Von Willebrand Disease E-Book

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

Seitenzahl: 739

Veröffentlichungsjahr: 2011

Beschreibung

Von Willebrand Disease: Basic and Clinical Aspects provides an insight into all aspects of the condition. Since its discovery, von Willebrand disease has been extensively studied and the causative factor deficiency, the understanding of the condition and its treatment has greatly improved. This book summarizes recent research and will help to optimize the management of patients with von Willebrand disease. This valuable book describes the important and complex role of von Willebrand factor in hemostasis and thrombosis. In addition to the current understanding of its molecular biology, this book gives particular focus to the association between genetic variants of von Willebrand factor and different von Willebrand disease phenotypes. It also reviews the important area of the obstetric and gynecological manifestations of von Willebrand disease, as well as the treatment of acute bleeding. Written by an international team of contributors it describes the progress in, and difficulties of diagnosing phenotypes and genotypes. Molecular diagnosis of type 1, type 2 and its subgroups, and type 3 von Willebrand disease are presented as well as discussion of the potential future role of gene therapy. Von Willebrand Disease: Basic and Clinical Aspects is a valuable resource for hematologists in practice and in training, and specialists in thrombosis and hemostasis.

Leseprobe

Table of Contents

Cover

Table of Contents

Half title page

Title page

Copyright page

Contributors

Foreword

Preface

1  Historical perspective on von Willebrand disease

Introduction

The scientist of the disease

First description of the disease: the Åland family

Other early clinical reports

The search for a new factor—the bleeding time factor

The end of the beginning

2  Biosynthesis and organization of von Willebrand factor

Introduction

Molecular biology of VWF

Cell biology of VWF

3  von Willebrand factor structure and function

Introduction

Four functions of VWF

Structure of VWF

VWF domain structure

Biochemistry of VWF

Summary

Acknowledgements

4  Modulation of von Willebrand factor by ADAMTS13

Pathogenesis of TTP and discovery of ADAMTS13

ADAMTS13 structure, synthesis, and secretion

ADAMTS13 gene mutations

Autoantibodies against ADAMTS13

ADAMTS13 activity and regulation

ADAMTS13 interaction with VWF

ADAMTS13 and VWD

Conclusions

5  Animal models in von Willebrand disease

Genetic and phenotypic characterization of the different VWD animal models

Contribution of VWD animal models in improving VWD therapy

Contribution of VWD animal models in improving knowledge of VWF functions

Contribution of VWD animal models to improving knowledge of VWF biology

Concluding remarks

6  Classification of von Willebrand disease

The old nomenclature of VWD: some historical steps

Classification of VWD based on structural and functional abnormalities of VWF (1987)

The revised classification of VWD (1994)

Current classification. The updated revised classification (2006)

Acknowledgements

7  The epidemiology of von Willebrand disease

Introduction

Historical studies on the prevalence of VWD

Prevalence of bleeding patients in the general population

Bleeding score: a new diagnostic tool to assess clinically relevant VWD

The problem of diagnosing mild VWD

Prevalence of intermediate VWD

Prevalence of severe VWD

Conclusions

8  Clinical aspects of von Willebrand disease: bleeding history

Introduction

Bleeding history in VWD

Bleeding symptoms in VWD

Specific situations

Bleeding assessment tools

Conclusion

9  Laboratory diagnosis of von Willebrand disease: the phenotype

Screening diagnostic tests

Extended diagnostic tests

Special and/or newer diagnostic tests and processes

Qualitative changes in VWF

Diagnosis in neonates and young children

Diagnosis in pregnancy

Desmopressin trials as an aid to the diagnosis and functional characterization of VWD

Future perspectives

10  Molecular diagnosis of von Willebrand disease: the genotype

Introduction

Molecular analysis

Range of genetic defects that contribute to VWD

VWF mutation analysis

Laboratory analysis

Mutation detection challenges

Mutation analysis resources

Acknowledgements

11  Clinical, laboratory, and molecular markers of type 1 von Willebrand disease

Introduction

The epidemiology of type 1 VWD

Clinical features of type 1 VWD

The laboratory diagnosis of type 1 VWD

The genetics of type 1 VWD

The role of ABO blood group and type 1 VWD

VWF gene mutations and type 1 VWD

Recurrent type I VWD candidate mutations

Noncoding sequence variants in type 1 VWD

Type 1 VWD and accelerated clearance of VWF

Future priorities in type 1 VWD characterization

12  Clinical, laboratory, and molecular markers of type 2 von Willebrand disease

Introduction

Clinical manifestations

Laboratory diagnosis

Molecular markers

Concluding remarks

13  Clinical, laboratory, and molecular markers of type 3 von Willebrand disease

General definition, history, and epidemiology

Clinical markers of type 3 VWD

Laboratory markers of type 3 VWD

Molecular markers of type 3 VWD

Treatment and prevention of bleeding in type 3 VWD

Future perspectives

Acknowledgements

14  Pediatric aspects of von Willebrand disease

Introduction

Diagnosis of VWD in childhood

Diagnosis of type 1 VWD versus low VWF levels as a risk factor for bleeding

VWD in neonates

Acquired VWD in childhood

VWD in adolescents

Treatment strategies in children

Conclusions

15  Women with von Willebrand disease

Epidemiology of VWD in women

The diagnosis of VWD in women

Clinical aspects of menorrhagia in women with VWD

Adolescent menorrhagia and VWD

Medical treatment of menorrhagia

Surgical treatment

Hemorrhagic ovarian cyst

Pregnancy

Treatment

Epidural anesthesia

Postpartum management

Neonates

Conclusion

16  On the use of desmopressin in von Willebrand disease

Summary

History

Mechanism of action

Modes of administration and dosage

Experience of desmopressin in VWD

Acquired von Willebrand syndrome

Pregnancy

Surgery

Menorrhagia

Conclusion

17  The use of plasma-derived concentrates

Introduction

VWF/FVIII concentrates

Thrombotic complications following VWF/FVIII concentrates

VWF concentrates devoid of FVIII

Conclusions

18  Prophylaxis in von Willebrand disease

Introduction

Prophylaxis in VWD

Experience in Sweden

Experience with prophylaxis in other cohorts

Planned or ongoing studies

The future of prophylaxis in VWD and recommendations

19  Pathophysiology, epidemiology, diagnosis, and treatment of acquired von Willebrand syndrome

General definition, history, and epidemiology

Pathophysiologic mechanisms for the management of AVWS

Lymphoproliferative diseases

Cardiovascular diseases

Thrombocythemia and other myeloproliferative disorders

Neoplasms

Immunologic diseases

AVWS in patients with other diseases

Current issues and future perspectives on AVWS

20  Gene therapy for von Willebrand disease

Introduction

Gene delivery approaches

Target cells

Conclusions and perspectives

Acknowledgements

Index

Von Willebrand Disease

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Library of Congress Cataloging-in-Publication Data

Von Willebrand disease : basic and clinical aspects / edited by Augusto B. Federici ... [et al.].

p. ; cm.

Includes bibliographical references and index.

