Hemostasis and Thrombosis E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 588

Veröffentlichungsjahr: 2014

Beschreibung

Hemostasis and Thrombosis: Practical Guidelines in Clinical Management

Edited by

Hussain I Saba MD, PhD

Professor of Medicine

Director of Hematology/Hemophilia/Hemostasis & Thrombosis Center

USF College of Medicine

Professor Emeritus

Department of Malignant Hematology

Moffitt Cancer Center and Research Institute

Tampa, FL, USA

Harold R Roberts MD

Emeritus Professor of Medicine and Pathology

Division of Hematology / Oncology

University of North Carolina

Chapel Hill, NC, USA

Hemostasis and thrombosis is a hugely important, changing and complex area that impacts many other medical specialties, affecting at least 10 million people in the United States alone. It is vital for physicians to recognize abnormalities of hemostasis and thrombosis and to be able to address these problems in their patients.

Hemostasis and Thrombosis: Practical Guidelines in Clinical Managementcovers all aspects of the practical
management of commonly encountered thrombotic and bleeding disorders, with emphasis on clinical diagnosis, treatment, and day-to-day management. The most clinically relevant material from the literature has been carefully compiled, along with:

 - Discussions of new and upcoming diagnostic and therapeutic modalities

- Practical clinical advice for all those dealing with coagulation, hemostasis and thrombosis

Written by world leading specialists contributing their expertise in succinct text and focusing on the latest clinical guidance, Hemostasis and Thrombosis: Practical Guidelines in Clinical Management will benefit hematologists in training and in practice working in the fields of coagulation, hemostasis and thrombosis.

Titles of related interest

Practical Hemostasis and Thrombosis, 2nd Edition

Key, ISBN 9781405184601

Hemophilia and Hemostasis: A Case-Based Approach to Management, 2nd Edition

Ma, ISBN 9780470659762

Quality in Laboratory Hemostasis and Thrombosis, 2nd Edition

Kitchen, ISBN 9780470671191

 www.wiley.com/go/hematology

Leseprobe

Table of Contents

Title page

Copyright page

Contributors

Preface

Acknowledgments

CHAPTER 1: Theories of Blood Coagulation: Basic Concepts and Recent Updates

Historical background

Cell-based model of coagulation

Clinical assays

Summary

CHAPTER 2: Vascular Endothelium, Influence on Hemostasis: Past and Present

Introduction

Function of endothelial cells

Antiplatelet factors

Anticoagulant factors

Fibrinolytic factors

Contribution of endothelium in hemostasis

Contribution of endothelium in thrombosis

Endothelial cell dysfunction: pathophysiology and biology

CHAPTER 3: Coagulation Testing: Basic and Advanced Clinical Laboratory Tests

Introduction

Blood sampling for diagnosis of hemostatic disorders

Screening for bleeding disorders

Specific blood coagulation factor analyses for diagnosis of bleeding disorders

Screening for thrombotic disorders

Synthetic peptide chromogenic substrates for hemostatic assays

Global assays

The fibrinolytic system

CHAPTER 4: Factor VIII Deficiency or Hemophilia A: Clinical Bleeding and Management

Introduction

Clinical manifestations of hemophilia A

Management of hemophilia A

CHAPTER 5: Factor IX Deficiency or Hemophilia B: Clinical Manifestations and Management

Introduction

Genetics and molecular biology of factor IX deficiency

Clinical manifestations of hemophilia B

Carrier detection and prenatal diagnosis

Laboratory findings

Differential diagnosis

Inhibitors to factor IX

Specific treatment of factor IX deficiency

Therapies in development

CHAPTER 6: Factor XI Deficiency or Hemophilia C

History

Factor XI structure and function

Congenital factor XI deficiency: inheritance pattern

Other congenital and acquired causes of factor XI deficiency

Clinical presentation

Laboratory diagnosis

Treatment

Factor XI and thrombosis

CHAPTER 7: Factor VIII and IX Inhibitors in Hemophilia

Introduction

Definition of inhibitors

Laboratory diagnosis of inhibitors in hemophilia

Kinetics of factor VIII inactivation

Factor VIII gene and inhibitor characteristics

Factor IX gene and inhibitor characteristics

Risk factors for inhibitor development

Management of patients with factor VIII and factor IX inhibitors

Immune tolerance induction for the eradication of inhibitors

Conclusion

CHAPTER 8: Treatment Options for Acquired Hemophilia

Introduction

Epidemiology

Etiology

Clinical presentation

Laboratory diagnosis

Treatment options for acquired hemophilia

Relapse

New approaches to the management of factor VIII autoantibodies

CHAPTER 9: Factor XII Deficiency or Hageman Factor Deficiency

Introduction

Factor XII structure

Regulation of factor XII expression

Role of factor XII in hemostasis and thrombosis

Role in inflammation

Summary

CHAPTER 10: Inherited Combined Factor Deficiency States

Introduction

Combined inherited factor V and factor VIII deficiency

Vitamin K-dependent clotting factor deficiency

Inheritance of multiple single-factor deficiencies

CHAPTER 11: Acute and Chronic Immune Thrombocytopenia: Biology, Diagnosis, and Management

Introduction

Primary ITP in adults

ITP in children

Treatment of ITP in emergency

ITP in pregnancy

CHAPTER 12: Disseminated Intravascular Coagulation: Diagnosis and Management

Introduction

Etiology

Pathophysiology

Diagnosis

Clinical consequences

Treatment modalities and evolving therapeutics

Evolving therapeutics

CHAPTER 13: Mechanisms of Fibrinolysis and Basic Principles of Management

Biochemical process of fibrinolysis and its regulation

Plasminogen and plasmin

Plasminogen activators

Modulators of fibrinolysis

Cell surface “fibrinolytic” receptors

Pathologic fibrinolysis

Thrombolytic therapy

CHAPTER 14: Post-thrombotic Syndrome

Definition and diagnosis of post-thrombotic syndrome

Incidence

Pathophysiology

Risk factors for post-thrombotic syndrome

Socioeconomic impact

Therapeutic management of post-thrombotic syndrome

CHAPTER 15: Von Willebrand Disease: Clinical Aspects and Practical Management

The von Willebrand factor and its laboratory measurement

Classification of von Willebrand disease

Clinical manifestations

Diagnosis of von Willebrand disease

Management of patients with von Willebrand disease

Conclusions

Acknowledgments

CHAPTER 16: Platelets in Hemostasis: Inherited and Acquired Qualitative Disorders

