 479

II. Bispecific IgG (Intact Antibodies) ...................... 479 III. Bispecific (Fab')2 ..................................... 480 IV. Bispecific Antibodies by Hybrid-Hybridoma Technique. . . . 480 V. Double Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481

E. Expression and Characterization of Recombinant Antifibrin-Plasminogen Activator Fusion Proteins. . . . . . . . . . . . . . . . . . . . . . . . 481

XXIV Contents

I. Antifibrin-SK Constructs II. Antifibrin-tPA Constructs ............................. .

III. Antifibrin-scuPA Constructs .......................... . F. Other Approaches of Targeting Plasminogen Activators

to Thrombi ............................................... . G. Targeting the Thrombin Inhibitor Hirudin to Fibrin ............ .

I. General Considerations .............................. . II. Chemical Conjugates ................................ .

III. Recombinant Fusion Protein .......................... . H. Conclusion and Outlook ................................... . List of Abbreviations and Acronyms ............................ . References ................................................. .

CHAPTER 19

The Hunt for the Ideal Thrombolytic Agent: Mutants of tPA and uP A, Chimera of Both Molecules, Fibrolase

481 481 482

484 486 486 486 486 487 488 488

M. VERSTRAETE. With 3 Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

A. Mutants and Variants of Single-Chain Urokinase-Type Plasminogen Activator. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

I. Plasmin-Resistant Mutants of sc-uPA . . . . . . . . . . . . . . . . . . . . 493 II. Low-Molecular-Weight sc-uPA ......................... 493

III. Mutant of sc-uPA Resistant to PAl-I. . . . . . . . . . . . . . . . . . . . 494 B. Mutants of Tissue-Type Plasminogen Activator (tPA) ........... 494

I. Mutants with Modified Epidermal Growth Factor Domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494

II. Mutants with Modified or Deleted Finger Domain ........ 495 III. Deletion of the Finger and Epidermal Growth

Factor Domains ...................................... 495 IV. Deletion of the Epidermal Growth Factor and Finger

Domains and of Glycosylation Sites .................... 496 V. Mutants Consisting of Kringle 2 and the

Protease Domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496 VI. Mutant Consisting of Kringle 2 with Point Mutation

in Linkage to Protease Light Chain .................... 501 VII. Mutant with Modifications in Kringle 1 . . . . . . . . . . . . . . . . . . 503 VII. Recombinant TNK-tPA ........... " ... " ....... , . . . .. . 503 IX. Unglycosylated Protease Domains of tPA . . . . . . . . . . . . . . . . 510

C. Chimeric Plasminogen Activators ............................ 510 D. Fibrolase.................................................. 512 E. Other Fibrinolytic Agents .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512 List of Abbreviations and Acronyms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512 References .................................................. 513

Contents xxv

CHAPTER 20

Agents which Increase Synthesis and Release of Tissue-Type Plasminogen Activator T. KOOISTRA and J.J. EMEIs. With 3 Figures ........................ 521

A. Introduction .............................................. 521 I. The Plasminogen/Plasmin System ...................... 521

II. Intravascular Fibrinolysis .............................. 521 III. Tissue-Type Plasminogen Activator (tPA) in

the Circulation ....................................... 522 IV. Involvement of the Endothelium in Plasma Fibrinolysis. . . . 523

B. Regulation of Constitutive tPA Synthesis. . . . . . . . . . . . . . . . . . . . . . 524 I. General ........................... . . . . . . . . . . . . . . . . . . 524

II. Intracellular Signaling and Activation of Protein Kinase C ............................................ 525

III. Retinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526 IV. Steroid Hormones .................................... 528 V. Sodium Butyrate and Other Inhibitors of Histone

Deacetylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529 VI. Triazolobenzodiazepines .............................. 530

C. Regulated Secretion of tPA ................................. 531 I. General............. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 531

II. The Endothelial tPA Storage Compartment and the Circulatory Regulation of tPA ......................... 532

III. Cellular Mechanisms Involved in Regulated tPA Secretion ......................... . . . . . . . . . . . . . . . 535

IV. Compounds Affecting Regulated tPA Secretion in Man. . . . 536 1. The Occurrence of Regulated tPA Secretion in Man. . . . 536 2. Adrenergic Agents ................................ 538 3. Vasopressin . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 539 4. DDAVP ......................................... 539 5. Acetylcholine and Methacholine. . . . . . . . . . . . . . . . . . . . 541 6. Bradykinin and Substance P . . . . . . . . . . . . . . . . . . . . . . . . 541 7. Coagulation Activation Products .................... 542 8. cAMP........................................... 542 9. Miscellaneous Compounds ......................... 542

10. Abnormal Release of tPA . . . . . . . . . . . . . . . . . . . . . . . . . . 543 D. Increase of tPA Activity by Inhibition of PAI-1 ................ 543 List of Abbreviations and Symbols .............................. 544 References .................................................. 544

XXVI Contents

Autifibrinolytic Agents

CHAPTER 21

Structure, Pharmacology, and Clinical Use of Antifibrinolytic Agents C.M. SAMAMA, W. DIETRICH, 1. HORROW, O. TABY, and M.M. SAMAMA. With 3 Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559

A. Introduction............................................... 559 B. Pharmacology and Clinical Use of EACA and TA ............. 559

I. c-Aminocaproic Acid (EACA) ......................... 561 1. Pharmacology ................................... 561 2. Non-Cardiac Surgery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561

a) Urologic Surgery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561 b) Oral Surgery .................................. 561 c) Liver Transplantation. . . . . . . . . . . . . . . . . . . . . . . . . . . 562

3. Use in Cardiac Surgery ............................ 562 4. Adverse Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562

II. Tranexamic Acid ..................................... 564 1. Pharmacology ................................... 564 2. Nonsurgical Applications .......................... 564 3. Noncardiac Surgery ............................... 565 4. Cardiac Surgery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565 5. Adverse Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565

C. Pharmacology of Aprotinin ................................. 567 I. Structure ............................................ 567

II. Pharmacology........................................ 567 III. Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568 IV. Clinical Use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568

1. History .......................................... 568 2. Mode of Action of Aprotinin

(Clinical Point of View) ............................ 569 3. Efficacy of Aprotinin Treatment .................... 571 4. Miscellaneous Uses ............................... 572

a) Orthotopic Liver Transplantation (OLT) and Elective Liver Resection ........................ 572

b) Orthopedic Surgery ............................ 574 5. Side Effects of Aprotinin Therapy .................. 574

D. Conclusion................................................ 575 List of Abbreviations and Acronyms ............................ 576 References .................................................. 576

Subject Index ................................................ 587