ISBN 978-1-4051-9512-6 (hardcover : alk. paper)

1. Von Willebrand disease. I. Federici, Augusto B.

[DNLM:1. von Willebrand Diseases. WH 312]

RC647.V65V66 2011

616.1'57–dc22

2010036377

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

This book is published in the following electronic formats: ePDF 9781444329933; Wiley Online Library 9781444329926; ePub 9781444329940

Contributors

Thomas C. Abshire MD

Senior Vice President

Medical Services and the Medical Science Institute;

Chief Medical Officer

BloodCenter of Wisconsin

Milwaukee, WI, USA

Luciano Baronciani PhD

Hospital Scientist

Angelo Bianchi Bonomi Haemophilia and Thrombosis Centre

Department of Medicine and Medical Specialities

IRCCS Maggiore Policlinico Hospital

Mangiagalli and Regina Elena Foundation and University of Milan

Milan, Italy

Jennifer Barr BS

Department of Anatomy and Cell Biology

University of Iowa Carver College of Medicine

Iowa City, IA, USA

Javier Batlle MD

Chairman Servicio de Hematología y Hemoterapia.

INIBIC. C. Hospitalario Universitario A Coruña;

Associate Professor of Department of Medicine

School of Medicine

University of Santiago de Compostela

A Coruña, Spain

Margareta Blombäck MD PhD

Professor Emeritus

Department of Molecular Medicine and Surgery

Division of Clinical Chemistry and Blood Coagulation Research

The Karolinska Institute

Karolinska University Hospital

Stockholm, Sweden

Ulrich Budde MD

Director

Department of Hemostaseology

Medilys Laborgesellschaft mbH

c/o Asklepios Klinik Altona

Hamburg, Germany

Giancarlo Castaman MD

Consultant Haematologist

Department of Cell Therapy and Hematology

Hemophilia and Thrombosis Center

San Bortolo Hospital

Vicenza, Italy

Olivier D. Christophe PhD

Senior Researcher

INSERM Unit 770

Le Kremlin-Bicêtre, France

Marinee K.L. Chuah PhD

Professor

Flanders Institute for Biotechnology (VIB)

Vesalius Research Center

University of Leuven

Leuven;

Faculty of Medicine and Pharmacy

University Hospital Campus Jette

Free University of Brussels (VUB)

Brussels, Belgium

Cecile V. Denis PhD

Director of Research

INSERM Unit 770

Le Kremlin-Bicêtre, France

Jorge Di Paola MD

Associate Professor of Pediatrics and Genetics

Postle Family Chair in Pediatric Cancer and Blood Disorders

University of Colorado Denver School of Medicine

The Children’s Hospital

Aurora, CO, USA

Jeroen C.J. Eikenboom MD, PhD

Associate Professor

Department of Thrombosis and Hemostasis

Leiden University Medical Center

Leiden, the Netherlands

Emmanuel J. Favaloro PhD

Senior Hospital Scientist

Department of Haematology

Institute of Clinical Pathology and Medical Research (ICPMR)

Westmead Hospital

Westmead, NSW, Australia

Massimo Franchini MD

Head

Immunohematology and Transfusion Center

Department of Pathology and Laboratory Medicine

University Hospital of Parma

Italy

Edith Fressinaud MD, PhD

Consultant Haematologist

Centre National de Référence de la Maladie de Willebrand

Service d’Hématologie biologique

Hôpital Antoine Béclère

Clamart, France

Anne Goodeve BSc, PhD

Reader and Head, Haemostasis Research Group

Department of Cardiovascular Science

Faculty of Medicine, Dentistry and Health

University of Sheffield;

Principal Clinical Scientist

Sheffield Diagnostic Genetics Service

Sheffield Children’s NHS Foundation Trust

Sheffield, UK

Sandra L. Haberichter PhD

Associate Professor

Department of Pediatrics – Hematology/Oncology

Medical College of Wisconsin

Milwaukee, WI, USA

Paula D. James MD, FRCPC

Associate Professor, Hematologist

Queen’s University

Kingston, ON, Canada

Rezan A. Kadir MD, FRCS (ed), MRCOG, MD

Consultant Obstetrician and Gynaecologist

The Royal Free Hospital

London, UK

Peter A. Kouides MD

Medical and Research Director

Mary M Gooley Hemophilia Center

Rochester General Hospital

Rochester, NY, USA

Peter J. Lenting PhD

Director of Research

INSERM Unit 770

Le Kremlin-Bicêtre, France

Stefan Lethagen MD, PhD

Director of Copenhagen Haemophilia Centre

Thrombosis and Haemostasis Unit

Department of Haematology

Copenhagen University Hospital

Copenhagen, Denmark;

International Medical Director

Director of Medical & Science Haemostasis Department

Global Development

Novo Nordisk

Søborg, Denmark

María Fernanda López-Fernández MD

Head, Hemostasis and Thrombosis

Servicio de Hematología y Hemoterapia

Complexo Hospitalario Universitario de A Coruña

A Coruña, Spain

Pier Mannuccio Mannucci MD

Professor of Medicine

Angelo Bianchi Bonomi Haemophilia and Thrombosis Centre

University of Milan and IRCCS Maggiore Hospital

Milan, Italy

Claudine Mazurier PhD

Head of Analytical Department, Preclinical Development

Laboratoire Français du Fractionnement et des Biotechnologies

Lille, France

Dominique Meyer MD

Professor of Haematology

Centre National de Référence de la Maladie de Willebrand;

University Paris-Sud

France

David Motto MD, PhD

Assistant Professor

Departments of Internal Medicine and Pediatrics

University of Iowa Carver College of Medicine

Iowa City, IA, USA

Ian Peake BSc, PhD

Sir Edward Mellanby Professor of Molecular Medicine

Department of Cardiovascular Science

University of Sheffield Medical School

Sheffield, UK

Almudena Pérez-Rodríguez PhD

Post-doctoral Investigator

Servicio de Hematología y Hemoterapia—INIBIC

Complexo Hospitalario Universitario de A Coruña

A Coruña, Spain

Inge Petrus PhD

Flanders Institute for Biotechnology (VIB)

Vesalius Research Center

University of Leuven

Leuven, Belgium

Jacob H. Rand

Hematology Laboratory

Department of Pathology

Montefiore Center

Bronx, NY, USA

Francesco Rodeghiero MD

Director

Department of Cell Therapy and Hematology

San Bortolo Hospital

Vicenza, Italy

Reinhard Schneppenheim MD, PhD

Director

Department of Pediatric Hematology and Oncology

University Medical Center Hamburg-Eppendorf

Hamburg, Germany

Alberto Tosetto MD

Senior Consultant

Department of Hematology

San Bortolo Hospital

Vicenza, Italy

Thierry VandenDriessche PhD

Group Leader

Flanders Institute for Biotechnology (VIB);

Vesalius Research Center

University of Leuven

Leuven;

Faculty of Medicine and Pharmacy

University Hospital Campus Jette

Free University of Brussels (VUB)

Brussels, Belgium

Foreword

I feel very honored to have been asked to write the foreword to this book on von Willebrand disease (VWD). I am now the oldest living scientist to have experience in this area, and thus it may be of interest for readers to learn about some early experiences that I shared with the late Dr. Inga-Marie Nilsson, which I have described below. Since I started working with VWD in the mid-1950s, there has been enormous progress in the management of the disease in terms of knowledge about mechanisms, treatment, and underlying genetics. We have been able to follow this development in Stockholm because the hemophilia center here is currently responsible for the treatment of 40 patients with type 3 VWD (i.e., the most severe form).