Thrombopoiesis

Platelet structure

Platelet physiology

Evaluation of platelet disorders

Disorders of platelets

Inherited platelet disorders

Acquired platelet disorders

Summary

CHAPTER 17: Contributions of Platelet Polyphosphate to Hemostasis and Thrombosis

Introduction

Platelet polyP

PolyP and the contact pathway of blood clotting

Acceleration of thrombin generation by polyP

Effects of polyP on fibrin clot structure and fibrinolysis

Platelet polyP and the role of factor XI in normal hemostasis

PolyP in thrombosis and inflammation

PolyP as a potential drug target

Conclusions and future directions

Acknowledgments

CHAPTER 18: Thrombotic Microangiopathy: Biology, Diagnosis, and Management

History and pathophysiology

Diagnosis

Treatment: thrombotic thrombocytopenic purpura

Treatment: hemolytic uremic syndrome

Thrombotic thrombocytopenic purpura in pregnancy

CHAPTER 19: Hemostasis and Aging

Introduction

Clinical definition of aging

Interactions of aging and hemostasis

Conclusions

CHAPTER 20: Hemostatic Problems in Chronic and Acute Liver Disease

Introduction

Laboratory tests of hemostasis in patients with liver disease

The concept of rebalanced hemostasis in liver disease

Bleeding in liver disease

Thrombosis in liver disease

Conclusion

CHAPTER 21: Cancer and Thrombosis

Introduction

Prevention of venous thromboembolism in cancer patients

Treatment of cancer-associated thrombosis

CHAPTER 22: An Update on Low-Molecular-Weight Heparins

Introduction

Available low-molecular-weight heparins

Approved clinical use of low-molecular-weight heparins

Generic low-molecular-weight heparins

Ultra-low-molecular-weight heparins

Synthetic heparin derivatives

Other related agents

Neutralization of low-molecular-weight heparin and fondaparinux

Contamination of low-molecular-weight heparin

Additional clinical investigation of low-molecular-weight heparin

Low-molecular-weight heparin in special patient populations

Low-molecular-weight heparins in elderly patients

Low-molecular-weight heparins in patients with inflammatory bowel disease

Monitoring of low-molecular-weight heparins: prophylactic and therapeutic dosages

Newer anticoagulants and low-molecular-weight heparin

Conclusion

Supplemental Images

Index

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Library of Congress Cataloging-in-Publication Data

Hemostasis and thrombosis (Saba)

Hemostasis and thrombosis : practical guidelines in clinical management / edited by Hussain I. Saba, Harold R. Roberts.

p. ; cm.

Includes bibliographical references and index.

ISBN 978-0-470-67050-7 (pbk.)

I. Saba, Hussain I., editor of compilation. II. Roberts, H. R. (Harold Ross), editor of compilation. III. Title.

[DNLM: 1. Blood Coagulation Disorders–therapy. 2. Hemostatic Disorders–therapy. 3. Thrombosis–therapy. WH 322]

