, I . I

Antithrombotics

Contributors

W. Bell, P. Carmeliet, L. Chi, D. Collen, R.J.G. Cuthbert,C. Esmon, K.P. Gallagher, D. Ginsburg, T. Hara, L.A. Harker,T.A. Hennebry, M. Hollenberg, R.D. Hull, J.A. Jakubowski,R.E. Jordan, F. Kee, D. Keeling, S.D. Kimball, S. Kunitada,M.A. Lauer, L. Leblond, A.M. Lincoff, B.Lucchesi, T.E.Mertz,T. Nagahara, J. Pearson, G.F. Pineo, S. Rebello, J.R. Rubin,A.C.G. Uprichard, S. Watson, H.F. Weisman, P.D. Winocour

Editors

Andrew C.G. Uprichard and Kim P. Gallagher

Springer

Contents

CHAPTER 1The Coagulation Pathway and Antithrombotic StrategiesL. LEBLOND and P.D. WINOCOUR. With 7 Figures 1

A. Introduction 1B. The Coagulation Pathway 1

I. The Cascade/Waterfall Model 2II. The Revised Model 3

III. Structure-Activity Relationships of Coagulation Proteases.. 41. Thrombin 62. Factor Xa 73. Factor VII/Tissue Factor Complex 8

C. Physiological Regulators 9I. Antithrombin-III (ATIII) and Heparin Cofactor-II

(HCII) 10II. Tissue Factor Pathway Inhibitor 11

III. Protein C/S-Thrombomodulin Complex 12D. Platelet and Cellular Contributions 13

I. Cell Surface Dependence 14II. Platelet Participation 14

III. Vascular Contributions 15E. Fibrinolysis 16F. Antithrombotic Strategies 17

I. Coagulation Factor Inhibitors 181. Direct Thrombin Inhibitors 202. Thrombin Generation Inhibitors 23

a) Factor Xa Inhibitors 23b) Inhibitors of Other Coagulation Factors 23

II. Antiplatelet Agents 251. Platelet Adhesion and Activation Inhibitors 252. Fibrinogen Receptor Antagonists 28

III. Thrombolytic Agents 29IV. Other Strategies 30

G. Conclusion 30References 31

XII Contents

CHAPTER 2

New Developments in the Molecular Biology of Coagulationand FibrinolysisP. CARMELIET and D. COLLEN. With 7 Figures 41

A. Introduction 41I. The Coagulation System 41

II. The Plasminogen System 42III. Targeted Manipulation and Adenovirus-Mediated

Transfer of Genes in Mice 43B. Embryonic Development and Reproduction 43

I. Coagulation System 431. Tissue Factor and Factor VII 432. Thrombomodulin 523. Thrombin Receptor and Factor V 534. Fibrinogen 54

II. Fibrinolytic System 54III. Integrated View of a Role for the Coagulation and

Fibrinolytic System in Vascular Development 56C. Health and Survival 56

I. Coagulation System 56II. Fibrinolytic System 58

D. Hemostasis 58I. Coagulation System 58

II. Fibrinolytic System 60E. Thrombosis and Thrombolysis 61

I. Coagulation System 61II. Fibrinolytic System 61

III. Fibrin Deposits and Pulmonary Plasma Clot Lysis inTransgenic Mice 62

IV. Adenovirus-Mediated Transfer of t-PA or PAI-1 63F. Neointima Formation 64G. Atherosclerosis 66H. Tissue Remodeling Associated with Wound Healing 68

I. Conclusions 70References 70

CHAPTER 3

Epidemiology of Arterial and Venous ThrombosisF. KFE. With 4 Figures 77

A. Epidemiology: Its Potential and Its Limitations 77B. The Epidemiological Study of Arterial and

Venous Thrombosis 78I. Arterial Thrombosis 78

II. Venous Thrombosis 81

Contents XIII

C. Risk Factors for Arterial Thrombosis 83I. Fibrinogen 84

II. Fetal-Infant Origins Hypothesis ofIschemic Heart Disease 85

III. Insulin Resistance 87IV. Hyperhomocysteinemia 88

D. Risk Factors for Venous Thrombosis 89E. Future Directions in Epidemiological Research 93

I. Genetic Epidemiology 94II. Evidence Synthesis 95

References 96

CHAPTER 4

In Vivo Models of ThrombosisL. CHI, S. REBELLO, and B.R. LUCCHESI. With 5 Figures 101