In the 1950s there were only a few known cases of the disease—which was mostly called “pseudohemo­philia”—in addition to those cases known in the Åland Islands, where the disease was first identified by Erik von Willebrand. This was probably because most patients with type 3 VWD died young, either in utero or, if the patient was female and survived until puberty, as a result of menstrual bleeding. I remember some touching letters written at the end of the 19th century from a businessman to his wife, who was mostly bedridden owing to menstrual bleedings. This woman was an ancestor of a young woman from Stockholm with type 3 VWD, who is currently living a normal family life thanks to therapy in early childhood with the Swedish fraction I-0 (which contained von Willebrand factor [VWF], factor VIII [FVIII], and fibrinogen) and later with commercial VWF-containing concentrates.

When taking a bleeding history for a female in the 1950s, it was useful to ask whether she had been scolded in school for dropping blood onto her handiwork after pricking her finger with a sewing needle. We learned that it was useful to analyze blood groups in family investigations, as we found that a healthy child who showed no sign of having inherited the disease did not share the same father as the sick sibling. We made several mistakes—one girl was transfused with platelets during a severe menstrual bleeding without success but, when treated with fraction I-0, the bleeding stopped. It is possible that the platelet treatment was the reason why she later developed antibodies to VWD. At that time there were no oral contraceptives, which have revolutionized the management of menorrhagia in patients with VWD. In this particular patient, we used testosterone and later hysterectomy (under prophylaxis of fraction I-0) to deal with the menstrual bleedings.

To persuade doctors that a patient had to be treated with a concentrate was a difficult task. I remember the case of a 13-year-old boy who developed severe head trauma as a result of falling from a bicycle. Despite the fact that the boy had a bleeding chart saying that he should be treated immediately in the event of a trauma and the fact that I informed the doctor that the usual signs do not develop in bleeders immediately but sometimes several days later, the doctor refused to treat the boy with concentrates and he died from severe brain hemorrhage.

In 1958 we started prophylactic treatment in patients with hemophilia to avoid joint destruction. However, it was not until many years later that we realized that patients with type 3 VWD also required prophylaxis; therefore, some of them developed joint disabilities. We also did not know that the concentrates with which we treated our patients could contain hepatitis C virus, which has led to the premature death of some patients.

This book has become a very comprehensive and useful work into which many of the authors have put great efforts to make their chapters not only informative but also easy to understand. Progress, difficulties, and alternative ways to diagnose phenotypes and genotypes are described. Molecular diagnosis of type 1, type 2 and its subgroups, and type 3 VWD are presented. In addition, a chapter on gene therapy looking into the future is stimulating to read. Furthermore, many authors have endeavored to include all relevant literature, which is very useful for students.

A problem with regard to historical aspects is that the nomenclature has changed from FVIII-related antigen to VWF antigen. Therefore, some of the early findings with regard to the level of VWF in patients with blood group O or A have not been observed. Nevertheless, the topic of how to proceed in diagnosis when the patient has blood group O or A has been thoroughly discussed. I have the impression that there still are problems with regard to diagnosis of the phenotypes, particularly with regard to the diagnosis of type 1 VWD, even if preanalytic problems are taken into account, for example the quality of methodology and the importance of telling the patient to rest and not to run or be stressed, etc, before blood sampling. I made a serious mistake once when analyzing changes in VWF during the menstrual cycle—the volunteers were not well informed about resting before sampling and we therefore misinterpreted the results; there are not such great variations in FVIII and VWF during the menstrual cycle as initially suggested.

When investigating families with type 3 VWD, we found that the parents and siblings who were genetic carriers of VWD only had a phenotypically mild bleeding disorder and often, but not always, the common analyses of VWD indicated a mild disorder. However, we recorded the usefulness of an increased ratio of FVIII/VWF:Ag for the diagnosis of what we called type 1 VWD in these families.

It must have been an enormous task for the editors to encourage all the authors to write, although possibly some welcomed the opportunity to put together their experience in a comprehensive chapter. The efforts on trying to collate experience in multicenter studies on prophylaxis and diagnostic scores is very valuable and, of course, needs to be supported in order to solve the many difficulties that remain in the diagnosis and management of VWD.

Margareta Blombäck

Professor Emeritus

Karolinska Institutet

Sweden

Preface

Erik von Willebrand described a novel bleeding disorder in 1926 and, in his original publication, he provided an impressive description of the clinical and genetic features of the von Willebrand disease (VWD). In contrast to hemophilia, the epitome of inherited bleeding disorders, both sexes were affected, and mucosal bleeding was the predominant symptom. The history of VWD is fascinating because it demonstrates how good clinical observations, genetic studies, and biochemical skills can improve the basic understanding of a disease and its management. The continuous efforts of scientists and clinicians over the last 85 years have significantly furthered the understanding of the structure and function of von Willebrand factor (VWF), the protein that is absent, reduced, or dysfunctional in patients with VWD. Such basic information about VWF will undoubtedly improve both the diagnosis and the treatment of VWD. Determination of both the phenotype and the genotype is now readily available in many countries, and treatment is becoming more specific and directed by the type and subtype of VWD. Therapeutic agents must correct the dual defect of hemostasis, i.e. the abnormal platelet adhesion due to reduced and/or dysfunctional VWF and the associated low level of factor VIII (FVIII). Desmopressin (DDAVP) is the treatment of choice for type 1 VWD because it induces release of VWF from cellular compartments. VWF concentrates that are virally inactivated, with or without FVIII, are effective and safe in patients unresponsive to DDAVP; a recombinant VWF is currently under evaluation in clinical trials. Retrospective and prospective clinical studies, including bleeding history and laboratory markers for diagnosis, as well as the use of DDAVP and VWF concentrates to treat or prevent bleeding in patients with VWD, have been essential to provide general guidelines for the management of VWD.

This book presents the most important basic and clinical aspects of inherited and acquired defects of VWF, and it includes the many advances that have been made in recent years. The editors hope that a book specifically devoted to VWD can be useful to the hematologists of the 21st century who would like to manage VWD patients in a more comprehensive way using the most updated and evidence-based recommendations.

The editors would like to dedicate this first VWD book to three pioneers on VWD research who made pivotal and original contributions on this field: Arthur Bloom, Inga Maria Nilsson, and Theodore S. Zimmerman. Their life-long devotion to research on VWD and on other bleeding disorders should stimulate further studies on these topics of hematology.

The Editors

Augusto B. Federici

Christine A. Lee

Erik E. Berntorp

David Lillicrap

Robert R. Montgomery

16 January 2011

1

Historical perspective on von Willebrand disease

Erik Berntorp1 and Margareta Blombäck2

1Malmö Centre for Thrombosis and Haemostasis, Lund University, Skåne University Hospital, Malmö, Sweden

2Department of Molecular Medicine and Surgery, Division of Clinical Chemistry and Blood Coagulation Reasearch, The Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden

Introduction

The history of von Willebrand disease (VWD) and its causative factor, the von Willebrand factor (VWF), spans almost a century and was recently comprehensively reviewed by the late professor Birger Blombäck, who described the first publication by Erik von Willebrand [1], the gene cloning in 1985, and the discovery of the specific metalloprotease, ADAMTS13 [2], that degrades VWF. The purpose of this review is to describe the early history of the understanding of the disease and the first steps in the replacement therapy for its severe forms. Also, we describe in greater detail the findings in a group of different families investigated on the Åland Islands.