RC647.C55

616.1'57–dc23

2013042714

A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

Cover image: iStock photo. File #16045826 © Eraxion

Cover design by Meaden Creative

Contributors

Louis Aledort MD, MACP

The Mary Weinfeld Professor of Clinical Research in Hemophilia

Division of Hematology and Medical Oncology

The Tisch Cancer Institute

Mount Sinai School of Medicine

New York, NY, USA

Noman Ashraf MD

Assistant Professor

Department of Hematology/Oncology

University of South Florida/James A. Haley VA Hospital

Tampa, FL, USA

Lodovico Balducci MD

Professor of Oncologic Sciences

H. Lee Moffit Cancer Center & Research Institute

Tampa, FL, USA

Charles E. Bane Jr. DVM

Department of Pathology, Microbiology, and Immunology

Vanderbilt University

Nashville, TN, USA

Margareta Blombäck MD, PhD

Professor Emeritus

Department of Molecular Medicine and Surgery

Division of Clinical Chemistry and Blood Coagulation

Karolinska Institutet

Stockholm, Sweden

Giancarlo Castaman MD

Consultant Hematologist

Department of Cell Therapy and Hematology

Hemophilia and Thrombosis Center

San Bortolo Hospital

Vicenza, Italy

Meera Chitlur MD

Associate Professor of Pediatrics and Director of Hemophilia/Hemostasis Clinic

Wayne State University

Children's Hospital of Michigan

Detroit, MI, USA

Samir Dalia MD

Fellow in Hematology and Oncology

H. Lee Moffitt Cancer & Research Center

University of South Florida

Tampa, FL, USA

Stephanie J. Davis, MD

Resident

Department of Internal Medicine

University of North Carolina Hospitals

Chapel Hill, NC, USA

Benjamin Djulbegovic MD, PhD

Distinguished Professor

University of South Florida & H. Lee Moffitt Cancer Center & Research Institute

Tampa, FL, USA

Nils Egberg MD, PhD

Associate Professor

Department of Molecular Medicine and Surgery

Division of Clinical Chemistry and Blood Coagulation

Karolinska Institutet

Stockholm, Sweden

Jawed Fareed PhD

Professor of Pathology & Pharmacology

Hemostasis & Thrombosis Research Laboratories

Loyola University Chicago

Maywood, IL, USA

Massimo Franchini, MD

Director

Department of Transfusion Medicine and Hematology

C. Poma Hospital

Mantova, Italy

David Gailani MD

Professor of Pathology, Microbiology and Immunology

Division of Hematology/Oncology

Vanderbilt University

Nashville, TN, USA

Jean-Philippe Galanaud MD

Assistant Professor of Vascular Medicine

Clinical Investigation Centre and Department of Internal Medicine

Montpellier University Hospital

Montpellier, France

David Green MD, PhD

Professor Emeritus of Medicine

Feinberg School of Medicine

Northwestern University

Chicago, IL, USA

Maureane Hoffman MD, PhD

Professor of Pathology

Duke University Medical Center

Durham, NC, USA

Debra Hoppensteadt PhD

Professor of Pathology and Pharmacology

Loyola University Medical Center

Maywood, IL, USA

Walter P. Jeske PhD

Professor of Thoracic and Cadiovascular Surgery

Loyola University Medical Center

Maywood, IL, USA

Susan R. Kahn MD, MSc, FRCPC

Professor of Medicine

Division of Internal Medicine

Lady Davis Institute, Jewish General Hospital

Department of Medicine

McGill University

Montreal, QC, Canada

Raj S. Kasthuri MD

Associate Professor

Department of Medicine

Division of Hematology/Oncology

University of North Carolina at Chapel Hill

Chapel Hill, NC, USA

Craig M. Kessler MD, MACP

Professor of Medicine and Pathology

Director, Division of Coagulation

Division of Hematology and Oncology

Lombardi Comprehensive Cancer Center

Georgetown University Medical Center

Washington, DC, USA

Asma Latif MD

Clinical Fellow

Division of Hematology and Medical Oncology

The Tisch Cancer Institute

Mount Sinai School of Medicine

New York, NY, USA

Agnes Y. Y. Lee MD, MSc, FRCPC

Medical Director, Thrombosis Program

Associate Professor of Medicine Division of Hematology

University of British Columbia

British Columbia Cancer Agency

Vancouver, BC, Canada

Ton Lisman PhD

Associate Professor

Section of Hepatobiliary Surgery and Liver Transplantation

Department of Surgery

University Medical Center Groningen

University of Groningen

Groningen, The Netherlands

Rustem I. Litvinov MD, PhD, DrSci

Senior Research Investigator

Department of Cell and Developmental Biology

University of Pennsylvania Perelman School of Medicine

Philadelphia, PA, USA

Jeanne M. Lusher MD

Distinguished Professor of Pediatrics and Marion I Barnhart Chair in Hemostasis Research

Wayne State University

Children's Hospital of Michigan

Detroit, MI, USA

Pier Mannuccio Mannucci MD

Scientific Director

IRCCS Cà Granda Foundation

Maggiore Policlinico Hospital

Milan, Italy

Paul E. Monahan MD

Associate Professor

Department of Pediatrics

Division of Hematology/Oncology

University of North Carolina at Chapel Hill

Chapel Hill, NC, USA

Dougald M. Monroe, PhD

Professor

University of North Carolina at Chapel Hill

School of Medicine

Division of Hematology/Oncology

Chapel Hill, NC, USA

James H. Morrissey PhD

Professor

Department of Biochemistry

University of Illinois at Urbana-Champaign

Urbana, IL, USA

Anne T. Neff MD

Associate Professor

Department of Medicine Division of Hematology/Oncology

Vanderbilt University

Nashville, TN, USA

Erica A. Peterson MD, MSc

Fellow, Thrombosis Program

Division of Hematology

University of British Columbia and Vancouver Coastal Health

Vancouver, BC, Canada

Robert J. Porte MD, PhD

Professor of Surgery

Section of Hepatobiliairy Surgery and Liver Transplantation

Department of Surgery

University Medical Center Groningen

University of Groningen

Groningen, The Netherlands

Francesco Rodeghiero MD

Director

Department of Cell Therapy and Hematology

San Bortolo Hospital

Vicenza, Italy

Sabiha R. Saba MD

Associate Professor

Department of Pathology and Cell Biology

USF College of Medicine

Tampa, FL, USA

Alvin H. Schmaier MD

Robert W Kellemeyer Professor of Hematology/Oncology

Division of Hematology and Oncology

Department of Medicine

Case Western Reserve University

Cleveland, OH, USA

Anjali A. Sharathkumar MD, MS

Assistant Professor

Director, Hemophilia and Thrombophilia Program

Northwestern University Feinberg School of Medicine

Chicago, IL, USA

Evi X. Stavrou MD

Assistant Professor

Division of Hematology and Oncology

Department of Medicine

Case Western Reserve University

Cleveland, OH, USA

Alberto Tosetto MD

Consultant Hematologist

Department of Cell Therapy and Hematology

Hemophilia and Thrombosis Center

San Bortolo Hospital

Vicenza, Italy

John W. Weisel PhD

Professor

Department of Cell & Developmental Biology

University of Pennsylvania PerelmanSchool of Medicine

Philadelphia, PA, USA

Preface

Since the discovery and early concept of hemostasis and thrombosis, there has been a progressive and remarkable change in the ongoing availability of knowledge in this area. The available knowledge has become extensive and dynamic. The information has led to the understanding of the formation of the steps involved in blood clotting reactions. Advances and understanding in the management of bleeding diseases has led to safe management of diseases such as hemophilia, von Willebrand Disease (VWD) and other hemophoid disorders; appropriate management of inhibitors has been achieved. Alterations in hemostasis and thrombosis have been investigated in the pathogenesis of liver disease as well as cancer. The important influence of platelet polyphosphates on hemostasis and thrombosis has also been explored. Significant advances have been made in the proper use of anticoagulation agents. Critical knowledge also led to advances in the area of disseminated intravascular coagulation (DIC) as well. This book presented here, Hemostasis and Thrombosis: Practical Guidelines in Clinical Management, represents the current understanding of important European and American academics on the subject.

Hussain I. Saba

Tampa, Florida, USA

Acknowledgments

First of all, I thank Almighty God who gave me the strength and wisdom to complete this book.

I wish to acknowledge my gratitude and sincere appreciation to Dr. Harold R. Roberts at UNC Chapel Hill, NC, my friend, advisor, and colleague, for his support in the completion of this book. Our long-term academic association has offered me the opportunity to learn not only some of the principles of scientific research but also, simultaneously, the value of critical thinking. I am also in debt to him for many conversations pertaining not only to science but also to the broad aspect of human life and the human condition. From these discussions it has become apparent, to me at least, that science cannot encompass the real world and part of reality lies also in the realm of metaphysics. The realization has, perhaps, influenced both of us more than we know. I would like to acknowledge Sabiha R. Saba, Hasan I. Zeya and John C. Herrion for their support of my pursuit of knowledge in the area of hematology and hematological research.

My sincere thanks to Genevieve Morelli for her kind support on this project. Her organizational skills and efforts in contacting, proofing, and coordinating the work of the authors, editors, and publisher of this work have been remarkable and appreciated.

I would also like to thank Rukhsana Azam and Monique Johnson for the help and support I received in the development and publication of this book.

