Tài liệu HANDBOOK OF PLATELET PHYSIOLOGY AND PHARMACOLOGY ppt

In this book, the chapters are organized into six major sections, including Introduction, Receptor Biology, Platelet Biochemistry, Experimental Physiology, Platelet Pathology and Platele

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Handbook of platelet physiology and pharmacology / edited by Gundu

H.R Rao

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Includes bibliographical references and index

ISBN 0-7923-8538-1 (alk paper)

1 Blood platelets Handbooks, manuals, etc I Rao, Gundu H.R

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Pathology & Pharmacology

Loyola University Med Center

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7 Robert W Colman, M D.Thrombosis Research CenterTemple Univ Sch of Medicine

3400 N Broad StreetPhiladelphia, PA19140,USA

8 Maribel Diaz-Ricart, Ph D.Servicio de HemoterapiaHospital Clinico ProvincialVillarroel 170, Barcelona

08036, Spain

9 Gines Escolar, M D Ph D.Servicio de HemoterapiaHospital Clinico ProvincialVillarroel 170, Barcelona

08036, Spain

10 Daniel Fareed, B.Sc

Departments ofPathology & PharmacologyLoyola University Med Center

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1 1 Jawed Fareed, Ph D

Departments ofPathology & PharmacologyLoyola University Med Center

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12 Deborah French, M D.Department of MedicineMount Sinai Hospital &Medical SchoolOne Gustave L Levy PlaceNew York, NY

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20 BeateKehrel,Ph.D

Experimental and ClinicalHaemostaseologyDepartment of Anaesthesiologyand Intensive Care MedicineUniversity of MuensterD-48149 Muenster, Germany

914 South Eighth Street, D-3Minneapolis, MN

55404 USA

23 Ellinor I Peerschke, Ph D.Cornell Medical CenterNew York University

525 E 68th Street, Rm F51 1 JNew York, NY

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08025 BarcelonaSpain

25 Marek W Radomski, M.D,D.Sc.Division of R and D

Lacer, S.A., 08025 BarcelonaSpain

26 Gundu H R Rao, Ph D.

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33 Jeanine M Walenga, Ph D.Departments of Pathology &Pharmacology

Loyola UniversityMedical Center

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34 Douglas J Weiss, D V.M., Ph D.Department of Pathobiologyand Veterinary SciencesUniversity of Minnesota

St Paul, MN55108,USA

35 Helmut Wolf, M D, Ph D.Departments of Pathology &Pharmacology

Loyola UniversityMedical Center

2 1260 South First Ave

Maywood, IL60153,USA

Despite my many years of research and teaching in platelet physiology andpharmacology at the University of Minnesota, I am often confronted with conflictingopinions as to the relevance of nonnucleated platelets in human health and disease It

is fascinating to think that how cells with no apparent nucleus, have such a toweringimpact on concepts, dealing with often overlapping physiological (i.e hemostasis,wound healing, etc.) and pathophysiological (i.e thrombosis, stroke, atherosclerosis,wound healing, diabetes, inflammation and cancer) components Although the idea ofcompiling new frontiers of platelet research in the form of a book was quite simple atthe beginning, the project turned out to be a major undertaking from my part At theend, I am elated that the contributors to this book were gracious enough to write chapters

in their area of research expertise despite their pressing and highly valuable time For

me, it has been an humbling experience as the chapters that I have compiled, are written

by people with incredible recognition for their relentless contributions over the years tostrengthen the understanding of platelet physiology and pharmacology In my opinion,this has added an immense value to the book I am proud to have been involved in thisundertaking despite several unexpected problems and delays during this project I amconfident that this book would be highly useful to the community of scientists, includinggraduate students, researchers, academicians, physicians and other health careprofessionals, and pharmaceutical industry scientists

Circulating platelets which lack nucleus neither adhere to the vessel wall nor aggregateunless they encounter a zone of injury Upon encountering such a zone of injury, theybecome almost instantly activated, which leads to their adhesion and aggregation, bothreactions are of fundamental importance to hemostasis and thrombosis Because of thisreason, platelet research has clearly led the way in the continuing development of newstrategies and drugs that can help prevent and treat arterial thrombosis, stroke andatherosclerosis Unquestionably, platelet research has also impacted concepts dealingwith many other diseases Nevertheless, considerable progress has been made in thedevelopment of new antiplatelet agents in recent years These newer agents are aimed

at interrupting specific sites and pathways of platelet activation Inhibitors of specificplatelet agonist-receptor interactions include antithrombins, thromboxane A2 receptorantagonists, and adenosine diphosphate receptor blockers (i.e ticlopidine, clopidogrel)

In addition, inhibitors of arachidonic acid metabolism and thromboxane A2 includeaspirin, newer COX-2 inhibitors, other NSAIDs, thromboxane A2 synthase inhibitorsand o>-3 fatty acids Moreover, long awaiting drugs that block ligand binding to theplatelet glycoprotein Ilb/IIIa complex (i.e tirofiban) have now entered the market

In this book, the chapters are organized into six major sections, including Introduction, Receptor Biology, Platelet Biochemistry, Experimental Physiology, Platelet Pathology and Platelet Pharmacology Authoritative chapters in each section have provided a

collective strength to our initial philosophy of accomplishing a comprehensive review

of current concepts in each discipline Although every attempt has been made to provide

an interdisciplinary discussion on the subject of platelets in this book, there may still besome gaps and lapses for which readers are urged to consult other articles and reviews

I have deliberately avoided going into any specific comments on reviews in order to letthe imagination of the readers flow freely I believe that the readers are intelligentenough to judge and form their own critical opinion

I must humbly express my deep gratitude to thirty five scientists in the field for theirinvaluable contributions I now honestly believe that this publication would not havebeen possible without their meritorious contributions.

I am deeply indebted to my dear friend and close research collaborator, MahadevMurthy, Ph D., Director of Research, Division of Endocrinology, Metabolism andNutrition, Department of Medicine, Hennepin County Medical Center, Minneapolis,

MN, USA, for his commitment and contribution to this project He has spent countlesshours during this project in reviewing and preparing camera ready manuscripts for finalsubmission to the Kluwer Academic Publisher In addition, he has written two excellentchapters for the book I must confess that this publication would not have beencompleted without his generous and truly dedicated efforts

I would like to take this opportunity to thank Charles W Schmieg, Jr., AcquisitionsEditor, Kluwer Academic Publishers, 101 Philip Drive, Assinippi Park, Norwell, MA,

02061, USA, for facilitating the publication of this book I am specially thankful for hiscooperation and patience even though this project was delayed by about four months

Finally, I would not be in this field today without my mentor, James G White, M D.,Regents' Professor & Associate Dean, Academic Health Center, University ofMinnesota, Minneapolis, MN, USA I humbly dedicate this publication to James G.White, M D., who has been my mentor, teacher, associate and dear friend, during mylong career in platelet research In the end, my academic success and accomplishmentsover the years, would not have been possible without the support of my wife Yashoda,

my daughter Aupama and my son Prashanth I sincerely acknowledge and appreciatetheir patience and support throughout my career