A. Introduction 101B. Vessel Wall Injury-Induced Model of Thrombosis 102

I. Photochemical Reaction 102II. Laser 104

III. Mechanically Induced Injury 1051. Pinching or Crushing 1052. Perfusion with Saline or Air 1083. Endarterectomy and Balloon Angioplasty 109

IV. Electrical Current-Induced Injury 109C. Stasis/Hypercoagulability-Induced Models of Thrombosis 112

I. Wessler Test and Its Variants 112D. Foreign Surface-Induced Thrombosis 113

I. Eversion Graft 113II. Wire Coils 114

III. Preformed Thrombi 114IV. Hollenbach's Deep Venous Thrombosis Model 115V. A Novel Veno-Venous Shunt Model in Rabbit 116

E. Transgenic Animal Models 117F. Conclusions 120References 121

CHAPTER 5

Monitoring Antithrombotic TherapyR.J.G. ClTHUERT 129

A. Introduction 129I. Balancing Antithrombotic Efficacy Against the Risk

of Bleeding 129B. Warfarin ." 129

I. Mechanism of Action 1291. Effect on Vitamin K-Dependent Clotting Factors 129

XIV Contents

2. Kinetics of Vitamin K-Dependent Clotting FactorsDuring Warfarin Therapy 130

3. Variation in Pharmacological Response 130II. Laboratory Monitoring 131

1. Prothrombin Time for Monitoring WarfarinTherapy 131

2. Standardisation of Thromboplastin Reagents 1323. Choice of Thromboplastins for Clinical

Monitoring 132III. Determinants of Bleeding Risk 133IV. Practical Aspects of Warfarin Dosing 134V. Maintenance Treatment 134

1. Anticoagulant Clinics 1342. Computer-Assisted Monitoring and

Patient Self-Monitoring 135VI. Alternative Methods of Monitoring

Warfarin Therapy 1351. Functional Prothrombin Assay 1352. Prothrombin Fragment Fl .2 1353. Prothrombin-Proconvertin Ratio 136

C. Heparin 136I. Heparin Structure 136

II. Mechanism of Action 136III. Unfractionated Heparin 137

1. Pharmacokinetics 1372. Laboratory Monitoring by the Activated Partial

Thromboplastin Time (APTT) 1373. Heparin Resistance 1384. Dose-Adjustment Nomograms 1395. Subcutaneous Heparin Regimens 1396. Determinants of Bleeding Risk 1407. The Activated Clotting Time for Monitoring High

Dose Heparin Therapy 140IV. Low Molecular Weight Heparin 141

1. Pharmacokinetics 1412. Laboratory Monitoring by Chromogenic

Anti-Factor Xa Assays 1413. Clinical Efficacy and Bleeding Risks 142

D. Direct-Acting Antithrombin Agents 142I. Role of Thrombin in Thrombogenesis 142

II. Mechanism of Action and Clinical Studies 142III. Laboratory Monitoring 143

E. Thrombolytic Agents 143I. Clinical Use 143

II. Monitoring Thrombolytic Therapy 144

Contents XV

F. Antiplatelet Agents 144I. Aspirin 144

1. Clinical Effects 1442. Mechanism of Action 1453. Laboratory Monitoring 1454. Other Antiplatelet Agents 146

G. Summary/Conclusion 146References 148

CHAPTER 6

Use of Transgenic Mice in the Study of Thrombosis and HemostasisJ.M. PEARSON and D. GINSBURG. With 4 Figures 157

A. Introduction 157B. Overview of Coagulation and Fibrinolysis 157C. Transgenic Technology 159

I. Generation of Standard Transgenic Mice byZygote Injection 159

II. Generation of Knockout Mice 159D. Transgenic Mice Deficient in Coagulation Factors 161E. Transgenic Approaches to the Study of the

Fibrinolytic System 164I. Plasminogen 165

II. Plasminogen/Fibrinogen 166III. t-PA, u-PA and t-PA/u-PA 166IV. u-PAR and t-PA/u-PAR 167V. PAI-1 168

F. Summary 169References 170

CHAPTER 7

Current Antiplatelet TherapyJ.A. JAKIBOWSKI. R.E. JORDAN, and H.F. WIISM AN.