Hussain I. Saba

Tampa, Florida, USA

CHAPTER 1

Theories of Blood Coagulation: Basic Concepts and Recent Updates

Dougald M. Monroe1 and Maureane Hoffman2

1 University of North Carolina at Chapel Hill, School of Medicine, Division of Hematology/Oncology, Chapel Hill, NC, USA

2 Duke University, Department of Pathology, Durham VA Medical Center, Durham, NC, USA

Historical background

Any mechanistic description of blood coagulation should account for a number of simple observations about the blood coagulation process. Blood that is circulating inside the body tends not to clot. However, blood that escapes from the vasculature does clot. This suggests that there is a material outside blood that is necessary for the clotting process. This point was emphasized by the study of Foà and Pellacani who showed that “tissue juice” (filtered saline extract of brain), when injected into the circulation of a rabbit, could cause intravascular thrombus formation [1]. This result was further clarified by Macfarlane and Biggs who showed that blood had all the factors needed to clot (intrinsic factors) but that this process was slow and that clotting was accelerated by the addition of tissue extracts (extrinsic factors) [2].

A clotted mass of blood was called a thrombus. When this thrombus was washed, a material, thrombin, could be eluted that would immediately clot fresh blood. It was further shown that there existed in blood an inactive agent, prothrombin, which could be converted to active thrombin. The agent responsible for this conversion was called thromboplastin (or thrombokinase). Attempts to discover the nature of thromboplastin led to much of our current mechanistic understanding of coagulation.

In 1875, Zahn made the important observation that bleeding from a blood vessel was blocked by a white (not red) thrombus [3]. Bizzozero and Hayem, working separately, studied a colorless corpuscle in blood called a thrombocyte or platelet [4,5]. This cell could be shown to be associated with fibrin and was postulated to be a major component of the white thrombus [4]. It was therefore suggested that there was a platelet thromboplastin that was critical for clotting (in modern usage, platelet procoagulant function is described as such and the term thromboplastin is used to mean the protein tissue factor [TF] which is the coagulation initiator in tissues).

It is known that in some families there is an inherited bleeding tendency (hemophilia). Eagle studied individuals with this disorder and showed that the platelet function in those patients was normal but that there was still a deficiency in prothrombin conversion [6]. This established that there was a plasma component required for clotting in addition to a requirement for platelets. Further studies in patients with different bleeding tendencies established that there are a number of elements that make up the plasma clotting component. Because these factors were discovered by multiple investigators in different parts of the world (and given a different name by each group), a systematic nomenclature was established using Roman numerals [7] (Table 1.1).

Table 1.1 Systematic nomenclature of clotting factors.

Factor*CommentsIFibrinogenIIProthrombinIIILipid, platelet surface, or Thromboplastin (not used)IVCalcium (not used)VVIActivated factor V (not used)VIIVIIIHemophilia A factorIXHemophilia B factorXXIHemophilia C factorXII

*Activated forms of the factor are indicated by appending the letter “a” to the name.

While studies on deficient plasmas had established what the important components were, the mechanisms of action and the interactions between these components were not immediately clear. Early coagulation schemes started from the model of prothrombin being converted to thrombin and visualized all of the circulating coagulation proteins as zymogens that were converted during coagulation into active enzymes [8,9]. Once the proteins involved in coagulation were isolated and their structure and functions were studied, it became clear that coagulation function was organized around a mechanism of an active enzyme being paired with a cofactor [10]. In the absence of the cofactor, the enzyme has limited activity; typically a cofactor will accelerate the activity of a coagulation enzyme as much as 1000-fold [11]. Thus, each step in coagulation is regulated at two levels: 1) activation of the zymogen to an active enzyme and 2) the presence of (and sometimes activation of) the requisite cofactor. Since some cofactors, such as thromboplastin (tissue factor) and thrombomodulin, are integral membrane proteins, the functions of these complexes can be limited to cells and tissues that express the protein (Table 1.2).

Table 1.2 Composition and physiologic location of coagulation complexes.

The coagulation factors show only weak activity in solution, and binding to an appropriate cell surface accelerates their activity up to 1000-fold. This surface binding is dependent on calcium and, therefore, blood can be anticoagulated by the addition of chelating agents such as citrate or EDTA that bind calcium [12]. This chelation does not alter protein properties and can be readily reversed by reintroduction of calcium in excess of the chelating agents. Clinical assays use plasma prepared from blood chelated with citrate to analyze clotting factor function by addition of an appropriate activator and calcium and measuring the time to clot formation.

Localization of the coagulation reactions to a desired surface represents a powerful mechanism for limiting coagulation to surfaces at the site of injury. One component of coagulation factor binding to cells is the phospholipid composition of the outer leaflet of the cell membrane. Phospholipids with acidic head groups, phosphatidic acid (PA) and phosphatidylserine (PS), promote binding of coagulation factors [13]. In addition, phosphatidylserine acts as an allosteric regulator of function and accounts for the ability of PS-containing membranes to enhance coagulation factor activity [14]. While generic phospholipid surfaces can support coagulation reactions (and are used in clinical assays), it is clear that cells, in addition to having appropriate lipid surfaces, have regulatory elements that control the coagulation reactions [15,16].

Functional platelets are required as a surface for hemostasis, and patients with low platelet counts (thrombocytopenia) or platelet function defects (thrombocytopathia) such as Bernard–Soulier syndrome or Glanzmann's thrombasthenia have a bleeding tendency. Circulating platelets, like essentially all cells in blood as well as endothelial cells, have outer membranes with low levels of acidic phospholipids. When platelets adhere at a site of injury the composition of their membrane changes such that acidic phospholipids, including phosphatidylserine, are now expressed on the outer surface of the membranes [17]. This change in surface lipid composition, along with changes in platelet surface proteins and release of procoagulant factors from platelet granules, provides a surface that supports robust coagulation.

Cell-based model of coagulation

In a mild injury, the coagulation process starts with hemostatic platelet aggregates which can be found at the ends of transected blood vessels [18]. Early in the process, these aggregates consist of activated (degranulated) platelets packed together. In time, small amounts of fibrin are deposited between the platelets. At longer times more fibrin becomes associated with the platelet masses. This fibrin extends into the tissues and provides stability to the area of injury [18,19].

In hemophilia A or B, the process is somewhat different [20]. Early in the process, the platelets are loosely associated but are not activated. Even at longer times platelets are only poorly activated and fibrin is not seen between the platelets. The result is that the platelet mass is not stabilized. Whereas normal individuals show extensive fibrin extending into the tissues, in hemophilia a thin layer of fibrin can be seen only at the margins of the wound area and does not extend significantly into the tissues [19,20].

These observations of hemostasis suggest that coagulation can be conceived of as a series of overlapping steps: initiation; amplification; and propagation.