Gundu H R Rao University of MinnesotaProfessor Minneapolis, MN

55455

v

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Contents

Contributors ix

Preface xiii

Introduction 1

1 Platelet Physiology & Pharmacology: an Overview 1

1.1 Introduction 1

1.2 Role of Platelets in Hemostasis and Thrombosis 2

1.3 Platelet Morphology and Biochemistry 2

1.4 Platelet Physiology 5

1.5 Altered Physiology and Function 6

1.6 Platelet Pharmacology 8

1.7 Platelet Function Inhibitory Drugs 9

1.8 Acknowledgements 14

References 15

Receptor Biology 21

2 Human Platelet Thrombin Receptors and the Two Receptor Model for Platelet Activation 21

2.1 Introduction 21

2.2 Binding Studies 22

2.3 Membrane Microviscosity 24

2.4 Candidate Receptors 26

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2.5 The GPIb-IX-V Complex 27

2.6 Two Receptor Model 31

References 33

3 Platelet Thromboxane Receptors: Biology and Function 38

3.1 Introduction 38

3.2 Biological Effects of TP Receptor Activation 39

3.3 Smooth Muscle Contraction 39

3.4 TP Receptor Structure 41

3.5 TP Receptor Function 49

3.6 Altered TP Receptor Function 58

References 66

4 Collagen Receptors: Biology and Functions 80

4.1 Introduction 80

4.2 Collagens 82

4.3 Von-Willebrand-Factor 83

4.4 P65 84

4.5 CD36 84

4.6 a2b1-Integrin (GPIa/IIa, VLA2, ECMRII) 87

4.7 GPVI/FcRg 89

4.8 Collagen-Induced Signal Transduction 90

References 92

5 Adenosine Receptors: Biology and Function 102

5.1 Introduction 102

5.2 Adenosine Receptors 103

5.3 Antiplatelet Action of Adenosine 104

5.4 Adenosine Production and Platelet Inactivation 106

5.5 Agents Affecting Adenosine Actions 109

Contents vii

This page has been reformatted by Knovel to provide easier navigation 5.6 Adenosine Effects on Intracellular Ca2+ Mobilization 113

5.7 Conclusions 114

References 115

6 Platelet Activating Factor and Platelets 120

6.1 PAF Discovery, Structure and Heterogeneity 120

6.2 PAF Biosynthesis in Platelets 121

6.3 Responses of Platelets to PAF 122

6.4 PAF Receptor and Signal Transduction Pathways in Platelets 123

6.5 Antagonist 124

6.6 PAF Receptor 125

6.7 Phospholipases 126

6.8 Platelet and PAF in Pathophysiological and Disease States 129

6.9 Acknowledgement 133

References 133

7 Platelet Glycoprotein Ib-V-IX: Biology and Function 142

7.1 Introduction 142

7.2 Structure 143

7.3 Post-Translational Modification of GPIb-V-IX 145

7.4 Basic Functions 146

7.5 Signal Transduction 148

7.6 GPIb-V-IX as a Target for Pharmacological Inhibition 149

7.7 Genetic Disorders Affecting GPIb-V-IX 151

7.8 Tissue Specific Expression of GPIb-V-IX Subunits 153

7.9 Future Developments 154

References 155

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8 Fibrinogen Receptors: Biology and Function 162

8.1 Introduction 162

8.2 Characterization of the Platelet Fibrinogen Receptor 163

8.3 Function 166

8.4 Post-Fibrinogen Binding Events 171

8.5 Conclusion 177

References 178

Platelet Biochemistry 188

9 Biochemistry of Platelet Activation 188

9.1 Function 188

9.2 Morphology and Subcellular Organelles 189

9.3 Platelet Activation and Responses 190

9.4 Signal Transduction Systems 194

9.5 Platelet Agonists and Their Signaling Systems 206

9.6 Inhibition of Platelet Activation 211

9.7 Autocrine Stimulation and Inhibition 213

9.8 Crosstalk Between Different Signaling Systems 214

9.9 Communication Between Platelets and Other Blood Cells 215

9.10 Summary 217

References 217

10 GTP Binding Proteins in Platelets 238

10.1 Introduction 238

10.2 G-Proteins and Signal Transduction 240

10.3 Low Molecular Weight GTP-Binding Proteins 243

10.4 Summary 247

Contents ix

This page has been reformatted by Knovel to provide easier navigation References 247

11 Platelet Cyclic Nucleotide Phosphodiesterases 251

11.1 Introduction 251

11.2 Regulation of Platelet Activation By cAMP and cGMP 252

11.3 Classification of Cyclic Nucleotide PDEs 252

11.4 Platelet cGI-PDE (PDE3A) 255

11.5 CGI-PDE Regulatory Domain 257

11.6 Platelet cGMP-Stimulated PDE (PDE2) 260

11.7 Platelet cGMP-Binding, cGMP-Specific Phosphodi-Esterase (cGB-PDE, PDE5) 262

References 263

12 Polyenoic Fatty Acids and Platelet Function 268

12.1 Introduction 268

12.2 Platelet Function and Its Relevance to Thrombosis 269

12.3 Polyunsaturated Fatty Acids (PUFAs) 270

12.4 Platelet Membranes and Their Lipid Composition 271

12.5 Arachidonic Acid and Platelet Elcosanoids 273

12.6 Omega-3 Fatty Acids 276

12.7 Omega-3 Fatty Acids and Platelet Function 279

12.8 Docoshexaenoic Acid and Platelets 280

12.9 PUFAs and Their Newly Discovered Roles 281

Concluding Comments 284

Acknowledgements 285

References 286

13 Phospholipase A2 in Platelets 293

13.1 Introduction 293

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13.2 Pathways of Arachidonic Acid Release in

Platelets 294

13.3 Phospholipid Breakdown Measurements in Stimulated Platelets 296

13.4 Phospholipase A2 in Platelets 296

13.5 Calcium and Phospholipase A2 298

13.6 Hydroperoxides and Phospholipase A2 302

13.7 Phosphatidic Acid and Platelets 302

13.8 PAF and Phospholipase A2 303

13.9 LDL and Platelet Function 304

Concluding Remarks 304

Acknowledgements 305

References 305

Experimental Physiology 315

14 Platelet Biorheology: Adhesive Interactions in Flow 315

14.1 Introduction: General Overview for Flow Studies of Platelet Aggregation 315

14.2 General Physiology of Platelet Activation and Aggregation in Flow 316

14.3 Range of Shear Rates in Normal and Pathological Settings 317

14.4 Flow Regimes and Corresponding Devices Used to Study in Vitro Platelet Aggregation 318

14.2 Ligands and Receptors Involved in Platelet Aggregation 319

14.3 Quantitation of Aggregation: Theoretical and Experimental Approaches 322

14.4 Platelet Aggregation in Non-Stirred Platelet Suspensions: Role of Pseudopods 323

Contents xi

This page has been reformatted by Knovel to provide easier navigation 14.5 Model Cell Aggregation in Near-Stasis Versus Stirred Suspensions 324

14.6 Dynamics of Soluble Fg Binding (Receptor Occupancy) and Platelet Aggregation as Function of Shear Rate (66,67) 324

14.7 Dynamics of Von Willebrand Factor-Mediated Platelet Aggregation 327

14.8 Some New Directions 330

14.9 Summary 332

Acknowledgements 333

References 333

15 Platelet Vessel Wall Interactions 342

15.1 Introduction 342

15.2 Interaction of Platelets with Vascular Subendothelium 343

15.3 Interaction of Platelets with Extracellular Matrices, Isolated Components of the Vessel Wall or Purified Plasma Proteins 350