With 8 Figures 175

A. Introduction 175B. Platelets: Physiological and Pathological Activities 175

I. Physiological Activities 176II. Pathological Activities 177

C. Current Antiplatelet Therapy 177I. Aspirin 177

II. Dipyridamole 179III. Ticlopidine 180IV. Abciximab ISO

1. Preclinical Development ISO2. Clinical Pharmacolouv 183

XVI Contents

3. Additional Consequences 191a) Inhibition of Platelet Release 191b) Inhibition of Mac-1 Upregulation 191c) Inhibition of Platelet-Mediated Thrombin

Generation 192d) Characterization of Abciximab Binding to av/33 193

4. Clinical Experience 194a) Early Human Efficacy Studies 194b) The Phase III EPIC Trial 195c) The EPILOG Trial 198d) The CAPTURE Trial 200e) Clinical Summary of Abciximab 202

D. Investigational Agents 202I. GPIIb/IIIa Antagonists 202

II. Clopidogrel 203References 203

CHAPTER 8

Platelet Membrane Receptors and Signalling Pathways:New Therapeutic TargetsS.P. WATSON, D. KEELING, and M.D. HOLLENBERG. With 7 Figures . . . 209

A. Introduction 209I. Platelet Activation 209

II. Platelet Inhibition 210III. Regulation of Platelet Activation 211

B. Signalling by Cell Surface Receptors 211I. G Protein-Coupled Receptor Signalling 213

1. Guanine Nucleotide Binding Proteins andEffector Regulation 213

2. The G Protein GTP/GDP Cycle andEffector Modulation 215

3. G Protein-Regulated Effectors 216a) Adenylyl Cyclase 216b) Phospholipase C 217c) Other /Jy-Regulated Effectors: src Family Kinases

and PI 3-Kinase 217II. Tyrosine Kinase-Linked Receptors 218

III. Ion Channels and Their Receptors 219C. Signal Enzymes and Mediators 219

I. Phosphoinositide Metabolism 220II. Phospholipase A, 221

III. PI 3-Kinase 223

Contents XVII

IV. Cyclic Nucleotides 2231. cAMP 2232. cGMP 224

D. Platelet G Protein-Coupled Receptors 224I. Thrombin (PAR,) 224

1. Thrombin Binding Sites 2242. PAR,, A G Protein-Coupled Receptor for

Thrombin 2253. Other Protease-Activated Receptors (PARs) 2264. PAR, and Human Platelet Activation 227

II. Thromboxane A, (TP Receptor) 227III. ADP Receptors 228IV. 5-Hydroxytryptamine (5HT2A Receptor) 229V. Vasopressin (V, Receptor) 229

VI. Platelet Activating Factor (PAF) 230VII. Adrenaline (a,-Adrenoceptor) 230

VIII. Prostacyclin (IP Receptor) 231IX. Other Seven Transmembrane Receptors 231

E. Tyrosine Kinase-Linked Receptors 231I. Collagen 231

II. FcyRIIA 234III. Thrombopoietin 235

F. Adhesion Receptors 235I. Integrins 235

1. GPIIb-IIIa 2362. Other Platelet Integrins 238

II. GP-IX-V (vWf Receptor) 238III. PADGEM (P-Selectin) 239IV. PECAM-1 239V. GPIV 240

G. Clinical Settings for Antiplatelet Drugs 240I. The Platelet as a Target in Thrombotic Disease 240

II. Overview of Currently Used Agents 242III. Possible Settings for New Antiplatelet Agents 242

H. New Targets for Drug Development 242I. Receptors as Targets 243

1. ADP Receptors 2432. Adhesion Receptors 2443. Thrombin Receptor 245

II. Signalling Pathways as Targets 2471. Protein-Protein Interfaces 2482. Enzyme Targets 249

III. Development of New Drugs: Aspirin's Legacy 249References 250

XVIII Contents

CHAPTER 9

Heparin and Other Indirect Antithrombin AgentsW.R. BELL and T.A. HENNEBRY. With 1 Figure 259

A. Introduction 259I. Magnitude of the Problem of Intravascular Thrombosis

and Thromboembolic Disease 259B. Unfractionated Heparin:

The Prototypical Indirect Antithrombin 259I. History 259

II. Source 261III. Structure 261IV. Mechanism of Action 262V. Pharmacokinetics 265

1. Administration 265a) Intravenous Route 265b) Subcutaneous Route 266c) Novel Methods of Administration 267

2. Distribution 2673. Clearance 2674. Effect of Physiological State 2685. Drug Interactions 268

VI. Clinical Indications 2691. Venous Thrombosis and Thromboembolic Disease . . . . 2702. Use of Heparin in Acute Coronary Syndromes 2713. Heparin for Trousseau Syndrome:

A Unique Therapy 2724. Novel Uses of Heparin 273

VII. Therapeutic Monitoring 274VIII. Toxicity 276

IX. Antidotes: Reversal of Anticoagulant Effect 278C. Heparinoids and Related Anticoagulants 279

I. Pentosans (Sulfonated Xylans) 2791. Source 2792. Mechanism of Action 2803. Administration 2804. Clinical Uses 2805. Toxicity 2816. Clinical Relevance 281

II. Dermatan Sulfate 2821. Introduction 2822. Source 2823. Mechanism of Action 2824. Pharmacokinetics 2835. Clinical Use 283

Contents XIX

6. Clinical Relevance 284III. Sulodexide 284

1. Introduction 2842. Source 2843. Mechanism of Action 2854. Pharmacokinetics 2855. Clinical Indications 2866. Toxicity 2867. Future 286

IV. Danaparoid (Organan 10172) 2861. Introduction 2862. Source 2873. Mechanism of Action 2874. Pharmacokinetics 2875. Clinical Uses 2876. Toxicity 2887. Antidotes 2888. Future 288

V. Other Indirect Antithrombins 288D. Conclusion 289References 289

CHAPTER 10Low Molecular Weight HeparinG.F. PINEO and R.D. HULI 305

A. Introduction 305B. Discovery and Development of Low Molecular Weight

Heparins 305I. Properties of Unfractionated Heparin 305

II. Antithrombotic Properties of Low Molecular WeightHeparin 307

III. Advantages of Low Molecular Weight Heparin overUnfractionated Heparin 309

C. Use of Low Molecular Weight Heparins 310I. Prevention of Venous Thromboembolism 310

II. Orthopedic Surgery 311D. Trauma \ 312E. General Surgery 313

I. Medical Patients 314II. Low Molecular Weight Heparinoid 314

F. Treatment of Venous Thromboembolism 314G. Out-of-Hospital Treatment of Venous Thromboembolism

with Low Molecular Weight Heparin 316H. Role of Low Molecular Weight Heparin in the Prevention and

Treatment of Arterial Thrombosis 317

XX Contents

I. Unstable Angina 317II. Thrombotic Stroke 318

III. Peripheral Vascular Disease 318IV. Hemodialysis 319V. Other Vascular Problems 319

I. Current Recommendations for the Use of Low MolecularWeight Heparin 319

I. Prevention of Venous Thromboembolism 319II. Treatment of Venous Thromboembolism 320

J. Summary and Conclusions 320References 321

CHAPTER 11

Parenteral Direct AntithrombinsM.A. LAUER and A.M. LINCOFE. With 3 Figures 331

A. Introduction 331B. Thrombin: Structure and Function 331C. Limitations of Current Antithrombotic Therapy 333D. Direct Thrombin Inhibitors 334

I. Hirudin 334II. Other Direct Thrombin Inhibitors 335

E. Potential Roles for Direct Thrombin Inhibition 335I. Acute Myocardial Infarction, Adjunct to

Thrombolysis 3361. Preclinical Studies 3362. Phase II Clinical Trials 3363. Phase III Clinical Trials 3374. Further Acute Myocardial Infarction Studies 339

II. Unstable Angina and Myocardial Infarction WithoutST Elevation 3421. Phase II Clinical Trials 3422. Phase III Clinical Trials 343

III. Adjunct to Percutaneous Revascularization 3431. Preclinical Studies 3442. Phase II Clinical Trials 3443. Phase III Clinical Trials 344

IV. Deep Venous Thrombosis 345V. Heparin-Induced Thrombocytopenia 346

F. Rebound Phenomenon 346G. Summary and Future Directions 348References 348

Contents XXI

CHAPTER 12

Anticoagulant Therapy with Warfarin for Thrombotic DisordersL.A. HARKER. With 2 Figures 353