Initiation

Blood coagulation is initiated by an injury to a blood vessel; this injury could be a denudement of some of the endothelium or a break in the vessel. In either case, two processes begin immediately. One process is that platelets quickly adhere to the site of injury. This adherence requires von Willebrand factor which binds to both collagen in the exposed subendothelium and the abundant platelet protein glycoprotein Ib. This adherence brings platelets into contact with collagen which, through the platelet collagen receptor glycoprotein VI, activates platelets [21]. This activation causes changes in platelet surface receptors and leads the platelets to degranulate. Degranulation releases a number of stored proteins including a partially activated form of factor V [22].

The second process that begins with a break in the vasculature is that plasma concentrations of coagulation proteins are brought into the area of injury and presented to extravascular cells. Cells surrounding the vasculature tend to be rich in the protein called tissue factor (thromboplastin); the high concentration of tissue factor around blood vessels has been described as contributing to a hemostatic envelope [23]. At least some of the tissue factor already has factor VII bound [24] and factor VII binds tightly to any free tissue factor. On cells this tissue factor-bound factor VII is rapidly converted to factor VIIa. This conversion can be via cellular proteases, by autoactivation by other factor VIIa molecules, or by factor Xa generated by factor VIIa–tissue factor complexes [25,26].

These factor VIIa–tissue factor complexes catalyze two reactions: activation of factor X and activation of factor IX [27]. The factor Xa that is formed can complex with the partially active factor V released from platelets; this factor Xa–Va complex converts at least some prothrombin to thrombin. Formation of factor Xa also starts the process of regulating coagulation. The inhibitor TFPI (tissue factor pathway inhibitor) can bind to factor Xa and factor VIIa to turn off the factor VIIa–tissue factor complex [28,29]. This inhibition requires factor Xa so that the factor VIIa–tissue factor complex is not turned off until some factor Xa has been formed. Factor Xa in a complex with factor Va is protected from the abundant plasma inhibitor antithrombin, but once released from the complex, factor Xa inhibition by antithrombin is rapid with an expected half-life of about 4 minutes.

Amplification

The initial thrombin formed during the initiation phase is probably not sufficient to provide for robust fibrin formation and hemostasis. However, the thrombin formed on the initiating cell can transfer to platelets where the initial hemostatic signal is amplified by activating platelets and cofactors. On the platelet surface, thrombin is relatively protected from inhibition by antithrombin (in plasma, thrombin has a half-life of just over 1 minute). Thrombin can bind to at least two receptors on the platelet surface: glycoprotein Ib and protease-activated receptor (PAR)-1 [30,31]. Thrombin binding to and cleavage of PAR-1 transmits signals that lead to platelet activation (outside-in signals) [32]. This activation results in changes in the surface lipid content with increased exposure on the outer leaflet of acid phospholipids [17]. Activation also leads to inside-out signals that alter the conformation and function of some surface proteins including the fibrin binding protein complex of glycoproteins IIb and IIIa [33]. Activation also results in release of internal stores of a number of components from alpha granules and dense granules. The released components include partially active factor V, fibrinogen, ADP which acts as signal for further platelet activation, and polyphosphates [34,35].

Thrombin bound to glycoprotein Ib can cleave PAR-1 and PAR-4 [36]. This thrombin can also cleave factor VIII, releasing factor VIIIa onto the platelet surface. Factor VIII circulates in a complex with von Willebrand factor [37]; since both thrombin and von Willebrand factor are bound to glycoprotein Ib, it suggests that factor VIII may be presented to thrombin in such a way as to allow for rapid activation. Thrombin on the platelet surface can also fully activate platelet surface factor V, a reaction that is enhanced by platelet-released polyphosphates [35]. Either the partially active factor V released from platelets or plasma-derived factor V can be activated by thrombin. Thrombin activation of platelets is augmented in platelets bound directly to collagen (as opposed to platelets aggregated onto other platelets or onto fibrin) [38]. These platelets, sometimes called COAT platelets, have higher levels of acidic phospholipids as well as significantly increased binding of factors X, IX, VIII, and V [17,39].

The amplification process leads to platelets which are primed to varying degrees for thrombin generation. These platelets have an appropriate lipid surface with activated receptors and activated cofactors bound to the surface.

Propagation

Factor IXa formed during the Initiation phase binds to the platelet surface. Factor IXa is available even in the presence of plasma levels of antithrombin since the half-life of activated factor IXa is about an hour in plasma. Factor IXa can bind either to platelet surface factor VIIIa [40] formed in the amplification phase or to a platelet receptor and be transferred to factor VIIIa [41]. The factor IXa/VIIIa complex activates factor X on the platelet surface. Factor Xa can then move quickly into a complex with factor Va. The resulting factor Xa/Va complex provides the rapid burst of thrombin critical to giving good fibrin structure and providing for a stable clot.

Formation of factor Xa on the platelet surface plays a critical role in regulating the clotting process [42]. The rate of factor X activation determines the rate and amount of thrombin generation. Deficiencies in platelet surface factor Xa generation caused by a lack of or reduction in factor VIII or IX levels (hemophilia A or B, respectively) result in reduced or absent factor Xa and thrombin generation. Therapies to treat hemophilia involve restoring a robust rate of platelet surface factor Xa generation [43]. Factor Xa generation appears to be regulated in part by release of TFPI from platelets, and agents that block TFPI are under consideration as possible therapeutic agents in hemophilia [44].

Thrombin generation on platelets initiates a positive feedback loop through factor XI. Factor XI can be activated on platelets by thrombin [45,46]. This thrombin activation is enhanced by polyphosphates released from platelets [35]. The platelet surface factor XIa can activate factor IX, leading to enhanced factor Xa and thrombin generation. The amount of enhancement from factor XI shows wide variations on platelets from different individuals and may account for some of the variable bleeding associated with factor XI deficiency (hemophilia C) [45].

In some cases, particularly in cases of intravascular injury where there is substantial blood flow across the injured surface, there may be a contribution to the propagation phase from circulating tissue factor [47]. While healthy individuals have little or no circulating tissue factor [48], in some pathologic conditions, such as pancreatic cancer, there are measurable levels of circulating tissue factor in the form of microparticles [49]. If the microparticles also have surface molecules that can associate with platelets or other cells at the site of injury, then tissue factor on these microparticles may contribute to factor X activation and thrombin generation at a site of injury [50].