The Scientist of the Disease

Erik Adolf von Willebrand (Figure 1.1) was born in Vasa, Finland, in 1870. He qualified as a medical doctor in 1896 and specialized initially in physical therapy and later in internal medicine at Helsinki. Erik von Willebrand devoted much of his professional life to an interest in blood, especially its coagulation properties. In 1899, he defended a doctoral thesis that dealt with his investigation of the changes that occur in blood following a serious hemorrhage. From 1908 until his retirement in 1935, Erik von Willebrand worked at the Deaconess Institute in Helsinki, where he headed the Department of Internal Medicine between 1922 and 1931. Erik von Willebrand was known for his modesty and integrity, and in his obituary it was said that he “usually preferred to discuss his observations of nature rather than his personal achievements.” He died in September 1949, at the age of 89 years.

Figure 1.1 Erik von Willebrand.

First Description of the Disease: the Åland Family

In 1926, Erik von Willebrand first described the inherited bleeding disorder in Finska Läkaresällskapets Handlingar (in Swedish). He identified features that suggested that this disease was distinct from classic hemophilia and other bleeding disorders known at the time, such as anaphylactoid purpura, thrombocytopenic purpura, and the hereditary thrombasthenia described by Glanzmann. What differentiated this bleeding disorder from classic hemophilia was that it was not frequently associated with muscle and joint bleeding, and it affected both women and men. He stressed that a prolonged bleeding time was its most prominent characteristic. He concluded that the condition was a previously unknown form of hemophilia, and called it “hereditary pseudohemophilia.” Erik von Willebrand also discussed the pathogenesis of the condition and felt that the bleeding could best be explained by the combined effect of a functional disorder of the platelets and a systemic lesion of the vessel walls.

The original observations leading to this new disease were made in several members of a large family (identified as family S) living on the island of Föglö in the Åland archipelago in the Baltic Sea. The index case was a girl aged 5 years, named Hjördis S, who had marked and recurrent bleeding tendencies and was brought to Helsinki for consultation. Both her mother and father were from families with histories of bleeding. The girl was the ninth of 11 children, of whom seven had experienced bleeding symptoms. Four of her sisters had died from uncontrolled bleeding at an early age. Hjördis herself had experienced several severe episodes of bleeding from the nose and lips and following tooth extractions, as well as bleeding in her ankle. At the age of 3 years she bled for 3 days from a deep wound in her upper lip. The bleeding was so severe that she almost lost consciousness and had to be hospitalized for 10 weeks. At the age of 14 years, Hjördis bled to death during her fourth menstrual period.

Hjördis came from a large family (Figure 1.2). Intrigued by their history, Erik von Willebrand stud­ied the family further with the help of coworkers. He published the pedigree and his clinical and laboratory evaluation in his 1926 paper. He found that 23 of the 66 family members had bleeding problems. The most prominent problem among the affected family mem­bers was mucosal bleeding: epistaxis, followed by profuse bleeding from oral lesions, easy bruising, and, in females, excessive bleeding during menstruation and at childbirth. Intestinal bleeding had been the cause of death at early ages in some family members.

Figure 1.2 The Åland pedigree as originally described in 1926 [1]. The index case, Hjördis, is the ninth sibling in family S (Fam S). unaffected male; unaffected female; male with mild bleeding disease; female with mild bleeding disease; • female with severe bleeding disease; † bled to death.

In further studies, Erik von Willebrand found two families related to Hjördis S and one unrelated family in whom bleeding symptoms similar to those observed in Hjördis were common [3,4]. In the 1930s, Jürgens, together with von Willebrand [5,6], reinvestigated the patients in Åland and concluded that the disease was due to some impairment of platelet function, including platelet factor 3 deficiency. This observation led to the disease being called von Willebrand–Jürgens thrombopathy, and, although this condition is not officially recognized today, von Willebrand did not dismiss the notion that factors in blood plasma might also be important in the pathogenesis of the disease.

Other Early Clinical Reports

In 1928, Dr. George R. Minot of Boston described five patients from two families with prolonged bleeding times and symptoms similar to the Åland family members. This may have been one of the first descriptions of VWD [7–9]. In the following years, numerous cases similar to those described by von Willebrand were reported, usually under the name of pseudohemophilia. In 1953, Alexander and Goldstein [10] found a dual defect in two patients with hereditary pseudohemophilia. They confirmed the earlier findings of prolonged bleeding time, normal platelet count and function, and abnormal nail bed capil­laries. However, they also found a decreased FVIII level (5–10% of normal) and they observed a prolonged coagulation time that was normalized by normal plasma. The prolonged bleeding time, however, was not normalized and this was later explained by the fact that infusion of a restricted volume of plasma does not provide a sufficient amount of VWF [11]. Larrieu and Soulier [12] also found low FVIII activity and a prolonged bleeding time in pseudohemophilia, but otherwise normal clotting factors and platelet parameters. They proposed the name of von Willebrand syndrome for the condition.

The Search for a New Factor—the Bleeding Time Factor

The first demonstration of the VWF was during the 1950s through a joint effort by Margareta and Birger Blombäck, working in Stockholm with the purification of fibrinogen, and Inga Marie Nilsson, who had established a clinical coagulation unit in Malmö. It was found that fibrinogen purified from Cohn fraction I of human plasma, when specifically obtained in fraction I-0 (AHF-Kabi), was heavily contaminated with an antihemophilic factor, that is plasma factor VIII (FVIII) [13].

At that time, Dr. Nilsson had a 15-year-old female patient named Birgitta who had a severe hemorrhagic diathesis. When she began to menstruate, the condition worsened and she received frequent blood transfusions. However, Birgitta developed serious side-effects from the transfusions and they were stopped. As a consequence, other treatment options had to be considered, and a hysterectomy was planned. Her coagulation evaluation had shown a prolonged bleeding time and a somewhat prolonged coagulation time but normal platelet count and function. FVIII activity was low. Since fraction I-0 had a high concentration of FVIII, it was decided that its effects should be tested in Birgitta. To the surprise of the treating physicians, not only did FVIII activity increase as expected but the bleeding time was also normalized [14]. Subsequently, a hysterectomy was successfully performed under the cover of fraction I-0. According to modern classification, this patient had type 3 VWD. She is now well, and has been on regular prophylaxis with VWF concentrate for many years.

In June 1957, Inga Marie Nilsson, Erik Jorpes, Margareta Blombäck, and Stig-Arne Johansson visited Åland and studied 16 patients who had been examined 25–30 years previously by von Willebrand. No patients who had severe forms of the disease were still living. In their investigation they found FVIII activity to be reduced in 15 of 16 cases [15]. The father of Hjördis had a normal level. The Duke bleeding time varied, with two patients having a definite prolongation and three patients a moderate prolongation. Platelet counts were normal and, in contrast to Jürgens’ earlier observation, the platelets themselves were normal with respect to platelet factor 3. One of the patients was given fraction I-0, which normalized the FVIII level and the bleeding time. It could be concluded that the Åland family had the same disease described by several other authors in Europe and the USA [16]. At the same time, Jürgens visited the islands (Erik Jorpes had told him of his team’s research plan) and took samples from many of the same patients, and confirmed the decreased FVIII levels [17].

