Luciano Baronciani Pier Mannuccio Mannucci

Introduction, 199 Von Willebrand disease and its classification, 200

Function of von Willebrand factor in primary hemostasis, 199 Genetic defects in von Willebrand disease, 203

Function of von Willebrand factor in blood coagulation, 199 Treatment of von Willebrand disease, 205

Gene organization, synthesis and multimeric structure of von Willebrand Von Willebrand disease resources on the Internet, 208

factor, 200 Further reading, 208

Introduction

Von Willebrand disease (vWD) is a common inherited bleeding disorder. Precise data regarding its prevalence are not available because of the extreme variability in clinical symptoms of mild vWD. However, population-based studies give an estimate of clinically significant vWD with a prevalence of at least 100 per million. vWD is caused by the deficiency or dysfunction of a multimeric plasma glycoprotein, von Willebrand factor (vWF). Because of its ability to bind to a number of ligands, vWF is involved in hemostasis via a variety of mechanisms but can be considered as having two main roles, in primary hemostasis and in intrinsic blood coagulation. vWF is directly involved in platelet binding to the subendo-thelium and in platelet-to-platelet interactions, and also acts as the carrier of procoagulant factor VIII (FVIII). Mutations at the VWF locus can affect vWF synthesis, its complex bio-synthetic assembly, its stability in the circulation, and its binding interactions with specific ligands.

Function of von Willebrand factor in primary hemostasis

At the time of a hemostatic challenge, vWF acts as a bridge between platelets and the subendothelium of blood vessels, and is involved in the formation of the platelet plug. The role of platelets in hemostasis is to become irreversibly attached at sites of injury. The primary physical factor that affects platelet binding to the vessel wall is the rate of blood flow in the vessel, which is faster at the center and slower close to the wall. These variations in velocity create a shearing effect, or shear stress, between layers of fluid. Disruption of the vascular endothelial surface leads to exposure of the subendothelium and results in an alteration in the rate of blood flow and an increase in shear stress. Plasma vWF binds rapidly and tightly to subendothelial collagen. vWF does not constitutively bind platelets, but the immobilized vWF becomes activated. Even in high blood flow conditions, vWF is able to tether platelets and to expose thrombogenic surfaces through the interaction of its A1 domain and platelet receptor Gplb. However, the vWF-GpIb interaction does not provide irreversible platelet adhesion because of the fast dissociation rate, and platelets tethered to the vessel wall still move constantly in the direction of the flow, but at a much slower rate. A second molecule on the platelet surface is required to obtain firm platelet adhesion: the integrin GpIIb-IIIa (aIIbP3). This molecule is responsible for the platelet-to-platelet interaction, which is mediated once again by vWF and, under slow-flow conditions, by fibrinogen. aIIbP3 does not appear to be involved in the first events of platelet adhesion, probably because its rate of binding to vWF is too slow to mediate the initial platelet attachment to the vessel wall under high-flow conditions. However, when platelets become activated as a consequence of the vWF-GpIb interaction, aIIbP3 increases its affinity for its ligand, vWF. This event, together with the slow motion of the platelets due to the vWF-GpIb interaction, allows aIIbP3 to bind platelets irreversibly to the vessel wall (Figure 17.1). In vessels with a high shear rate, vWF is the primary mediator of platelet binding to the vessel wall and of platelet aggregation. This function of vWF is of great importance in primary hemostasis.

Function of von Willebrand factor in blood coagulation vWF circulates in the plasma as a complex associated with

Fluid flow

Initial tethering

Translocation

Torque nonactivated Activation

o, \ / i , F/ (->, Pi y , \ i Ù, \ / y ; \ / > , FTnmw, FT H v ,

Fluid flow

Initial tethering

Firm adhesion activated

Fig. 17.1 Schematic representation of platelet adhesion to immobilized von Willebrand factor

At first, platelets are tethered to the subendothelium through the interaction of their GPIba with von Willebrand factor (vWF; A1 domain), and the inactivated — aIIbP3 does not bind to the RGDS sequence of vWF. Because of the torque imposed by the flowing fluid, the platelets begin to roll. New bounds are formed as different regions of the membrane of the rolling platelets come into contact with the surface, and the translocation continues until the aIIbP3 becomes activated and binds firmly to the RGDS of the vWF. Adapted with permission from Ruggeri ZM (1997). von Willebrand factor. Journal of Clinical Investigation, 99, 559-564.

FVIII. The binding of vWF to FVIII is required to stabilize FVIII in the circulation, preventing cleavage by activated protein C (APC) or factor Xa. This interaction with vWF is crucial in prolonging the half-life of FVIII and concentrating FVIII at the point of bleeding. When patients with severe vWD (type 3) are treated with purified FVIII, this is cleared with a half-life of around 2 hours, whereas the vWF-FVIII complex infused in the same patient has a half-life of about 20-24 hours. vWF binds to FVIII via regions within the first 272 residues of the mature polypeptide, in the D' and D3 domains of vWF.

by disulfide bonding between cysteine residues in the cystine knot (CK) carboxyl-terminal region of the monomer (tail-to-tail dimerization). The signal peptide is cleaved before it enters the Golgi apparatus. The tail-to-tail glycosylated dimers are then transported to the Golgi apparatus where multimer-ization and further glycosylation occur. The propeptide of vWF mediates the assembly of vWF multimers. This process requires the presence of the D1 and D2 domains (propeptide) and the D' and D3 domains of the mature polypeptide. This is followed by cleavage of the propeptide sequence (Figure 17.3) and secretion of both the mature polypeptide and the pro-peptide into the circulation, or storage within the Weibel-Pal-ade bodies of endothelial cells or the a-granules of platelets. The molecular weight of the mature subunit is 220 kDa but circulates as multimers of up to 20 000 kDa.