A. Introduction 353B. Pathogenesis 353C. Pharmacology 354

I. Mechanism of Action 354II. Assessment of Clinical Efficacy 354

III. Optimal Therapeutic Regimens 357IV. Benefits of Monitoring 359

D. Prevention and Management of VenousThromboembolism 359

I. Prevention of Venous Thrombosis FollowingOrthopedic Surgery 359

II. Prevention of Stroke and Venous Thromboembolism inAcute Myocardial Infarction 359

III. Treatment of Deep Venous Thrombosis 360E. Antithrombotic Therapy for Atrial Fibrillation 360F. Antithrombotic Therapy for Prosthetic Heart Valves 360G. Other Indications for Oral Anticoagulant Therapy 361H. Complications of Warfarin Therapy 361References 363

CHAPTER 13

Oral Thrombin Inhibitors: Challenges and ProgressS.D. KIMBALL. With 3 Figures 367

A. Introduction 367I. Role of Thrombin in Hemostasis and Thrombosis 367

II. Medical Need for Anticoagulant and AntithromboticDrugs 3701. Acute Anticoagulation 3712. Chronic Anticoagulation with Warfarin 372

B. Potential Advantages of Direct. Small Molecule Inhibitors 373C. Pharmacological and Pharmacokinetic Issues 374

I. Safety" 374II. Selectivity and Fibrinolytic Compromise 374

III. Pharmacodynamics: Efficacy and Kinetics ofInhibition 375

IV. The Rebound Phenomenon 377V. Oral Bioavailability and Pharmacokinetics 378

D. Thrombin Inhibitors in Development 379I. Bivalent Direct Thrombin Inhibitors 379

1. Hirudin 379

XXII Contents

2. Hirulog 381II. Reversible Inhibitors of Thrombin 381

1. Argatroban (Novastan) 3822. Napsagatran 3823. Inogatran 383

III. Covalent Inhibitors of Thrombin 3831. Efegatran 3842. Corvas: CVS 1123 3853. DuPont Merck: DuP 714 385

E. Summary and Conclusions 386References 386

CHAPTER 14

Inhibitors of Factor XaS. KUNI'I ADA, T. NAGAHARA, and T. HARA. With 6 Figures 397

A. Introduction 397B. Rationale of Factor Xa Inhibitors 397C. Pharmacological Profile of Factor Xa Inhibitors 399

I. ATIII-Dependent Inhibitors 400II. Direct Inhibitors 401

1. Naturally Occurring Inhibitors 4012. Synthetic Small Molecule Inhibitors 403

a) Peptidomimetics 403b) Benzamidine Derivatives 404c) Bisamidine Derivatives 405d) Argininal Derivatives 408e) Piperidinylpyridine Derivatives 410

D. ATIII-Independent Inhibition of Factor Xaon Prothrombinase 410

E. Comparative Antithrombotic Efficacy ofDirect Factor Xa Inhibitors 414

F. Summary and Conclusions 415References 415

CHAPTER 15

Inhibitors of Tissue Factor/Factor VilaK.P. GALLAGHER. T.E. MERTZ, L. CHI, J.R. RUBIN.

and A.C.G. UPRICHARD. With 4 Figures 421

A. Introduction 421B. Role in Hemostasis 422C. Structural Biology of Tissue Factor and Factor Vila 423D. Endogenous Regulators of Tissue Factor 429

I. Tissue Factor Pathway Inhibitor 429

Contents XX11I

II. Antithrombin 430E. Pathophysiology of TF/VIIa 430F. Experimental Inhibitors of TF/VIIa 432

I. Recombinant TFPI (rTFPI) and Truncated rTFPI 433II. Inactivated Factor Vila: FVIIai 435

III. Recombinant Nematode Anticoagulant Peptide(rNAPc2) 436

IV. TF Antibodies 437G. Summary and Conclusions 438References 439

CHAPTER 16

Natural Anticoagulants and Their PathwaysC.T. ESMON. With 5 Figures 447

A. Introduction 447I. The Protein C Activation Complex 450

II. The APC Anticoagulant Complex 453III. Inhibition of the Anticoagulant Complex 454

B. Modulation of the Protein C Pathway in Disease 455I. APC Resistance and Factor V Leiden 458

C. Thrombomodulin as an Antithrombotic Agent 458D. Protein C as an Antithrombotic Agent 459

I. Protein C and Arterial Thrombosis 461II. Reperfusion Injury 462

E. Protein S as an Antithrombotic Agent 463F. Mutations to Modulate Natural Anticoagulant Responses 463

I. Mutations in Protein C 463II. Mutations in Thrombin 464

G. Inactive Coagulation Factors as Antithrombotics 464H. Summary 466References 466

Subject Index 477