15.4 Concluding Remarks 354

Acknowledgements 355

References 355

16 Platelet-Biomaterial Interactions 362

16.1 Introduction 362

16.2 Contribution of Platelets to Thrombus Formation 363

16.3 Platelet Adhesion on Biomaterials 364

16.4 Role of Plasma Proteins on Platelet Adhesion 364

16.5 Effect of Shear on Platelet-Surface Interaction 370

16.6 Role of Erythrocytes and White Cells on Platelet-Biomaterial Interactions 370

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16.7 Platelet Activation and Morphological

Changes 371

16.8 Concluding Remarks 374

Acknowledgements 375

References 375

17 Comparative Physiology of Platelets from Different Species 379

17.1 Introduction 379

17.2 Horse 380

17.3 Ruminants 382

17.4 DOG 383

17.5 CAT 385

17.6 PIG 386

17.7 Rabbit 387

17.8 Rat and Mouse 387

17.9 Guinea PIG 388

Conclusions 389

References 389

Platelet Pathology 394

18 The Molecular Pathology of Glanzmann’s Thrombasthenia 394

18.1 Introduction 394

18.2 Glanzmann Thrombasthenia 395

18.3 Genetics and Expression of the Platelet GPIIb/IIIa Receptor 397

18.4 Molecular Identification of Mutations 399

18.5 Mutations Resulting in Biosynthetic Defects 408

18.6 Mutation Hotspots 412

Contents xiii

This page has been reformatted by Knovel to provide easier navigation 18.7 Prenatal Diagnosis, Carrier Detection, and Gene Therapy 413

18.8 Conclusions 414

References 414

19 Congenital Disorders of Platelet Signal Transduction and Secretion 424

19.1 Introduction 424

19.2 Signal Transduction Mechanisms (Fig 1) 425

19.3 Role of Platelets in Blood Coagulation 426

19.4 Congenital Disorders of Platelet Function 426

19.5 Disorders of Platelet Secretion and Signal Transduction 429

19.6 Signal Transduction Defects and Activation of GPIIb-IIIa 433

19.7 Abnormalities in Thromboxane Production and Arachidonic Acid Pathways 433

19.8 Relative Frequency of Various Platelet Abnormalities 434

19.9 Conclusions 435

Acknowledgments 435

References 435

20 Biochemistry of Altered Platelet Reactivity in Hypertension 439

20.1 Introduction 439

20.2 Platelet Adhesion and Aggregation Responses in Hypertension 441

20.3 Role of Phosphoinositide in Platelet Reactivity Inhypertension 443

20.4 Thrombin- and PGE1-Receptor Mediated Signal Transduction Mechanisms in Hypertension 448

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20.5 Role of Nitric Oxide and Cyclic GMP in Platelet

Reactivity in Hypertension 450

20.6 Role of Thromboxane A2 and Lipoxygenase Metabolites in Hypertension 450

20.7 Summary 451

20.8 Acknowledgements 452

References 452

Platelet Pharmacology 458

21 Nitric Oxide-Mediated Regulation of Platelet Function 458

21.1 Introduction 458

21.2 Enzymology 459

21.3 Molecular Targets and Metabolism of NO 460

21.4 Nitric Oxide as Physiological Regulator of Platelet Function 460

21.5 Mechanisms of NO Action on Platelets 461

21.6 Peroxynitrite 462

21.7 Nitric Oxide and Vascular Disorders 463

21.8 Nitric Oxide, Platelets and Septicemia 464

21.9 Pharmacology of NO Generation and Action in the Platelet Microenvironment 465

Acknowledgements 469

References 469

22 Aspirin, Prostaglandins and Platelet Function: Pharmacology and Thrombosis Prevention 478

22.1 Introduction 478

22.2 Prostaglandin Structure and Function 480

22.3 Effect of Aspirin on Prostaglandin Synthesis 481

22.4 Aspirin's Unique Effect on Platelet Physiology 483

Contents xv

This page has been reformatted by Knovel to provide easier navigation 22.5 Molecular and Clinical Correlates: How Aspririn-Mediated Cyclooxygenase Inhibition Prevents Arterial Thrombosis 485

22.6 Prevention of Clinical Thrombosis by Aspirin 486

22.7 The Pharmacology of Aspirin Dose and Its Antithrombotic Effect: How Lower Doses May Minimize Prostacyclin Synthesis Inhibition 487

22.8 Side Effects of Aspirin 488

References 489

23 Current Developments in Anticoagulant and Antithrombotic Agents 495

23.1 Overview 495

23.2 Newer Applications of Unfractionated Heparin 498

23.3 The Development of Low Molecular Weight Heparins 499

23.4 Low Molecular Weight Heparins in the Management of Thrombosis 501

23.5 Glycosaminoglycan Related Antithrombotic Agents 503

23.6 Non-Heparin Glycosaminoglycans 504

23.7 Recombinant and Synthetic Antithrombin Drugs 507

23.8 Antiplatelet Drugs in Development 515

23.9 Synopsis 518

References 520

24 Randomized Trials of Antiplatelet Therapy 526

24.1 Introduction 526

24.2 Antiplatelet Therapy in the Prevention of Vascular Events Among Patients at High Risk of Occlusive Arterial Disease 528

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24.3 Antiplatelet Therapy in the Prevention of

Vascular Events Among Patients at Low Risk

of Occlusive Arterial Disease 536 24.4 Antiplatelet Therapy in the Maintenance of

Vascular Graft or Arterial Patency Among

Patients at High Risk of Occlusive Arterial

Disease 539 24.5 Antiplatelet Therapy in Patients at Risk of

Venous Thromboembolism 540 24.6 The Risks of Serious Bleeding with Antiplatelet

Therapy 543 24.7 Conclusions and Recommendations for

Practice 544 References 545

Index 549

PLATELET PHYSIOLOGY AND PHARMACOLOGY:

AN OVERVIEW

Gundu H R Rao, Ph D.Departments of Laboratory Medicine & Pathology

and Biomedical Engineering Institute

Academic Health CenterUniversity of Minnesota, Minneapolis, MN

55455, USA

1.1 INTRODUCTION

Dr Gundu Rao has spent the last 25

years studying and teaching platelet

physiology and pharmacology at the

University of Minnesota Over the

years, he has made significant

contributions to the understanding of

platelet function and its impact on the

pathogenesis of atherosclerosis and

thrombosis In addition, he has been

vocal in the national and international

activities that advocate prevention,

early diagnosis and treatment of

Coronary Artery Disease (CAD)

More recently, he led the

establishment of South Asian Society

for Atherosclerosis and Thrombosis

(SASAT) He continues to champion

activities on the prevention of CAD, in

the International arena

Clinical, experimental and epidemiologicalstudies have demonstrated a ubiquitous role forplatelets in the pathogenesis of thromboembolicevents and hemorrhagic disorders (1-19).Therefore, there is great need for developingspecific and effective drugs capable ofmodulating platelet function A thoroughunderstanding of the mechanisms involved inregulating platelet function will facilitate thedevelopment of better antiplatelet drugs.Agonists interact at specific sites on the plasmamembrane of platelets and initiate a series ofsignaling events (20-37) Activation signals arecommunicated to intracellular effector enzymesthrough transmembrane receptors that arecoupled to GTP-binding proteins (29)