Very small amounts of thrombin (less than 1 nM or 0.1 U/mL) are required to promote fibrin formation, and much of the thrombin generation occurs after a clot has formed [51]. The thrombin formed binds to fibrin where it can remain active for many hours; the binding of thrombin to fibrin resulted in fibrin being described as antithrombin I [52]. Furthermore, platelet factor Xa/Va complexes appear to be active long after clot formation (hours) and can rapidly generate thrombin when presented with fresh plasma as a substrate [51]. It is likely that the persistence of fibrin-bound thrombin and the prolonged ability to generate thrombin are protective mechanisms to stabilize clots. Disruption of a clot would mean that thrombin is immediately available to cleave fibrinogen and refresh the fibrin clot. Also, new prothrombin present in plasma could be activated to further replenish thrombin stores and provide for clot stability.

Control and localization

Multiple mechanisms exist to prevent a clot from spreading into healthy vasculature. Flow is an important control mechanism and reduced flow is associated with venous thrombosis. Flow removes procoagulant proteins from the area of active thrombin generation, reducing their concentrations below the threshold required to maintain coagulation. Once thrombin, factor Xa, and other procoagulant proteases are removed from the relatively protected area of the clot, they are subject to inhibition by antithrombin, TFPI, and other plasma inhibitors. This inhibition is enhanced by the carbohydrate components of proteoglycans found on endothelial cells [53]. Also, endothelial cells have surface-associated TFPI that promotes rapid inactivation of factor Xa [54].

Platelet activation also represents a control mechanism. Thrombin cleavage of PARs is important for platelet activation, but the final activated state is dependent on signaling through other platelet receptors [55]. Platelets bound to collagen (and that thus signal through glycoprotein IV) have very high levels of procoagulant factors and are associated with enhanced thrombin generation and fibrin formation. Other platelets appear to have less procoagulant activity and have a more structural role in stabilizing the fibrin clot [56]. It appears that growth of a clot may in part be constrained by structural platelets that do not strongly support thrombin generation and, therefore, do not strongly support the positive feedback loop that generates the burst of thrombin.

Platelets support a thrombin-driven positive feedback loop that activates additional platelets, promotes thrombin generation, and leads to fibrin formation. In contrast, thrombin on healthy endothelial cells leads to a negative feedback loop that shuts off further thrombin generation. Endothelial cells express the thrombin-binding protein, thrombomodulin [57]. Thrombin bound to thrombomodulin can no longer cleave fibrinogen; however, thrombin bound to thrombomodulin gains the ability to activate protein C [58]. Activation of protein C is enhanced by another endothelial cell protein called EPCR (endothelial cell protein C receptor) [59].

Activated protein C cleaves and inactivates both factor VIIIa and factor Va in a reaction that is enhanced somewhat by protein S [60]. This inactivation is more efficient on the endothelial cell surface than on platelets and suggests that the protein C pathway localizes thrombin generation rather than strictly shutting it down [61]. One of the sites on factor V that is cleaved by activated protein C is altered by the common factor V Leiden mutation [62]; this mutation is associated with venous thrombosis, suggesting that the negative feedback loop on healthy endothelium is a critical component of maintaining vascular patency.

Clinical assays

Coagulation function is generally measured in clinical assays that incorporate the elements of either the Initiation phase (prothrombin time or PT) or the propagation phase (activated partial thromboplastin time or aPTT). These assays are done on platelet-poor plasma so that the platelet contributions to clotting are not studied. The normal controls of these assays tend to be very reproducible, and the cell-free plasma can be frozen and stored. Other assays integrate the initiation and propagation phases and may include platelet function, such as thrombin generation (thrombogram or calibrated automated thrombography (CAT) [63] or whole blood clotting (thromboelastogram) [64].

The PT assay is done by adding very high levels of thromboplastin (TF) to plasma. Factor VII binds to this TF and is activated. The factor VIIa/TF complex activates factor X and the factor Xa/Va complex generates thrombin which clots the plasma. The endpoint of the assay is the time required for clot formation. Because thromboplastin is generally external to the blood, the assay is sometimes referred to as assaying the extrinsic pathway.

The aPTT assay is done by adding, in the absence of calcium, a negatively charged activator to plasma. The negative charge assembles a complex of high-molecular-weight kininogen and factor XII that converts all of the factor XI in the sample to factor XIa [65]. When recalcified, the factor XIa activates factor IXa which forms a complex with factor VIIIa. This factor IXa/VIIIa complex activates factor Xa which, in complex with factor Va, cleaves prothrombin to thrombin and clots the plasma. The endpoint of the assay is the time required for clot formation. Since all of the protein components are found in plasma, this assay is sometimes referred to as assaying the intrinsic pathway.

Since thrombin generation in the aPTT requires factors IX and VIII, it is used to monitor factor levels during therapy in hemophilia. The assay is very sensitive to the levels of the contact factors, factors XI and XII. However, patients with factor XI deficiency have a variable bleeding diathesis that is not strictly correlated with plasma levels [66]. This may be a function of how factor XI interacts with platelets and would not be assayed by the aPTT. Factor XII deficiency is not associated with any bleeding, nor is the deficiency protective of thrombosis in humans [67]. Bacterial polyphosphates are able to promote factor XII activation and may play a role in coagulation associated with the innate immune response; platelet polyphosphates are shorter than bacterial polyphosphates and do not promote activation of factor XII [35].

Summary

This overview provides a conceptual model of hemostasis as being initiated by injury leading to exposure of collagen and thromboplastin. Platelets adhere and are activated. Coagulation factors are activated, assemble on the platelet surface, and give robust thrombin generation leading to stable clot formation and clot retraction. Subsequent chapters will detail mechanisms of the processes involved in promoting hemostasis. Many of the same players (thromboplastin, platelets, coagulation factors), albeit with slightly different roles, are also involved in pathological coagulation leading to thrombosis or bleeding. Dysregulation resulting in thrombosis and other coagulation abnormalities will also be discussed in subsequent chapters.