The findings by the Swedish group confirmed what had been documented in a number of Swedish families [18]. The observation was also made that FVIII increased during the first 24 h after infusion of fraction I-0 in patients with VWD, in contrast to what is seen in hemophilia [19]. The results of fraction I-0 infusion in a patient with severe VWD are shown in Figure 1.3. The bleeding time is reduced or normalized; factor VIII clotting activity (VIII : C) increases steadily during the first 24 h whereas the VWF (VIIIR:Ag and VIIIR:RC according to old nomenclature) displays a pharmacokinetic profile as expected and as later shown. Control experiments and further studies [11,20,21] revealed that the bleeding time factor was a plasma factor not earlier described. Fraction I-0 prepared from patients with severe hemophilia A not only corrected the bleeding time in VWD, but also stimulated the production of FVIII activity, whereas fraction I-0 prepared from patients with VWD had no such effect. Purified fibrinogen had no effect on the bleeding time. Still, there was the possibility that the shortening of the bleeding time was due to platelets or platelet factors contaminating fraction I-0. This turned out to be unlikely, since the effect on bleeding time was the same whether the fraction had been prepared from platelet-rich or platelet-poor plasma. Infusion of a platelet suspension from a normal donor to a patient with VWD had no effect on either bleeding time or bleeding tendency, nor did injection of fraction I-0 into a patient with thrombocytopenia. From these findings, it was concluded that the impaired hemostasis in VWD was due to lack of a plasma factor, the bleeding time correcting factor, or the VWF, which occurs not only in normal plasma but also in hemophilia A plasma. This factor not only corrected the prolonged bleeding time, but apparently increased the level of FVIII. Thus, platelets or platelet factors were not identical with the bleeding time factor, which had been proposed by both Rudolf Jürgens and Erik von Willebrand to be responsible, together with a vascular defect, for the bleeding diathesis. These findings have since been widely confirmed. The claim that a previously unknown factor in plasma had been discovered was communicated at the Congress of the International Society of Hematology in Rome in 1958 (see also [20]).

Figure 1.3 VIII:C, VWF:Ag (VIIIR:Ag), VWF:RCo (VIIIR:RCF), and Duke bleeding time (BT) in a patient with severe von Willebrand disease after infusion of human fraction I-0 (AHF-Kabi) [16]. Bleeding time is shortened and VIII:C is successively increased after the initial post-infusion peak during the first 24 h, whereas the von Willebrand factor (VIIIR:Ag and VIIIR:RC) displays a pharmacokinetic profile as expected.

Reproduced from Nilson and Holmberg [16].

At first, it was not understood how a plasma factor could affect primary hemostasis and shorten the bleeding time. However, Borchgrevink [22] found decreased platelet adhesiveness in vivo, and Salzman [23] demonstrated decreased platelet adhesiveness to glass in VWD. Borchgrevink employed the method suggested by Hellem [24], which used a slow flow and could not discriminate between samples from patients with or without VWD. Salzman modified this method and introduced a higher flow, making it more specific for VWD. It was also shown that normal or hemophilic plasma can normalize the reduced platelet adhesiveness as well as the bleeding time in VWD [23,25,26]. In studies using electron microscopy, Jörgensen and Borchgrevink [27] demonstrated a decreased adhesion of platelets to disrupted endothelium in VWD. This observation indicated that the plasma factor lacking in VWD exerted its action in primary hemostasis via the platelets by enhancing their adhesiveness.

During the 1960s, cases of VWD were reported from several countries. The disease was thought to be uniform and was defined as an autosomal dominant inheritable hemorrhagic disease characterized by a prolonged bleeding time, decreased FVIII clotting activity, decreased platelet adhesiveness as measured by the Salzman method, and progressive increase of FVIII activity after infusion of plasma and FVIII concentrate [16].

However, returning to the earlier papers by Erik von Willebrand and Rudolf Jürgens, the findings on the Åland islands showed what appeared to be a discrepancy between the original family S and some of the others investigated; the original von Willebrand family having “pure” VWD while in other families there were also platelet function defects. Thus, in 1977, Dag Nyman (originally from Åland) and collaborators [28] traveled from Stockholm to Åland to undertake a thorough investigation using new laboratory methods [28]. They found that the families described as having VWD could be divided into four categories: (i) the survivors with a mild disorder from the original family S had the characteristics of type 1 VWD, that is they had similarly decreased levels of VWF:Ag and ristocetin cofactor activity in addition to normal or decreased levels of FVIII, and the platelet aggregation was normal; (ii) one family had a platelet function defect (pure cyclooxygenase defect); (iii) one family had a mixture of VWD and a cyclooxygenase defect; and (iv) one family had a platelet function defect of the aspirin type. These findings, of course, made it easier to investigate the genetic defects of the original VWD (family S).

In the beginning of the 1990s, Zhang and collaborators [29] investigated the DNA sequence from 24 patients with type 3 VWD living in Sweden. They found a cytosine deletion in exon 18 of the VWF gene in most of those of Swedish origin and an insertion in exon 28 in those of Finnish origin. Most patients with type 3 VWD were homozygous or double heterozygous for the mutations. Most of the parents had type 1 VWD and were heterozygous. As the Åland population is primarily of Swedish origin, the researchers also investigated family S and found that the surviving members who had type 1 VWD were heterozygous with respect to the mutation in exon 18. There was a small boy with severe VWD whose family was related long ago to another family with VWD from Åland. He was homozygous for the mutation in exon 18 [30].

The End of the Beginning

After the publication by Erik von Willebrand in 1926, it took some 30 years until it was clear that a new plasma factor responsible for the hemostatic impairment in VWD had been detected. In that time, a factor concentrate had been produced that was effective in the replacement of VWF: fraction I-0 (or, later, AHF-Kabi). Studies using this concentrate and concen­trates purified from different types of bleeding disorders, helped scientists to find and prove the presence of the VWF. This was the end of the beginning.

In 1971, VWF was first detected immunologically and named “FVIII-related antigen” [31]. Since 1985, the VWF has been cloned [32–35], the primary amino-acid sequence has been determined [36], and the complex molecular structure and multiple functions are becoming understood in detail. The metalloprotease ADAMTS13 that cleaves VWF was discovered in 2001 [2]. VWD is no longer a uniform disease [37]. The treatment armamentarium has been developed and includes prophylactic treatment with concentrates in type 3 VWD. It includes desmopressin for most milder cases, new concentrates [38,39], and we are now anticipating development of recombinant VWF for therapeutic use.

References

1 von Willebrand EA. Hereditär pseudohämofili. Finska Läkaresällskapets Handlingar 1926; LXVII: 87–112.

2 Fujikawa K, Suzuki H, McMullen B, Chung D. Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family. Blood 2001; 98: 1662–6.

3 von Willebrand EA. Uber hereditäre pseudohämophilie. Acta Med Scand 1931; 76: 521–50.

4 von Willebrand EA. Uber ein neues vererbbares Blutungsubel: Die konstitutionelle Thrombopathie. Dtsc Arch Klin Med 1933; 175: 453–83.

5 von Willebrand A, Jürgens R. Über eine neues vererbbares Blutungsübel: die konstitutionelle Thrombopathie. Deutsches Archiv für Klinische Medizin: München, Leipzig, 1933, vol. 175, pp. 453–483.

6 von Willebrand E, Jürgens R, Dahlberg U. Konstitu­tionell trombopati, en ny ärftlig blödarsjukdom. Finska Läkaresällskapets Handlingar 1934; LXXVI: 193–232.

7 Minot GR. A familial hemorrhagic condition associated with prolongation of the bleeding time. Amer J Med Sci 1928; 175: 301.