Stimulation of intracellular enzymes facilitatethe formation of second messengers capable of

mobilizing cytosolic free calcium from internal membrane stores Ionized calcium is theprimary bioregulator of various platelet responses (31,32) Agonists as well as antagonistsare capable of modulating cytosolic calcium levels Elevation of cytosolic calciumstimulates phospholipase A2, liberates arachidonic acid from membrane phospholipids,promotes assembly of filamentous actin, granule mobilization and secretion of granulecontents (31) The mechanisms involved in platelet activation facilitate the expression

of binding sites for fibrinogen on platelet glycoprotein (GP) 1 lb/11 Ia receptor (38-45).Activated GP 1 lb/11 Ia receptor binds fibrinogen and this binding seems to contribute tothe formation of platelet aggregates and growth of the thrombus Major pharmacologicalapproaches have focused on developing inhibitors capable of preventing one or moresignaling mechanisms (33) Currently available antiplatelet drugs are quite effective inpreventing platelet activation, including aggregation and secretion However, they areless effective in preventing platelet interaction with biomaterials and cell matrixcomponents Intense research is in progress to develop newer antiplatelet drugs A recentreview of antiplatelet clinical trials concluded that use of any antiplatelet drug significantlyreduces the development of cardiovascular events relating to coronary artery disease Thearticles in this book which are written by experts, provide a comprehensive review onvarious aspects of platelet physiology and pharmacology

1.2 Role of platelets in hemostasis and thrombosis

Platelets contribute significantly to the normal hemostatic process (1-6) They play acritical role in the recognition of injured vasculature, formation of hemostatic plugs,prevention of bleeding, retraction of clots and wound healing (6-9) When they developsevere dysfunction they contribute to the pathogenesis of hemorrhagic disorders.Whereas, when they are hyperactive, they can initiate events leading to clinicalcomplications associated with cardiovascular disorders (10-16) Platelets circulate in theblood as nonadhesive disc shaped cells When activated they undergo a series of discretetransformations The degree of activation depends upon the strength of the activatingstimuli and the information available on the interactive domains of the cell matrixcomponents For instance, interaction with laminin will result in minimum activation andformation of focal adhesion However, fibronectin will promote complete spreading ofplatelets Whereas, collagen will induce formation of aggregates as well as secretion ofgranule contents Platelet responses that are well recognized include development ofstickiness, adhesion, change in shape, irreversible aggregation and secretion of granulecontents Platelet activation is a prerequisite for the formation of a hemostatic plug andarrest of bleeding at the injured site Although formation of a hemostatic plug is a naturalresponse to injury, the role of platelets in atherosclerosis, thrombosis and stroke arepathological manifestations (1-19)

1.3 Platelet morphology and biochemistry

Blood platelets have a discoid form in their resting state A circumferential band ofmicrotubules support this shape This characteristic shape facilitates their movement inthe circulating blood at the periphery of the flow close to the vessel wall (Fig 1)

Ih order to relate cell structure to the functional responses, the work by White has dividedanatomy of the platelet into three distinct zones, the peripheral zone, the sol-gel zone andthe organelle zone (20) The peripheral zone consists of membranes and closely associated

structures including lipid bilayer, cytoskeletal proteins, transmembrane proteins, various

Figure 1 Scanning electronmicrograph of resting platelets The characteristic shape of circulating discoid

platelets is critical for their passage in the vessel wall with smooth blood flow (Courtesy: James G White, MD).glycoprotein-rich interactive domains, transmembrane receptors for soluble agonists such

as epinephrine, adenosine diphosphate (ADP), thrombin, thromboxane and plateletactivating factor (PAF), GTP-binding proteins, integrin (GP lib/11 Ia, GPla/lla,GPlc/1 Ia) and non-integrin (GPlV, GPV, GPlb/lX) receptors and ion channels (reviewed

in subsequent chapters)

Figure 2 Electronmicrograph of a discoid platelet (sectioned in the equatorial plane) Microtubules that support

the discoid form are seen beneath the plasma membrane; Organelles such as dense bodies, granules (alpha, lysosomal) and mitochondria can also be seen (Courtesy: James G White, MD).

Figure 3 Schematic representation of discoid platelets: Features of a discoid platelet: EC, exterior coat; CM,

cell membrane; CS, channels of the surface connected canalicular system; SNfF, submembrane filaments; gel zone contains actin microfilaments, microtubule(MT), and glycogen (GIy), formed elements; a-granules (G), dense bodies (DB) and mitochondria (M); DTS and OCS are part of the membrane system (Courtesy: James G White, MD)

Sol-The morphological features described above support the discoid shape in nesting plateletsand thus, providing a contractile system that facilitates shape change, pseudopodformation, contraction and secretion The major portion of the contractile system involvesactin Other proteins of the platelet contractile system include, myosin, tropomyosin,acting binding protein, cc-actinin, gelsolin, profilin, vinculin and spectrin The organellezone consists of granules, dense bodies, peroxisomes, lysosomes, mitochondria andglycogen This zone serves as the storage site for various enzymes, non-metabolic adeninenucleotides, serotonin, a variety of proteins, calcium, and antioxidants such as ascorbicacid, taurine, and glutathione The unique complex membrane system (DTS & OCS) plays

a very critical role in platelet pathophysiology The dense dense bodies(DB) are the sitefor calcium, adenine nucleotides and serotonin sequestration The DTS is the site whereenzymes of arachidonic acid metabolism and prostaglandin synthesis are localized Thesol-gel zone constitutes components of the cytoplasm It contains fibers, filaments andproteins in various states of polymerization (Fig 2)

The surface-connected open canalicular system (OCS) provides access to the interior forplasma-borne substances and it serves as a conduit for products secreted during the releasereaction (Fig 3)

1.4 Platelet physiology

Physiological agonists cannot penetrate the plasma membrane barrier and therefore, theymust first coiq)le to specific interactive domains on the platelet surface membrane in order

to trigger the sequence of activation signals (21 -29, reviewed by Ryningen and Holmsen

in this book) The physiological agonists that are known to activate platelets includeadenosine diphosphate (ADP), epinephrine (adrenaline), thromboxane A2 (Tx^ )>thrombin, and platelet activation factor (PAF) In addition, cell matrix components such

as laminin, fibronectin, collagen and von Willebrand factor also trigger platelet activation.Platelet adherence to surfaces and resulting activation leads to the development ofstickiness, change in shape, formation of pseudopods, adhesion, followed by aggregationand secretion of granule contents (Fig 4)

Receptors for ADP, epinephrine, thromboxane, thrombin and PAF have been wellcharacterized (27, 31, reviewed in other chapters) Membrane spanning receptors ofepinephrine, thrombin and thromboxane are coupled to the ubiquitous GTP-bindingproteins Platelets contain monomeric, low molecular weight G proteins as well asheterotrimeric membrane associated G-proteins GTP binding to the a-subunit of G-proteins facilitates the interaction with effector enzymes, resulting in the hydrolysis ofGTP to GDP, which terminates its stimulatory role (29)

Figure 4 Scanning electronmicrograph of activated platelets Platelet activation resulting in shape change and

formation of pseudopods (PS) (Courtesy: James G White, MD).