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CHAPTER 2

Vascular Endothelium, Influence on Hemostasis: Past and Present

Hussain I. Saba1 and Sabiha R. Saba2

1 Hematology/Hemophilia/Hemostasis & Thrombosis Center, USF College of Medicine, Tampa, FL, USA

2 Department of Pathology and Cell Biology, USF College of Medicine, Tampa, FL, USA

Introduction

William Harvey was the first scientist to offer a new radical concept of blood circulation, in the year 1628 [1]. It led to immediate controversy in the medical community at that time, as it contradicted the usually unquestioned teaching of the Greek philosopher, Galen, regarding the theory and concept of blood movement. Galen's theory was based on the ideas that blood was formed in the liver, absorbed by the body, and flowed through the septum of the heart (dividing walls). Although Harvey's contradicting concept was based upon human and animal experiments, his findings were ridiculed and not well accepted. Later in the year 1661, Marcello Malpighi published his discovery of capillaries which then gave unwavering, factual evidence to support Harvey's concept of blood and circulation [2]. By the year 1800, Von Recklinghausen established that blood vessels were not merely a tunnel-like membrane structure similar in shape of cellophane tube, but had primary and important function of maintaining the vascular permeability [3]. Heidenhain (1891) introduced the concept that endothelium possessed an active secretory system [4]. In 1959, Gowans described the interaction between lymphocytes and endothelium at postcapillary venules. By 1959, electron microscopic studies by Palade [5] and the physical studies by Gowans [6] led to the current concept that endothelium is a dynamic heterogeneous disseminated organ which possesses vital secretory, metabolic, and immunologic activities.

In adult human subjects, the total endothelial surface consists of approximately 1–6 × 1013 cells, weighing about 1 kg and covering an area of approximately 4–7 × 103 square meters. Endothelial cells line the blood vessel of every inner human organ, are responsible for regulation of the flow of nutrients, and possess diverse biologically active molecules such as hormones, growth factors, coagulant and anticoagulant proteins, lipid transporting particles (LDL), and metabolites such as nitrous oxide. Protective and receptive endothelium also governs cell and cell matrix interaction. Endothelial cells that make up the lining of the inner surface of blood vessels wall are called vascular endothelial cells. These cells line the entire circulatory system from the heart to the smallest capillaries, and have very distinct and unique functions that are of importance to the vascular biology. Their functions include fluid filtration, such as that seen in the glomeri of the kidney. They maintain vascular tone and are, therefore, involved in the maintenance of blood pressure. The cells are also involved in mediation of hemostatic responses and trafficking of the neutrophil in and out of the lumen of the blood vessel to the tissue space. Endothelial cells are involved in many aspects of vascular biology. These are biologically of paramount importance. Their role has included the development of early and late stages of atherosclerosis. One of the very important functions of endothelial cells is their role in the maintenance of a non-thrombogenic surface apparently due to the presence of heparan sulfate, which works as a cofactor for activating antithrombin, a protease that inactivates several factors in the clotting cascades.

Function of endothelial cells

Endothelial cells are involved in many aspects of vascular biology, and play a role in the development of atherosclerosis. They also function as a selective barrier between blood cells and surrounding tissue, controlling the passage of material and the transit of white cells in and out of the bloodstream. Excessive and prolonged increase in the permeability of the endothelial cells monolayer such as that seen in cases of chronic inflammatory process may lead to accumulation of inflammatory fluid in the tissue space. Endothelial cells are involved in maintaining a nonthrombogenic and thromboresistant surface. This physiologic activity inhibits platelets and other cells from sticking to endothelium and is related to the presence of heparan sulfate on the endothelial surface, which works as a cofactor for activating antithrombin, a protease that inactivates several factors responsible for activating the clotting cascades. Vascular endothelium, because of its strategic location interfering between tissue and blood, is in an ideal situation to modulate and influence functions of various organs. Endothelial cell function includes transport of nutrients and solutes across the endothelium, maintenance of vascular tone and maintenance of the thromboresistant surface, and the activation and inactivation of various vasoactive hormones. Under normal conditions the endothelial cells provide a nonthrombogenic surface which does not allow platelets and other blood cells to adhere and to stick to the surface of endothelium. This nonthrombogenic nature of endothelium is unique for the flow of the blood as well as for the flow of blood cells.

The mechanism of the thromboresistance of endothelium has not been fully understood but is considered to be related to the interaction of anticoagulant, fibrinolytic, and antiplatelet factors. The endothelium confers strong defense mechanisms against these insults by expressing a series of molecules. With successful culture of the endothelial cells, a myriad of molecules have been identified and characterized. The accepted view at this stage is that the main function of endothelial cells is to produce vasoprotective and thromboresistant molecules. Some molecules are constitutively expressed, while others are produced and respond to stimuli. Some are expressed on the interior endothelial surface and others are released. Molecules physiologically important in suppressing platelet activation and platelet vessel wall interaction include prostacyclin (PGI2), nitric oxide (NO), and ecto-adenosine diphosphatase (ADPase). Molecules involved in controlling coagulation include the surface-expressed thrombomodulin (a heparin-like molecule), von Willebrand factor (VWF), protein S, and tissue factor pathway inhibitor (TFPI). Endothelial cells synthesize and secrete tissue plasminogen activator (TPA) and urokinase-type plasminogen activator to promote fibrinolysis. To control TPA activity, the endothelium produces plasminogen activator inhibitor-1 (PAI-1), which serves to neutralize the TPA activity.

Antiplatelet factors

Prostacyclin

Prostacyclin (PGI2) is a multifunctional molecule it is an important inhibitor of platelet activation, aggregation, and secretion [7–10]. It induces vascular smooth muscle relaxation and blocks monocyte endothelial cell interaction. It also reduces lipid accumulation in smooth muscles. Its platelet inhibitory activity is mediated via guanosine nucleotide-binding receptor with subsequent activation of adenylate cyclase and elevation of platelet adenosine monophosphate (cAMP).

cAMP levels will result in inhibition of platelet activation. Its actions on other cells are thought to be mediated by a similar receptor-mediated signal transduction pathway. Prostacyclin is primarily synthesized by vascular endothelial cells and smooth muscle cells. Its synthesis is catalyzed by a series of enzymes. When stimulated by diverse physiologic agonists including thrombin, histamine, and bradykinin, endothelial cell cytosolic phospholipase (PLA-2) is activated. Activated PLA-2 catalyzes the liberation of arachidonic acid (AA) primarily from phosphatidylcholine. The released free AA serves as a substrate for prostaglandin H synthase (PGHS), also known as cyclooxygenase.