8 Kasper C. Von Willebrand Disease. An Introductionary Discussion for Young Physicians. World Federation of Hemophilia: Montreal, Canada, 2004.

9 Owen JCA, ed. Inherited coagulation factor deficiencies. Mayo Foundation for Medical Education and Research: Rochester, 2001.

10 Alexander B, Goldstein R. Dual hemostatic defect in pseudohemophilia. J Clin Invest 1953; 32: 551.

11 Blombäck B. A journey with bleeding time factor. Compr Biochem 2007; 45: 209–55.

12 Larrieu M-J, Soulier J-P. Deficit en facteur antihe’mophilique A, chez une fille associe’a’un trouble du saignement. Revue d’hematologie 1953; 8: 361–70.

13 Blombäck B, Blombäck M. Purification of human and bovine fibrinogen. Ark Kemi 1956; 10: 415–43.

14 Nilsson IM, Blombäck B, Blombäck M, Svennerud S. Kvinnlig hämofili och dess behandling med humant antihämofiliglobulin. Nordisk Medicin 1956; 56: 1654–62.

15 Nilsson IM, Blombäck M, Jorpes E, Blombäck B, Johansson SA. Von Willebrand’s disease and its correction with human plasma fraction I-O. Acta Med Scand 1957; 159: 179–88.

16 Nilsson IM, Holmberg L. Von Willebrand’s disease today. Clin Haematol 1979; 8: 147–68.

17 Jürgens R, Lehmann W, Wegelius O, Eriksson AW, Hiepler E. Mitteilung uber den Mangel an antihämophilem Globulin (Faktor VIII) bei der Aaländisschen Thrombopatitie (von Willebrand–Jurgens). Thromb Diath Haemorrh 1957; 1: 257–60.

18 Nilsson IM, Blombäck B, von Francken I. On an inherited autosomal hemorrhagic diathesis with antihemophilic globulin (AHG) deficiency and prolonged bleeding time. Acta Med Scand 1957; 159: 35–57.

19 Nilsson IM, Blombäck M, Blombäck B. von Willebrand’s disease in Sweden. Its pathogenesis and treatment. Acta Med Scand 1959; 164: 263–78.

20 Blombäck B. Studies on fibrinogen and antihaemophilic globulin, XIIth Conference of the Protein Foundation on blood cells and plasma proteins, Albany, NY, November 1957. Vox Sang 1958; 3: 58.

21 Blombäck M, Blombäck B, Nilsson IM. Response to fractions in von Willebrand’s disease. In: Brinkhous KM (ed). The Haemophilias, pp. 286–94. University North Carolina Press: Chapel Hill, 1964.

22 Borchgrevink CF. A method for measuring platelet adhesiveness in vivo. Acta Med Scand 1960; 168: 157–64.

23 Salzman EW. Measurement of platelet adhesiveness. A simple in vitro technique demonstrating an abnormality in von Willebrand’s disease. J Lab Clin Med 1963; 62: 724–35.

24 Hellem AJ. The adhesiveness of human blood platelets in vitro. Scand J Clin Lab Invest 1960; 12 (Suppl. 51): 1–115.

25 Larrieu MJ, Caen JP, Meyer DO, Vainer H, Sultan Y, Bernard J. Congenital bleeding disorders with long bleed­ing time and normal platelet count. II. von Willebrand’s disease (report of thirty-seven patients). Am J Med 1968; 45: 354–72.

26 Meyer DO, Larrieu MJ. von Willebrand’s factor and platelet adhesiveness. J Clin Pathol 1970; 23: 228–31.

27 Jörgensen L, Borchgrevink CF. The haemostatic mechanism in patients with haemorrhagic diseases. Acta Athol Microbiol Scand 1964; 60: 55–82.

28 Nyman D, Blombäck M, Lehmann W, Frants RR, Eriksson AW, eds. Blood Coagulation Studies in Bleeder Families on Åland Islands. Academic Press: New York and London, 1980.

29 Zhang ZP, Blombäck M, Egberg N, Falk G, Anvret M. Characterization of the von Willebrand factor gene (VWF) in von Willebrand disease type III patients from 24 families of Swedish and Finnish origin. Genomics 1994; 21: 188–93.

30 Zhang ZP, Blombäck M, Nyman D, Anvret M. Mutations of von Willebrand factor gene in families with von Willebrand disease in the Åland Islands. Proc Natl Acad Sci U S A 1993; 90: 7937–40.

31 Zimmerman TS, Ratnoff OD, Powell AE. Immunologic differentiation of classic hemophilia (factor 8 deficiency) and von Willebrand’s disease, with observations on combined deficiencies of antihemophilic factor and proaccelerin (factor V) and on an acquired circulating anticoagulant against antihemophilic factor. J Clin Invest 1971; 50: 244–54.

32 Ginsburg D, Handin RI, Bonthron DT, et al. Human von Willebrand factor (vWF): isolation of complementary DNA (cDNA) clones and chromosomal localization. Science 1985; 228: 1401–6.

33 Lynch DC, Zimmerman TS, Collins CJ, et al. Molecular cloning of cDNA for human von Willebrand factor: authentication by a new method. Cell 1985; 41: 49–56.

34 Sadler JE, Shelton-Inloes BB, Sorace JM, Harlan JM, Titani K, Davie EW. Cloning and characterization of two cDNAs coding for human von Willebrand factor. Proc Natl Acad Sci U S A 1985; 82: 6394–8.

35 Verweij CL, de Vries CJM, Distel B, et al. Construction of cDA coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene. Nucleic Acids Res 1985; 13: 4699.

36 Titani K, Kumar S, Takio K, et al. Amino acid sequence of human von Willebrand factor. Biochemistry 1986; 25: 3171–84.

37 Sadler JE, Budde U, Eikenboom JC, et al. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thromb Haemost 2006; 4: 2103–14.

38 Mannucci PM. How I treat patients with von Willebrand disease. Blood 2001; 97: 1915–19.

39 Federici AB, Mannucci PM. Management of inherited von Willebrand disease in 2007. Ann Med 2007; 39: 346–58.

2

Biosynthesis and organization of von Willebrand factor

Sandra L. Haberichter

Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA

Introduction

von Willebrand factor (VWF) is a large multimeric adhesive plasma glycoprotein that is synthesized in megakaryocytes and endothelial cells [1–3]. Decreased levels or defective function of VWF cause the most common inherited bleeding disorder, von Willebrand disease (VWD) [4–10], which has a prevalence estimated by some to be as high as 1% [11,12]. The primary function of VWF is to promote platelet binding to subendothelial tissue at the site of a vascular injury. VWF also mediates platelet–platelet interactions, promoting further clotting. A second critical role for VWF is that it serves as the carrier protein for coagulation factor VIII (FVIII), protecting it from proteolytic degradation in plasma. The biosynthesis and organization of VWF involves a com­plex intracellular pathway; defects at any point in this pathway may contribute to the decreased plasma VWF level or dysfunction that causes VWD.

Terminology

It was not known until the 1970s that VWF and FVIII are different proteins with distinct functions [13–16]. VWF is the carrier protein for FVIII in plasma; thus, these two proteins are intimately associated with one another and may copurify when isolated from plasma, leading to erroneous identification. Earlier reports referred to VWF as “FVIII-related antigen,” and this confusion in terminology occasionally still occurs in texts today [17].