Agonist-mediated activation of platelets stimulates phospholipase C (PLC) and it thentriggers the hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2), the formation ofsecond messengers such as 1, 2-diacylglycerol (DAG) and inositol 1,4, 5-trisphosphate(IP3) (30-38, also reviewed in this book) Diacylglycerol (DAG) is a substrate for proteinkinase C (PKC), which is recognized as a multifunctional enzyme (25) This lipidintermediate is also a substrate for phosphatidic acid Diacylglycerol (DAG) inducestranslocation of cytosolic PKC to membranes, which acts as a trigger mechanism for itsactivation On the other hand, IP3 is known to mobilize ionized calcium from internal

membrane stores However, thrombin and PAF can also mobilize calcium from theexternal milieu Ionized calcium plays a central role in all platelet functional responsesand it is therefore considered as an important bioregulator in platelet pathophysiology (31 -

36, also reviewed elsewhere in this book) Elevated cytosolic calcium is essential for theassembly of filamentous actin Furthermore, it is also considered essential for theactivation of phospholipase A2, a key enzyme to mobilize arachidonic acid (AA) frommembrane phospholipids for further metabolism (reviewed elsewhere in this book) It iswell documented that free AA then is converted to cyclic endoperoxides such as PGG2 andPGH2 in the presence of cyclooxygenase (27, 11, 23) These transient metabolites arefurther transformed into novel thromboxanes by thromboxane synthetase Thromboxane

A2 is the major metabolite of AA metabolism in platelets (31) It is a vasoconstrictor andpotent platelet agonist In endothelial cells, AA is converted to cyclic endoperoxides bycyclooxygenase, and these metabolites are further transformed to prostacylcin (PGI2) byprostacyclin synthetase Prostacyclin is a vasodilator and a potent platelet antagonist (31).Thus both phosphoinositol pathway and AA metabolism, contribute significantly to theactivation of platelets by soluble agonists (27, 2, 10, 11, 14) These events promote theexpression of an activation dependent epitope on the platelet glycoprotein GPl lb/11 Iareceptor (39-46) Activation of this receptor promotes fibrinogen binding and facilitatesplatelet adhesion, aggregation and growth of the thrombus However, activation of thisreceptor is not essential for its interaction with surface bound fibrinogen Apart from theagonists mentioned above, shear force also can induce platelet activation (discussedelsewhere in this book) It is believed that fibrinogen plays a role in the adhesion andaggregation of platelets under low shear rates Furthermore, at high shear forces vonWillebrand factor interaction with GP Ib/IX seems to be important (31, also discussed inother chapters of this book) Circulating adhesive proteins such as fibrinogen, cell matrixcomponents, bacterial membrane proteins, certain tumor cells and biomaterial surfacesalso interact with the platelet plasma membrane at discrete domains Binding of ligands

to integrinandnon-integrin receptors induce activation signaling mechanisms (27, 31, 62,also discussed in other chapters of this book) Binding results in the activation andstimulation of various effector enzymes and formation of second messengers, leading toaggregation and secretion of granule contents Specific mechanisms involved in theprocess of centralization of granules and release of their contents are poorlyunderstood(26,27, 31)

1.5 Altered physiology and function

Many investigators have attempted to correlate the in vitro functional response of platelets

to clinical manifestations of thrombotic episodes or bleeding diathesis (8,10,12, 15, 30,46,47, also discussed in other chapters) Yet, it remains a difficult task to establish a clearrelationship between specific functional responses and their role in normal hemostasis.However, the presence of functional glycoprotein GPlb/lX and GPl lb/11 Ia receptors andthe ability of platelets to undergo shape change, spread, become sticky, irreversiblyaggregate, or release granule contents, are considered essential for normal hemostaticfunction Drug-induced impairment of signal transduction and biochemical lesions,resulting from procedures such as surgery, dialysis, angioplasty, may also lead to plateletdysfunction Platelets may also develop biochemical lesions during storage and thus, maycarry dysfunctional characteristics making them less suitable for transfusion Althoughintracellular calcium elevation is considered important for eliciting platelet responses such

as contraction and secretion, its role remains questionable in platelet shape change and

other processes such as the development of stickiness, fibrinogen binding, the adhesion

or interaction with the vascular subendothelium (48-49, also discussed in other chapters).Action of weak agonists for mediating secretion of granule contents depends uponavailability OfTxA2 However, bovine and equine platelets which do not aggregate inresponse to the action of the arachidonic acid metabolite, thromboxane, support normal

in vivo hemostasis in these animals Similarly, the majority of dogs have platelets that donot respond to AA metabolites (50) On the other hand, epinephrine exposure restores thesensitivity of these refractory platelets to the action of AA (51) The observations in ourlaboratory have also led to the development of a novel concept of membrane modulation(17) The concept seems to offer a reasonable explanation for observed functionalresponse of platelets with no detectable cyclooxygenase activity, which can still supportnormal hemostasis in adults These findings raise the possibility that cyclooxygenase maynot be obligatory for normal hemostasis, but may likely play an important role inthrombosis Other laboratories have also shown such phenomenon, particularly foradrenaline and noradrenaline Some reports also suggest that adrenaline stimulation of a-receptors may in fact amplify or restore signal transduction mechanisms in platelets.Studies by Scrutton et al and Stormorken and his associates, have shown compromisedfunctional response to epinephrine in apparently normal individuals (52, 53) Weiss et

al have described secretion defects from patients with bleeding disorders (54) White et

al have followed functional response of platelets of patients with diabetes and Pudlak Syndrome (HPS) whose platelets lack dense bodies Platelets of patients with HPSexhibit compromised response to the action of agonists (55) Hardisty et al., and Wareand associates, have provided further evidence for altered signal transduction mechanisms.These and other studies seem to suggest that an impaired intracellular calcium flux may

Hermasky-be the chief cause of platelet dysfunction (56-58, discussed in other chapters)

A brief review of the literature on platelet disorders indicates that there is still a great deal

of confusion in establishing a clear correlation between specific signal-driven responsesand platelet function despite voluminous information on the biochemistry of signaltransduction, second messengers and calcium pools and fluxes It is further complicated

by discrepancies between human and animal models For instance, the dogma that cows,horses, sheep and goat, could still maintain normal hemostasis despite their plateletsinability to respond to AA metabolites or epinephrine (17, also discussed in other chapters

in this book) On the contrary, platelets obtained after the cardiopulmonary by-passsurgery (CPBS), not only have diminished number of glycoprotein Ib/lX receptors butalso are highly refractory to thrombin This functional defect seems to offer a reasonableexplanation for reported post-CPBS bleeding Therefore, a general hypothesis can bereadily advanced that a compromise of one or more signaling events may clearly lead to

a defective or diminished response, which then could impact platelet-relatedpathophysiology For instance, there is evidence that altered membrane phospholipidcomposition may lead to the inactivation of a-adrenoreceptor function (59) Studies fromour laboratory have shown that exogenously added phospholipase A2 indeed adverselyaffect the platelet response, particularly to epinephrine (60) Recent studies have shownthat in many malignant and inflammatory conditions plasma phospholipase A2 activitymay be elevated (61) It is therefore reasonable to rationalize that membrane phospholipidand fatty acid changes brought about by disease processes, oxidative stress and agingprocess could indeed contribute to the altered signal-driven cellular mechanisms and thus,

impacting the overall pathophysiology For example, the a-adrenergic receptor whichmediates catecholamine responses, may be compromised by membrane modulation (17).