PGHS is a bifunctional enzyme with two distinct enzymatic activities. Cyclooxygenase catalyzes the oxygenation of AA from prostaglandin to prostaglandin G2 (PGG2) and peroxidase catalyzes the reduction of PGG2 to prostaglandin H2 (PGH2). PGH2 is a common precursor for the synthesis of prostaglandin, prostacyclin, and thromboxane. In endothelial cells, PGH2 is primarily converted to PGI2 by the specific enzyme PGI2 synthase. PGI2 synthesis is regulated at each enzymatic step. The exact regulatory mechanisms are not entirely clear, but it is generally believed that PGHS is the key step due to autoactivation of this enzyme during catalysis. Several studies have shown that PGI2 synthesis by arterial segments of cultured endothelial cell stimulated with thrombin or histamine has a short duration of activity of 15–30 minutes. Overexpression of PGHS type 1 in an endothelial cell line by retrovirus-mediated transfer of the human PGHS-1 gene is accompanied by a 10- to 100-fold increase in PGI2 synthesis. Two isoforms of PGHS have been identified in human endothelial cells. PGHS-1 is constitutively expressed and its synthesis may be augmented by shear stress, cytokines, and mitogenic factors. PGHS is thought to be primarily responsible for synthesizing the vasoprotective PGI2 under physiologic conditions. Endothelial cells possess type 2 PGHS (PGHS-2) which is expressed in smaller quantities in resting cells but is highly inducible by mitogenic factors and cytokines. The inducible PGHS-2 is present on both inflammatory and neoplastic cells. This PGHS isoform has been considered to be primarily involved in cell inflammation and cell proliferation. However, there is suggestive evidence that PGHS-2 may play an important role in producing vasoprotective PGI2 when the endothelium is under severe stress and cellular PGHS-1 levels are depleted because of autoactivation. Human PGHS-1 and PGHS-2 genes have been mapped to chromosomes 9 and 1 respectively; the structure and promoter activities of both genes have been characterized.

Prostacyclin synthase provides a final enzymatic step for this specific PGI2 synthesis. This enzyme is membrane bound to cytochrome P450 enzymes [11]. Its complementary DNA has recently been cloned [12,13]. One report reveals that this enzyme is inducible and hence may play a role in determining the extent and duration of PGI2 synthesis. Furthermore, PGI2 synthesis, like PGHS-1, is autoactivated during catalysis. It is likely that this enzyme plays a major role in controlling the extent of PGI2 production as well. PGI2 may also be synthesized by a transcellular mechanism [14]. Hence the production of PGI2 is tightly regulated and an alternate synthesis pathway exists to ensure a sufficient PGI2 level for vasoprotection when the vessel is under stress.

Nitric oxide

Nitric oxide (NO) is elaborated as hetero atomic radical production guaranteed through the oxidation of L-arginine and L-citrulline by nitric oxide synthetase. NO is the mediator of vasorelaxation, immunomodulation, cytotoxicity, and neurotransmission [15]. NO also inhibits platelet activation. The role of NO in vasorelaxation was discovered by Furchgott and Zawadzki. [16] These investigators noted that blood vessels depleted of endothelium failed to relax when treated with acetylcholine [16]. They postulated that endothelium elaborates the factor(s) endothelium-dependent relaxing factor (EDRF) which is responsible for acetylcholine-induced vasorelaxation. The major component of EDRF was subsequently found to be nitrous oxide.

Biosynthesis of nitrous oxide is catalyzed by nitric oxide synthase (NOS). NOS converts L-arginine to L-citrulline and NO. NO is diffusible and is thought to be released primarily into the albuminal side where it activates smooth muscle cell guanylate cyclase and increases cytosolic cyclic guanosine monophosphate. NO may also diffuse into the luminal side where it enters into the platelets and inhibits platelet adhesion, activation, and aggregation via activation of guanylate cyclase. Nitrous oxide and prostacyclin act synergistically not only to inhibit platelet adhesion and aggregation, but also to reverse platelet aggregation [17]. The synergistic inhibition of platelet activation by these two molecules has been considered to be of importance in maintaining blood fluidity and controlling thrombus formation. Three isoforms of NOS have been identified and characterized. Vascular endothelium possesses a constitutive NOS (NOS-III), which shares about 50–60% of amino acid sequences identified with neuronal constitutive NOS (NOS-I) and inducible NOS (NOS-II).

Like NOS-I and NOS-II, NOS-III is a bifunctional enzyme containing a reductase domain and an oxygenase domain [5]. Although NOS-III is constitutively expressed in endothelial cells, the enzyme is inactive in resting cells and little NO is synthesized. When endothelial cells are activated by physiologic agonists, elevated cellular calcium binds to calmodulin (CaM) and the Ca2+/CaM complex binds to CAM, binding sites on NOS thereby activating the enzyme. An elevated cytosolic calcium is also pivotal in PGI2 synthesis. PGI2 and NO are produced simultaneously during endothelial cell activation via calcium elevation.

The human NOS-III gene has been mapped to chromosome 7q35-36. Its 5' flanking region lacks canonical TATA or CAAT and is guanine plus cytosine (G+C) rich, consistent with the feature of “housekeeping” gene [18]. However, this gene is regulated at the transcriptional level. It has been shown that high shear stress and lysophosphatidylcholine produced during minimal low density lipoprotein oxidation augment NOS-III transcription [19] and increase NOS-III enzyme activity in endothelial cells. NOS-III levels and activity may, hence, be regulated tightly at multiple steps transcriptionally and post-transcriptionally.

Basal production of PGI2 and nitric oxide in vivo

Normal endothelium probably produces a constant basal level of PGI2, as evidenced by urinary excretion of its metabolite, 2,3-dinor-6-keto-PGF1a [20]. The evidence of basal production of NO is less clear, but inhibitor studies do suggest a basal level of NO production. The stimulating factors responsible for maintaining constitutive PGI2 and NO synthesis are probably multiple, including thrombin, histamine, shear stress, mechanical force, and lipid mediators. It should be noted that the endothelium may be stimulated by shear stress via two separate mechanisms: 1) activations of endothelium with resultant intracellular calcium elevation and, consequently, PLA2 and NOS activation leading to PGI2, and NO synthesis; and 2) inductions of PGHS-1 and NOS-111 transcription. The 5' flanking region of both genes contains putative shear stress response elements. However, binding of nuclear transcription activators to this element to promote PGHS-1 and NOS-III gene expression under controlled shear stress is a major, if not the only, factor responsible for sustained PGI2 and NO synthesis. The levels of PGI2