A second antigen is absent from plasma and platelets in patients with severe VWD [18]. This additional antigen was historically called von Willebrand antigen II (VW AgII) but is now termed the VWF propeptide (VWFpp) [18,19]. VWFpp was subsequently found to be synthesized in endothelial cells together with VWF [20]. When the VWF gene was later cloned, it was discovered that the N-terminal sequence of pro- VWF was identical to that of VWFpp [19,21]. It is now well established that VWFpp is the 741-amino acid propeptide of VWF that is cleaved from VWF, stored with it in Weibel–Palade bodies of endothelial cells and α-granules in megakaryocytes, and released with VWF into plasma from these storage organelles [19,20,22–25].

Molecular Biology of VWF

The VWF Gene

The coding sequence for VWF was first identified in 1985 by four independent groups [26–29]. The VWF mRNA was shown to be approximately 9 kb in size. After the coding sequence for VWF was identified, the entire VWF gene was cloned [30]. The gene has been localized to chromosome 12 [27,31]. The complete intron/exon sequence has been determined, and the 52 exons span approximately 178 kb [30]. The size of exons varies between 40 bp and 1.4 kb for exon 28. A second, partial VWF sequence has been identified on chromosome 22. This pseudogene shows 97% homology with the authentic VWF gene on chromosome 12. However, the occurrence of several stop codons within the coding sequence indicates that this gene is not expressed in humans. The presence of this second pseudogene can cause problems in identifying sequence abnormalities in patients with VWD, although this can be overcome with proper design of sequencing primers [32–34].

VWF Domain Structure

The open reading frame of the VWF cDNA predicts a 2813-amino acid protein as the primary translation product (Figure 2.1a). The transcriptional start site is located 245 nucleotides upstream of the initiator methionine [35]. Using the current numbering system, the initiating methionine codon is defined as nucleotide number one and the corresponding methionine residue is defined as amino acid 1. The N-terminal segment of VWF includes a hydrophobic 22-amino acid signal peptide and a 741-amino acid propeptide (VWFpp) followed by a 2050-amino acid mature VWF molecule (Figure 2.1a). The propeptide is proteolytically removed from the mature VWF protein in the Golgi, presumably by the enzyme furin (PACE). The VWF protein is composed of a series of repeated homologous domains that are termed A, B, C, and D domains (Figure 2.1b) [25,36–43]. The propeptide contains two D domains, D1 and D2. The mature VWF protein is composed of D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK domains. Comparison of VWF domain sequences with other known protein and DNA sequences identifies several sequence homologies. The A domains have been found to be similar to complement factors B and C2, type VI collagen, chicken cartilage matrix protein, and the α-chains of the leukocyte adhesion molecules Mac-1, p150, 95, and LFA-1 [44,45]. A sequence similarity between small segments of the VWF C1 and C2 domains and thrombospondin has also been identified [46,47]. The D domains of VWF show homology to vitellogenins [48]; however, the importance of these homologies to VWF function is unknown.

Figure 2.1 Domain structure of von Willebrand factor (VWF). (a) VWF is synthesized as pre-pro-VWF containing a 22-amino acid signal peptide (SP), 741-amino acid propeptide (VWFpp), and 2050-amino acid mature VWF molecule. (b) The VWF protein consists of a series of repeated homologous A, B, C, and D domains. VWFpp consists of the D1 and D2 domains. Mature VWF comprises D1-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK domains. VWFpp is proteolytically cleaved from mature VWF at amino acid 763 by the enzyme furin. The D3 domains contain cysteine residues that participate in the multimerization of VWF. In plasma, VWF is proteolytically cleaved by the ADAMTS13 and the cleavage site is located in the A2 domain, between amino acids 1605 and 1606. The last 150 amino acids of VWF, including the CK domain, are important in the C-terminal dimerization of VWF that precedes multimerization. (c) Various functional domains have been identified in the mature VWF protein and contain sites for interaction with coagulation factor VIII, heparin, platelet glycoprotein GPIb, collagen, and platelet glycoprotein GPIIb/IIIa.

This research was originally published in Blood [119] © the American Society of Hematology.

Several functions of VWF have been mapped to specific VWF domains (Figure 2.1c). The D′-D3 domains are important in binding FVIII to VWF [49–51]. The VWF A1 domain is essential in binding VWF to platelets through the platelet receptor glycoprotein, GPIbα [52–56], and also contains binding sites for heparin and collagen [52,57–59]. The A2 domain contains the cleavage site for post-secretion processing of VWF by the VWF-cleaving protease, ADAMTS13 [60–65]. The A3 domain has been reported to contain a binding site for collagen [66–69]. The C1 domain contains an Arg-Gly-Asp-Ser (RGDS) sequence that may bind the platelet glycoprotein GPIIb/IIIa [70].

Mutations in these binding domains have been found in patients with VWD. Patients with type 2N VWD have decreased plasma VWF levels as a result of substantially impaired FVIII binding to VWF [8,10]. Mutations have been identified in the D′-D3 domains in several of these individuals [71–77]. Type 2B VWD is characterized by a loss of plasma high-molecular-weight multimers resulting from the spontaneous binding of VWF to platelets. Mutations in patients with type 2B VWD have been identified in the A1 domain, which contains a binding site for the platelet glycoprotein GPIb [78–82]. Other mutations in the A1 domain prevent the binding of VWF to platelets, characteristic of type 2M VWD [83–85]. Individuals with type 2A VWD have decreased high-molecular-weight multimers and a platelet-binding function. Type 2A VWD results from at least two distinct mechanisms, defective multimerization and secretion, or increased susceptibility to proteolysis by ADAMTS13 [86,87]. Mutations causing increased cleavage by ADAMTS13 are likely to be identified in the A2 domain, which contains the cleavage site for ADAMTS13 proteolysis [88–92]. Mutations causing defective multimerization and secretion have been identified in the D1, D2, D′-D3, A1, A2, and CK domains of VWF [93–98]. Mutations associated with impaired binding of VWF to collagen have also been identified [99].

VWF Promoter

The VWF promoter is complex, and several upstream regulatory elements controlling VWF expression have been identified. A number of consensus sequences for cis-acting elements has been defined in the upstream promoter region and in the first exon, including two GATA-binding sequences [100]. The endothelial cell-specific regulation of VWF expression has been investigated and found to be controlled by a repressor–derepressor mechanism involving an NF1 binding site, an Oct-1 binding site, and Ets transcription fac­tors [35,100–105]. In addition to endothelial cell-specific expression, there are also complex pathways of transcriptional regulation through vascular bed-specific regulation. This vascular bed-specific regulation of the endothelial cell VWF gene is controlled by the tissue microenvironment [106]. The E4BP4 transcriptional repressor has also played a role in the cell type-specific regulation of VWF expression [107]. Additionally, single nucleotide polymorphisms (SNPs) in the VWF regulatory region have been identified that are associated with plasma VWF Ag levels, including the nucleotides –1793, –1234, –1185, and –1051. The regulation of VWF expression is com­plex and controlled by cell-specific and vascular bed-specific regulatory elements.