It is not unreasonable to assume that lipid changes which could readily affect thestereospecific lipid bilayer assembly that may be important for pathophysiologicalresponses However, further studies may be required to document such relationships moreprecisely, linking to signal transducing mechanisms, second messenger systems, calciumfluxes in both human and animal models Furthermore, a careful correlation betweensignal-driven responses and platelet function will lead to a better understanding of plateletpathophysiology (27,31, also discussed in other chapters)

1.6 Platelet pharmacology

Earlier studies on platelet biochemistry, physiology and function suggest that release ofgranule contents is essential for recruitment of platelets, irreversible aggregation andformation of thrombi (31, 62) Discovery of novel vasoactive thromboxanes andprostacyclins from AA, established a crucial link between these AA metabolites andplatelet-endothelial interactions (63,64) As discussed earlier, agonist-mediated activationleads to the stimulation of phospholipases and formation of second messengers thatfacilitate mobilization of cytosolic free calcium, centralization of granules, assembly offilamentous actin, expression of binding sites for adhesion molecules, contraction,secretion of granules and irreversible aggregation (27, 31, 62, 12,13, 14, also discussed

in other chapters) These observations clearly support that the initial receptor-couplingtriggers a complex series of biochemical events that eventually transforms into a functionalresponse Based on these new developments and understanding on specific plateletactivation pathways, many antiplatelet drugs that prevent discrete steps have beendeveloped (62) Early pharmacological approaches have focused on three specific areas:1) the development of drugs that prevent release of AA, and its conversion to variousprostanoids, including receptor antagonists; 2) the development of drugs that stimulateadenylyl and guanylyl cyclase enzymes and that inhibit phosphodiesterase; 3) thedevelopment of calcium antagonists (62, also discussed in other chapters) Studies fromour laboratory have demonstrated that the inhibition of AA metabolism alone isinsufficient to block platelet function (62-79) Furthermore, these studies demonstrate thatirreversible platelet aggregation can be accomplished independent of prostanoids (PGG2,PGH2 and TxA) or released granule contents (ADP, serotonin) (62-75) In separatestudies it has been shown that epinephrine induced membrane modulation can restore thesensitivity of drug-induced refractory platelets to the action of agonists (17) Studies withcalcium specific chelators suggest that platelet responses such as shape change, stickiness,irreversible aggregation and activation caused by extracellular matrix components do notrequire elevation of cytosolic calcium (49)

Some of the clinical complications attributed to increased platelet activity include acutemyocardial infarction, stroke (hemorrhagic and thrombotic), unstable angina, reocclusionfollowing coronary thrombolysis, occlusion during thromboplasty and coronary restenosisand tumor metastasis (8, 10, 12-15, 30, 47) In addition to these clinical situations,platelets are known to contribute to atrial fibrillation, pulmonary embolism, and leftventricular dysfunction in a highly significant manner In this regard, inhibitors of plateletfunction have proven effective in the secondary prevention of clinical vascularcomplications in patients with cardiovascular and cerebrovascular disease (80-87) Theencouraging results obtained with low to moderate doses of aspirin, a known inhibitor of

cyclooxygenase, in patients with coronary artery disease have prompted the use ofantiplatelet drugs in the secondary prevention of cardiovascular events associated withcoronary artery disease A recent review of over 400 clinical trials revealed that use of anyantiplatelet (hug significantly reduced the risk for developing cardiac events associatedwith coronary artery disease (86, also discussed in other chapters) These clinicalobservations have prompted a greater need and urgency to develop new effectiveantiplatelet drugs.

1.7 Platelet function inhibitory drugs

Screening for drugs that prevent platelet activation

Promising new drugs that could affect/prevent one or more platelet responses (i.e.adhesion, spreading, aggregation and secretion of granule contents) can be readilyscreened One of the extensively used methods involves measurement of plateletaggregation by aggregometry Furthermore, one can monitor the secretion of adenosinetriphosphate from the granules concurrent with platelet aggregation response, with theluciferin-luciferase system Alternately, one can use flow cytometry to follow theexpression of granule associated protein (GMP-140; P-selectin; CD 62) on activatedplatelets with fluorescent coupled monoclonal antibodies (88) By using fluorescentcoupled monoclonal antibodies for human fibrinogen one can detect bound fibrinogen onactivated platelets The presence of fibrinogen and the absence of P-selectin wouldindicate platelet activation independent of secretion One can also follow platelet microparticle formation using flow cytometry This newly emerging technique could be usedfor rapid drug screening very effectively

In addition to these techniques developed and used for measuring platelet activation insuspension, one can follow platelet adhesion, spreading and aggregation on various naturaland synthetic surfaces Various test materials can be exposed to platelets in plasma, buffer

or in whole blood If the use of anticoagulants are not preferred, one can draw blooddirectly on to the surface to be tested After the appropriate incubation period, the degree

of activation can be measured by using standard microscopical techniques One can alsouse platelet-specific FITC-coupled monoclonal antibodies or phalloidin rhodamine to stainplatelets on surfaces Such preparations can be imaged using fluorescence microscopy

To some extent, platelet interaction on surfaces is mediated by shear force Therefore it

is essential to screen antiplatelet drugs under flow conditions Two different techniquesare used to monitor platelet interaction on surfaces The classical Baumgartner techniqueemploys denuded rabbit aorta to evaluate platelet interaction with basement membranecomponents (89) The "flat chamber^ technique uses a chamber in which cover slipscoated with various test materials can be exposed to flowing blood (90) One can get alayer of cell matrix components for testing, by growing endothelial cells on cover slips andstripping them off the glass surface after they reach confluency

Once the drugs are screened using appropriate methods, promising new compounds need

to be tested in animal models for their safety, dose and efficacy It is very important toremember that platelets of different species vary widely in their responses to agonists (91,also discussed in other chapters) For instance, majority of dogs have platelets that do notrespond to AA with aggregation (50) However, when these AA refractory platelets are

exposed to epinephrine first they regain their sensitivity to the action of AA and aggregateirreversibly (51) This phenomenon of membrane modulation observed in dog and humanplatelets does not seem to work in many other species Platelets of sheep, goat, cow andhorse do not respond with aggregation when exposed to epinephrine or AA The largevariations observed in platelet responses to aggregating agents that have been reported fordifferent animal species warrant serious consideration in the choice of animal model for

in vivo drug testing

Platelet activation inhibitory drugs

Inhibitors of platelet activation are commonly described as antiplatelet or antithromboticdrugs They can be grouped under the following headings: 1) Cyclooxygenase inhibitors,2) Thromboxane synthetase inhibitors, 3) Receptor antagonists, 4) Adenylyl cyclase/guanylyl cyclase stimulators, 5) Phosphodiesterase inhibitors, 6) Serine proteaseinhibitors, 7) Calcium antagonists, 8) Miscellaneous drugs Some of the knownantiplatelet drugs are listed in Table 1

Most commonly used antiplatelet/antithrombotic drugs are aspirin, nitric oxide donors,dipyridamole, ticlopidine, adenylyl/guanylyl cyclase stimulators, phosphodiesteraseinhibitors, calcium antagonists, heparin and coumadin (62 80-86, also discussed in otherchapters) Aspirin is the most widely used drug for the prevention, treatment andprophylaxis of ischemic heart disease (80-86) Several major clinical trials have beenconducted with various doses of aspirin, ranging from 80 mg to several grams per day (62,86) The outcome of these studies seem to indicate conclusively that antiplatelet drugsprovide significant protection against clinical complications The results with antiplateletdrugs are highly significant in clinical terms even though the protection is not 100 %.However, complex drug-disease interactions could easily explain the effectiveness ofantiplatelet drugs on clinical outcomes