Gene organization, synthesis and multimeric structure of von Willebrand factor

The gene encoding vWF has been mapped on chromosome 12, has a length of approximately 178 kb, with 52 exons, and transcribes a messenger RNA of about 8.2 kb. Analysis of the VWF gene is complicated by the existence of a partial unprocessed pseudogene on chromosome 22. The pseudogene extends from exon 23 to exon 34 and presents a high degree of homology with the gene (97%). vWF is synthesized in megakaryocytes and endothelial cells as a precursor of 2813 amino acids, the prepro-vWF. It is composed of a 22-residue signal peptide, a 741-residue propeptide and a 2050-residue mature subunit. More than 95% of the sequence accounts for structural domains that are arranged in the following order: D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (Figure 17.2). The biosynthesis of vWF is a complex process, involving post-translational processing of the protein prior to storage or release into the circulation. The initial dimerization occurs

Von Willebrand disease and its classification

The most common symptoms of mild vWD are mucosal bleeding (epistaxis, gingival bleeding and menorrhagia) and prolonged bleeding after surgical procedures and dental extractions. Hemarthroses and soft-tissue hematomas are rare, but they occur in severely affected individuals. The diagnosis of vWD is suspected in individuals with these symptoms and a family history of bleeding. Several vWF assays are used in the diagnosis of vWD and its subtypes, such as those that measure the plasma levels of vWF antigen (vWF:Ag), vWF binding to type I or type III collagen (collagen binding activity, vWF:CB) and vWF interactions with the antibiotic ristocetin and platelet glycoprotein Ib (vWF ristocetin cofactor activity, vWF: RCo). This large number of measurements reflects the fact

The molecular basis of von Willebrand disease 201

Gene chromosome 12 51 intron 178 kb

Pseudogene chromosome 22

2N VWD

2B 2M 2A VWD VWD VWD

D'

D3

A1

A2

FVIII multimer GPIb heparin S-S heparin collagen collagen

COOH

GPIIb/IIIa dimer 2813 a.a. S-S

pro-peptide VWF mature polypeptide

Fig. 17.2 Structure of the vWF gene, pseudogene and protein

The schematic structure of the prepro-vWF is shown, along with the homologous repeated domain. Also shown are the locations of intersubunit disulfide bonds involved in dimerization and multimerization, and the binding sites for several ligands.

Fig. 17.3 Processing steps of vWF multimer synthesis and subcellular localization of the post-translational modification events

The propeptide is shown in blue and the mature subunit in gray. The N and C prefixes represent the amino- and carboxyl-terminal ends of the protein respectively. Monomers are linked together at the C-termini by disulfide bonds to form dimers (endoplasmic reticulum), which further multimerize in the Golgi apparatus. Cleavage of the propeptide occurs before secretion.

mRNA

Translation pre-pro-VWF

sp VWAgll

I glycosylation c 2813 a.a. (260 Kda)

VWF C-terminal s's dimerization /v 520kDa

Carbohydrate I N-terminal pro-VWF multimer pr°cessing T

multimerization

NN up to 20000 kDa Propeptide cleavage

VWF multimer

Rough endoplasmic reticulum

Golgi apparatus

Golgi storage bodies or secretion

that none of them is by itself sensitive and specific enough for a diagnosis of vWD.

vWD is a highly heterogeneous disease in which there are quantitative and qualitative abnormalities of vWF, usually resulting from mutations at the VWF locus. The classification identifies three basic types (Table 17.1). Type 1 is characterized by the partial quantitative deficiency of vWF. Type

2 is characterized by qualitative abnormalities of the moiety and is further categorized into subgroups (2A, 2B, 2M and 2N) depending on the nature of the qualitative defect. Type

3 is the most severe form of the disease and is characterized by the complete absence of vWF in plasma and platelets. Transmission of type 1 vWD is usually dominant, type 2 is dominant in the majority of cases, whereas transmission of

Table 17.1

Current classification of von Willebrand disease.

Type 1

Partial quantitative deficiency of vWF

Type 2

Qualitative deficiency of vWF

2A

Decreased platelet-dependent vWF function, with

lack of HMWM

2B

Increased vWF platelet-dependent vWF function,

with lack of HMWM

2M

Decreased platelet-dependent vWF function, with

normal multimeric structure

2N

Decreased vWF affinity for FVIII

Type 3

Complete deficiency of vWF

FVIII, factor VIII; HMWM, high molecular weight multimers; vWF, von Willebrand factor.

type 3 is recessive (Figure 17.4). The penetrance of disease due to the same mutation can be variable, even within the same family. In the last decade, many molecular defects of the VWF gene have been identified in vWD patients, mainly in the functional variants (types 2A, 2B and 2N). The unique phenotypes of these variants made it possible to restrict the genetic analysis to specific structural domains, such as the A1 domain for the variant 2B, A2 for the variant 2A and D'-D3 for the variant 2N. On the other hand, characterization of molecular defects in types 1 and 3 vWD requires extensive screening, since mutations are not restricted to specific regions and are usually scattered throughout the VWF gene. In most type 2 cases the mutations consist of amino acid substitutions (missense mutations), although small in-frame deletions or insertions have been reported. In type 3 vWD

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