Cell Biology of VWF

von Willebrand factor is synthesized exclusively in endothelial cells and megakaryocytes [108–110]. The processing of VWF involves a very complicated sequence of events. Most of the studies on VWF processing, assembly, and secretion have utilized cultured endothelial cells or transfected mammalian cells, although some studies on VWF expression in megakaryocytes have also been reported [3,19,25,40, 41,108,110,111–124]. The processing of VWF in endothelial cells and meg­akaryocytes appears to be similar, as VWF from both sources is structurally alike. In both endothe­lial cells and platelets produced by megakaryocytes, VWF forms high molecular weight multimers and is packaged in secretory vesicles; Weibel–Palade bodies in endothelial cells and α-granules in megakaryocytes [3,122]. Much of our understanding of VWF processing comes from expression studies using a variety of mammalian cells, as summarized in the following paragraphs.

VWF Processing and Dimerization in the Endoplasmic Reticulum

When moving through the cell’s secretory pathway, VWF undergoes extensive intracellular modifications (Figure 2.2). VWF is initially synthesized as pre-pro-VWF containing a signal peptide, propeptide, and mature VWF polypeptide. In the endoplasmic reticulum, the signal peptide is removed, the pro-VWF protein is folded, and disulfide bonds are formed (Figure 2.2). VWF is a cysteine-rich protein with 64 cysteine residues in VWFpp and 170 cysteines in the mature VWF protein [21]. In the secreted VWF protein all cysteines appear to be involved in disulfide bonds, as historically no free sulfhydryls have been detected. However, some recent studies employing more sensitive techniques suggest that there may indeed be some reactive unpaired cysteines in plasma VWF [112,125]. While the mapping of disulfide bonds has been accomplished for some cysteines in VWF, the majority of disulfide mapping is unresolved [126–129]. Given the number of cysteines in the full-length VWF, the process of protein fold­ing and disulfide bonding must be exceptionally complicated.

Figure 2.2 Intracellular processing of von Willebrand factor (VWF). The pathway of VWF biosynthesis and organization is depicted in this illustration. The VWF protomer is synthesized as pre-pro-VWF. In the endoplasmic reticulum (ER), VWF is folded, disulfide bonds are formed, glycosylation occurs, the signal peptide is removed, and pro-VWF forms C-terminal dimers. Upon transport to the Golgi apparatus (Golgi), the carbohydrates are processed into complex oligosaccharides, sulfation occurs, and C-terminal pro-VWF dimers are assembled into high molecular weight multimers. Before exiting the Golgi, VWFpp is proteolytically cleaved from VWF, but remains noncovalently associated with the mature VWF multimers. Both VWFpp and mature VWF multimers are either constitutively secreted or routed to regulated secretory granules, Weibel–Palade bodies in endothelial cells, or α-granules in platelets. Once secreted into plasma through either the constitutive or regulated secretory pathway, VWFpp and mature VWF multimers cease to be noncovalently associated and circulate in plasma independently of one another.

This research was originally published in Blood [119] © the American Society of Hematology.

In the endoplasmic reticulum, the pro-VWF subunits form carboxyl-terminal dimers (Figures 2.2 and 2.3). This dimerization involves the last 151 amino acids of the mature VWF protein [117,130]. Voorberg et al. [117] have demonstrated that recombinant VWF which lacks these 151 amino acids fails to dimerize and is proteolytically degraded in the endoplasmic reticulum. Thus, these carboxyl-terminal sequences may serve a role in retaining monomers in the endoplasmic reticulum until they are either dimerized or degraded. The last 90 residues of VWF comprise the CK domain, which contains a sequence homologous to the cysteine knot family of proteins. The common characteristic of this family of proteins is the tendency to dimerize through the formation of disulfide bonds. Further evidence of the importance of this region to VWF dimerization has been provided by an investigation of patients with VWD who have VWF structural abnormalities. Several mutations have been identified in this region of VWF, including C2362F, C2739Y, C2754W, C2773R, and A2801D variants. Expression studies using mutated recombinant VWF variants demonstrated a defective formation of VWF dimers, indicating the critical role of the carboxyl-terminal region in dimerization [97, 131–134]. While the importance of the C-terminal portion of VWF in dimerization is clear, the N-terminal has been found to be less important. The large propeptide VWFpp (pro-VWF) is not necessary for dimerization. Expres­sion of a propeptide-deleted mature VWF (signal peptide sequence followed by mature VWF sequence) results in the secretion of a dimeric VWF protein, indicating that VWFpp is not necessary for the formation of dimers or for exit from the endoplasmic reticulum [41,135,136].

Figure 2.3 von Willebrand factor (VWF) forms high molecular weight multimers. VWF forms a C-terminal dimer in the endoplasmic reticulum, which is generally the smallest form of VWF secreted into plasma. In the Golgi, these C-terminal dimers form N-terminal disulfide bonds to create tetramers, hexamers, octomers, and other such high molecular weight oligomers. The multimeric structure of expressed VWF constructs was analyzed nonreduced on a 2% agarose-SDS gel. Expressed wild-type VWF (lane 2, “Wild-type VWF”) shows a full range of multimers. The mock transfected control is shown in lane 2 (Mock). Expression of propeptide-deleted VWF results in loss of multimerization and only a dimeric VWF species is secreted (lane 3, “Dimeric VWF”). The highest molecular weight VWF multimers are the most active in platelet binding and clot formation.

This research was originally published in Blood [119] © the American Society of Hematology.

In addition to protein folding, disulfide formation, and dimerization, the large pro-VWF is also extensively modified in the endoplasmic reticulum by addition of high-mannose carbohydrate side chains. The mature VWF protein contains 12 N-linked and 10 O-linked glycosylation sites, and VWFpp contains three potential N-linked sites [137]. The O- and N- linked carbohydrates account for approximately 18–19% of the total VWF protein mass. Interestingly, the N-linked oligosaccharides of the plasma VWF protein contain ABO blood group oligosaccharides [138]. Wagner and colleagues [123] found that when human endothelial cells were metabolically labeled, it took approximately 120 minutes for VWF with complex-type oligosaccharide chains to be detected. As soon as metabolically labeled VWF was detected in the cell, it was also found to be constitutively secreted. The exit of pro-VWF from the endoplasmic reticulum appears to be the rate-limiting step in VWF biosynthesis, as it is for other proteins [139,140]. The exit of VWF from the endoplasmic reticulum is dependent upon both glycosylation and dimerization. When N-linked glycosylation is blocked by the addition of tunicamycin to the culture medium of endothelial cells, as reported by Wagner et al., pro-VWF monomers accumulate in the endoplasmic reticulum. These results also indicate that glycosylation is required for dimerization to occur [111]. In addition to glycosylation and dimerization, exit from the endoplasmic reticulum is also dependent upon proper folding of the VWF protein. Misfolded proteins are selected in the endoplasmic reticulum and targeted for degradation, although many cells may retain misfolded proteins in the endoplasmic reticulum [141,142]. A number of mutations have been identified in patients with VWD that result in impaired VWF secretion or endoplasmic reticulum degradation [143–146]. Defec­tive VWF processing within the endoplasmic reticulum may contribute to the VWD phenotype observed in patients.

VWF Processing in the Golgi

When pro-VWF dimers reach the Golgi, the high-mannose glycans are trimmed and galactose and sialic acid are added to form complex-type carbohydrates. Some of the N-linked oligosaccharides are sulfated during transport through the Golgi [147]. Here, the carboxyl-terminal pro-VWF dimers form amino terminal-linked multimers that may exceed 20 million a in size (Figures 2.2 and 2.3). VWF multimerization will be discussed in more detail below. An additional modification that occurs in the Golgi is the proteolytic removal of the 741-amino acid VWFpp (Figure 2.2