Studies from our laboratory have consistently demonstrated the existence of an intrinsicmechanism that can restore the sensitivity of drug-induced refractory platelets to the action

of agonists We have termed this mechanism as "membrane modulation." This uniquemechanism allows platelets to aggregate irreversibly independent of prostanoids, PAF,

or dense granule contents (17, 49, 65-69) It is mediated by a-adrenergic receptorstimulation and it is calcium dependent It facilitates calcium uptake and fibrinogenbinding Epinephrine-induced restoration of sensitivity seems to be independent ofphosphatidyl inositol metabolism, formation of second messengers and elevation ofcytosolic calcium Studies with calcium chelators (Quin 2 free acid, chlortetracycline,Quin 2 AM) have shown that elevation of cytosolic calcium is not critical for adhesion,spreading or aggregation of platelets (49)

It is apparent that platelets have multiple activation signaling mechanisms Interestingly,currently available antiplatelet drugs do not inhibit platelet activation on cell matrixcomponents or biomaterial surfaces (27, 3, 62) Because, activation of GPIIbMIIa is notrequired for its interaction with surface bound fibrinogen Furthermore, platelets caninteract with other lignads such as laminin, fibronectin, and von Willebrand factor.Therefore, platelet activation inhibitory drugs will only prevent aggreation, secretion ofgranule contents and growth of thrombus formation and not platelet-surface interaction

Table 1 Antiplatelet/Antithrombotic drugs

Benzydamine, Lnidazole congeners, 9, ll-azo-13 oxa-15hydroxy prostanoic acid, 9, ll-azoprosta-5-13 dienoic acid (U-51605), 9, !!(epoxmethano) prostanoic acid, l(isopropyl-2-indloy(l)-3 pyridyl-3-ketone (L-8027)

(n-penylamino)caibonyl 2-Oxazolyl-7-Oxa bicyclo Hept-2-ylmethyl benzenpropanoic acid (BMS 180,291), Vapiprost (SN 309),

13 azaprostanoic acid, Isoprostane, (3 pyridinyl) alkanoic acids,(arylsulfonylamino) alkanoic acid

Prostacyclin, PGEi, PGD2, adenosine, forskolin, coleonol;endothelium derived relaxing factor (EDRF, nitric oxide), cyclases:nitroglycerine, nitroprusside, Sin-1, nitrosoglutathione

Dipyridamole and related compounds, RA233, RA433, VK744,VK774, caffeine, papaverine, aminophylline, theophylline,methylxanthines

Heparin, hirudin, recombinant hirudin, hirudin analogues, peptideantagonists of thrombin receptor Peptide aldehydes (D-phe-pro-arg-H; D-phe-pro-arg-CH2Cl)

cyclic adenine nucleotides (cAMP.cGMP), simulators of adenylyl/guanylyl cyclases, inhibitors of cAMP/cGMP hydrolysis,Verapamil, Nifedipine, Diltiazem, Quin-2AM, BAPTA-AM.Antibiotics, immune suppressive agents, antibodies for specificagonist receptors, synthetic peptide mimetics of interactivedomains of cell matrix components, such as collagen, fibronectin;fibrinogen, ticlopidine, disintegrins, proteins from venoms, proteinsfrom saliva of blood sucking animals and insects

The knowledge gained over the years on platelet biochemistry, physiology andpharmacology has clearly led the development of antiplatelet drugs that can be effective

on specific key biochemical events and thus, in the treatment of thrombotic and otherplatelet-related disorders Earlier pharmacological approach seems to have focused oncompounds that are effective, specifically on platelet aggregation and secretion (62) Thisapproach was used because of the heavy emphasis on the role of secretion of granulecontenets for achieving irreversible aggregation In addition, not much was known about

the mechanisms involved in platelet activation on surfaces.

In the absence of a clear understanding of the various biochemical mechanisms thatunderlie discrete platelet activation sequences, earlier studies concentrated on drugs thatprevented agonist-mediated aggregation and secretion However, there has beenconsiderable progress made over the last few years in the understanding of thebiochemistry of platelet activation (27, 31, also discussed in other chapters) This newknowledge on cell signaling pathways, molecular events associated with ligand receptorinteractions, has given impetus for the development of specific inhibitory compounds andreceptor antagonists For instance, it has been shown that the n-terminal sequence ofamino acids of thrombin receptor can mimic the action of thrombin (92,93) Similarly,

a synthetic "mini collagen" derived from Type IV collagen sequence has been shown topromote platelet activation (94) The amino acid sequence of the recognition site onvarious adhesive proteins for the integrin receptor contains arginine, glycine, and asparticacid (RGD) (39-41, 95) Based on the known sequence of biologically active domainsseveral inhibitory peptides have been synthesized and targeted to block, specifically theaction of thrombin and collagen

Antiplatelet drugs

Aspirin is the most thoroughly studied antiplatelet drug (80-86) It was used as anantiplatelet drug even before its inhibitory effect on cyclooxygenase was recognized (96,97) Aspirin acetylates this enzyme and causes irreversible inhibition of its biologicalactivity (98) Polyenoic acids such as eicospentaenoic acid (EPA) and docosahexaenoicacid (DHA) also impair AA metabolism by cyclooxygenase (99, 100) In addition toaspirin, there are also several other drugs that inhibit this enzyme in platelets Apart fromthe inhibition of this enzyme, prostanoid synthesis could be prevented by blocking therelease of the free fatty acid from the phospholipids Since phospholipase A2 liberates AA,inhibitors of this enzyme can be effective antiplatelet drugs Other commonly usedantiplatelet drugs include dipyridamole (persantin) and ticlopidine (thienopyridines: Ticlid,Clopidogrel), nitric oxide donors, and calcium antagonists (62,101-110, also discussed inother chapters) Dipyridamole is a vasodilator and it has been used with aspirin in severalclinical trials This drug has also been used with warfarin (coumadin) in antithrombotictherapy Advances in biotechnology and in biochemical separation techniques have ledthe way in understanding the nature of ligand-receptor interacting domains and cellactivation signaling mechanisms Undoubtedly, these new appreaches in drug designinghave impacted the development of newer antiplatelet and antithrombotic drugs (110) Theuse of antiplatelet drugs has become an important therapeutic modality for the prevention

of acute arterial thromboembolic occlusions (112) There is sufficient scientific base now

to conclude that antiplatelet drugs are clinically effective in significantly lowering the risk

of developing cardiovascular events associated with coronary artery disease (86,) Aspirinremains as the drug of choice for the treatment of coronary artery disease (CAD) There

is sufficient evidence that low-to-moderate dose of aspirin is as effective as any otherantiplatelet drug in reducing the risk and clinical complications associated with CAD (80-83,109)

Antithrombotic drugs

Antithrombotic drugs have been in use clinically for over half a century (110-114,116)

The first clinical trial was reported in 1948 In the same year, the American HeartAssociation recommended the use of anticoagulant therapy for the treatment of coronarythrombosis.(l 12) Heparin was one of the earliest antithrombotic drugs available fortherapeutic use Dicoumarol and warfarin became available for therapeutic use later Inthe early years, there was considerable use of anticoagulants However, there has beenconsiderable shift towards the use of antiplatelet drugs in the treatment of CAD, in view

of the large body of evidence available to implicate platelet hyperactivation in thepathophysiology of cardiovascular events Coronary thrombus is by and large platele-rich.Earlier studies from our laboratory demonstrated that preformed aggregates can bedissociated by antiplatelet drugs(117) In these studies none of the thrombolytic drugscaused dissociation of platelet aggregates After several clinical trials and thedevelopment of newer atniplatelet drugs( GPl lb\l 1 Ia antagonists) our earlier observationhas been confirmed (118) It is important to note that platelet activation, thrombingeneration and fibrin polymerization play a critical role in the development of thrombus.Therefore, antiplatelet drugs, antithrombotic compounds and thrombolytic agents areimportant in the treatment of coronary thrombosis (62,109-114,117)

New antithrombotic drugs represent a wide spectrum of natural, synthetic, semisyntheticcompounds produced by new biotechnology techniques, with marked differences in theirchemical composition, properties, biochemical and pharmacological actions (110-114).Hirudin is a single chain carbohydrate free polypeptide Unlike heparin it can inactivatethrombus bound thrombin It is a potent inhibitor of thrombin and binds with high affinity

at the substrate binding site Hirulog (bivalirudin) is a bifunctional 20-amino acid peptidedeveloped to mimic the interactive domain of hirudin Argatroban is an arginine-derivative which inhibits thrombin with great affinity and is considered a better inhibitor

of thrombin action than heparin Many more inhibitors of thrombin action are availableincluding napasgatran (R046-6240), inogatran (pINN), efegatran sulfate (Ly294468), DuP714 and low molecular weight heparins (110-114) Badimon et al in a recent reviewconcluded that the unique effects of specific thrombin inhibitors, including thrombinaction on platelets and blood coagulation, demonstrated in experimental and preliminaryclinical trials, suggest that specific thrombin inhibitors may represent a new generation ofdrugs in antithrombotic therapy of acute coronary syndromes (113)

Newer antiplatelet drugs include several receptor antagonists (62,109) Platelet activationleads to the expression of binding sites for fibrinogen binding on platelet GPIIb/IIIareceptors(42-45) Activated GPl lb/11 Ia receptors bind fibrinogen and promote adhesion

on surfaces as well as aggregation of platelets This receptor is promiscuous and binds notonly fibrinogen but also vonWillebrand factor, fibronectin, vitronectin laminin andthrombospondin These adhesion molecules have a common recognition site containingarginine-glycine-aspartic acid (RGD) This observation has prompted the development

of a variety of antagonistic peptides Monoclonal antibodies, murine or chimeric 7E3(c7E3 Fab: abciximab, ReoPro) are the first receptor antagonists that have been studied

in humans A variety of RGD mimetics have been synthesized and tested in animals andhumans Blockade of the GPl lb/11 Ia receptor leads to effective inhibition of adhesion,platelet aggregation and thrombus formation promoted by fibrinogen (42,43,95) Thesedrugs are specific for the GPl lb/111 a receptor and do not interfere with the action ofreceptors for collagen, or von Willebrand factor

Newer orally active non-peptide antiplatelet/antithrombotic drugs are currently being

developed (109-116) Lamifiban (R044-9883) and tirofiban(MK-383) have been shown

to inhibit platelet aggregation at micromolar concentrations Tirofiban has been tested inhuman volunteers The compound has been shown to be four orders of magnitude moreselective for antagonizing GPl lb/11 Ia than the natural ligand, vitronectin Lamifiban andtirofiban are the first non-antibody/non-peptide antagonists that have been subjected tolarge human clinical trials (109) When thrombin binds to fibrin or is associated withthrombus, it is relatively impervious to the action of heparin, the widely usedanticoagulant Direct thrombin inhibitors like D-Phe-L-Pro-L-Arg-CH2Cl(PPACK),hirudin, hirulog and others inhibit bound thrombin as effectively as free thrombin Recentclinical trials suggest that direct thrombin inhibitors and specific platelet receptorantagonists may be the choice of drugs for antiplatelet and antithrombotic therapy (62,109-116)

In conclusion, the concept of platelet activation and its relevance to health and disease, hasevolved methodically over the last 2-3 decades, with greater understanding of biochemicalpathways These developments have clearly led to the defining of two major biochemicalpathways, which are now impacting the CAD prevention and treatment strategies throughnew drug discoveries in a highly significant way The first being the phosphoinositolmetabolism leading to the formation of second messengers, DAG, IP3, and elevation ofcytosolic calcium The second pathway is mediated by the increase in ionized calcium,causing phospholipase A2 activation and liberation of AA Free AA is then converted tocyclic endoperoxides and thromboxanes The second messengers produced by these twobiochemical pathways appear to be involved in the modulation of a number of functionalresponses, including assembly of filamentous actin, centralization of granules, secretion

of granule contents and expression of binding sites for fibrinogen Based on theknowledge and understanding, many antiplatelet drugs that inhibit prostanoid synthesis,platelet aggregation and secretion of granule contents, have been developed over the lastfew years However, the relationship between signal-driven biochemical events andplatelet interaction with cell matrix components, injured vascular surface and biomaterialsurfaces, remains a highly challenging field for further investigations In any event,studies on newer antiplatelet and antithrombotic drugs have provided a great deal ofexcitement and have led to the discovery of many specific antagonists Although thebiochemistry of platelets remains highly complex, we now have well defined pathwaysthat could impact platelet functional responses It is very likely that further understanding

of the role of these pathways and the specific mechanisms that underlie platelet activation

on surfaces will lead to the development of newer and more specific anti-platelet drugswith greater efficacy and safety The future for newer antiplatelet and antithromboticdrugs looks bright In this regard, the combined therapeutic approaches should prove to

be highly valuable in the treatment of coronary thrombosis, which is still a number onekiller in the United States and in other industrialized countries The compiled chapters inthis book are comprehensive reviews written by authorities in their own area and therefore,the book should serve as an invaluable source of information for readers, includingbiochemists, pharmacologists, pharmacists, drug companies, academicians and many othergroups

1.8 Acknowledgements

The author is thankful to thank Mrs Anupama R Tate and Dr Mahadev Murthy for theirhelp in the preparation of this manuscript This work was supported by grants from NIH

HL-18880, Laboratory Medicine & Pathology and Biomedical Engineering Institute.

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HUMAN PLATELETTHROMBIN RECEPTORS AND THE

TWO RECEPTOR MODEL FOR

PLATELET ACTIVATION

G A Jamieson, Narendra N Tandon* and

Nicholas J GrecoAmerican Red CrossOtsuka America Pharmaceutical, Inc.*

2.1 INTRODUCTION

The interaction of proteolytically-active a-thrombin with platelets and other cells of thevasculature, such as endothelial cells and smooth muscle cells, plays a major role in bothnormal hemostasis and atherosclerosis.1'2 Despite extensive studies in numerouslaboratories extending back over thirty years, major questions regarding the mechanism

of these interactions remain unresolved Furthermore, since thrombin can also inducechemotaxis and adhesion of inflammatory cells, and fibroblast mitogenesis, the importance

of elucidating the nature of its receptor, or receptors, extends far beyond its role in plateletactivation However, this review will be restricted mainly to considerations of thrombinreceptors in human platelets

The authors have collaborated for

many years in studies on platelet

activating receptors, including not only

receptors for a-thrombin but also

those for collagen (CD36 and GPVI,

laminin (67kDA), and adenosine

diphosphate.

The interaction of thrombin with platelets causesincreases in cytoplasmic Ca2+, shape change andthe conversion of prothrombin to thrombin viathe prothrombinase complex leading to furtherplatelet activation, aggregation and secretion.The concentration of free a-thrombin in plasmafollowing physiological activation has beendetermined to be in the range of 0.5-2nM due to