Thrombosis Research 128 Suppl.

1 (2011) S3S7

Contents lists available at ScienceDirect

Thrombosis Research
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t h r o m r e s

The pathophysiology of von Willebrand disease: therapeutic implications

Reinhard Schneppenheim*
Department of Pediatric Hematology & Oncology, University Medical Center, Hamburg-Eppendorf, Hamburg, Germany

ARTICLE INFO
Keywords: von Willebrand disease von Willebrand factor Pathology Desmopressin von Willebrand factor concentrate Genotype

ABSTRACT
von Willebrand disease (VWD) is a bleeding disorder characterized by quantitative or qualitative defects in von Willebrand factor (VWF), a multimeric glycoprotein that is essential for platelet-dependent primary hemostasis. High molecular-weight multimers of VWF circulate and bind to collagen and platelets to induce primary hemostasis. The activity of VWF and its eventual proteolytic degradation are dependent on shear stress, ensuring that, under normal conditions, VWF is active in a high shear stress environment only. Deficiency in VWF results in mucocutaneous bleeding, including epistaxis, menorrhagia, and excessive bleeding after trauma or surgery. Classification of VWD is based on the combined results of multiple laboratory tests related to VWF amount and activity as well as the relative amounts of large VWF multimers as determined by gel electrophoresis. Recently, specific mutations in the gene encoding VWF have been linked to characteristic multimer profiles and may aid in subtyping patients with VWD and predicting response to therapy. These genotypephenotype correlations are improving our understanding of the pathophysiology of VWD and helping to provide a more accurate diagnosis and classification with important treatment-related implications. 2011 Elsevier Ltd. All rights reserved.

Abbreviations aPTT: activated partial thromboplastin time DDAVP: 1-desamino-8-D-arginine vasopressin (desmopressin) ER: endoplasmic reticulum FVIII: factor VIII GP: glycoprotein PFA100: platelet function analyzer 100 RIPA: ristocetin-induced platelet agglutination VWD: von Willebrand disease VWF: von Willebrand factor VWF:Ag: VWF antigen VWF:CB: VWF:collagen binding VWF:GPIb: VWF:glycoprotein Ib binding VWF:RCo: VWF:ristocetin cofactor von Willebrand disease (VWD) is a congenital bleeding disorder characterized by reduced amounts of von Willebrand factor (VWF) or abnormal forms of VWF in the circulation [1]. High molecular-weight VWF is essential for platelet-dependent primary hemostasis. VWF is also a carrier of factor VIII (FVIII) and protects FVIII from proteolytic degradation while delivering it to sites of vascular damage for secondary hemostasis. Deficiency in VWF can result in mucocutaneous bleeding, including epistaxis, menorrhagia, and excessive bleeding after trauma or surgery [2]. Patients with severe forms of VWD may also develop joint and muscle bleedings similar to what is seen in patients with hemophilia.
*Corresponding author. Dr. R. Schneppenheim, University Medical Center Hamburg-Eppendorf, Department of Pediatric Hematology & Oncology, Martinistrasse 52, 20246 Hamburg, Germany. *Tel.: +49 40 74105 4270; fax: +49 40 74105 4601 E-mail address: schneppenheim@uke.de (R. Schneppenheim).
0049-3848 /$ see front matter 2011 Elsevier Ltd. All rights reserved.

Patients with VWD are classified into one of three main disease types based on partial (type 1) or complete (type 3) quantitative deficiency in VWF or qualitative VWF deficiency (type 2) [1]. Diagnosis and classification of VWD remains a challenge, but recent advances in diagnostic evaluation and additional insights into the molecular and genetic events that lead to VWD are helping to improve the clinical management of patients with VWD. VWF structure and function Genetic structure To understand the pathophysiology of VWD, it is important to first understand the biosynthesis, function and molecular genetics of VWF. VWF is synthesized predominantly in endothelial cells and either secreted constitutively or stored in WeibelPalade bodies within endothelial cells before stimulated release into the circulation [3,4]. The VWF monomer is comprised of 2,813 amino acids arranged in repeating domains (Figure 1) [4-8]. These domains may be characterized as structural or functional, depending on whether their primary role is related to VWF structure or its interaction with other factors. The cysteine knot domain, for example, is responsible for dimerization of VWF monomers and can therefore be considered a structural domain. Similarly, the D3 domain is required for proper multimerization of VWF dimers. All D domains contain a consensus sequence of disulfide isomerases (CGLC) that are necessary for proper multimerization involving D3 [6,7]. Mutations in these sequences (with the exception of D4) will prevent multimerization in D3. Functional domains include those that contain cleavage sites for proteolysis (domain A2) and binding sites for collagen (domains A1 and A3), platelets (domain A1 for glycoprotein [GP] Ib [GPIb] receptors and domain C1 for GPIIb/IIIa receptors), and factor VIII (FVIII; domain D). Multimerization The VWF monomer has a molecular weight of about 250 kDa. VWF monomers dimerize in the endoplasmic reticulum (ER), and only dimers are usually

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Fig. 1. Structural and functional domains of von Willebrand factor [6-8]. CGLC, amino acid consensus sequence of disulfide isomerases; Collagen, collagen binding site; FVIIIB, factor VIII binding site; GPIb, glycoprotein Ib binding site; RGD, arginine-glycine-aspartic acid sequence; SP, signal peptide.

released from the ER. In the Golgi apparatus and post-Golgi compartments, dimers undergo multimerization to form tetramers, hexamers, and so on, eventually creating very large multimers containing up to 100 monomers [4,5]. Multimerization is an important step because, while VWF multimers of any size can bind to FVIII, high molecular-weight multimers have more efficient binding capacity for collagen and the platelet receptors GPIb, and GPIIb/IIIa [9,10]. Thus, large multimers are necessary for adequate VWF function in hemostasis. Proteolysis While deficient VWF can lead to bleeding complications, excessive VWF activity can create large hemostatic plugs that may lead to thrombotic thrombocytopenic purpura [11,12]. An important regulator of VWF activity is the VWF-specific protease ADAMTS13 [13]. This protease cleaves large VWF multimers at their A2 domains, creating smaller multimers with limited activity. Our laboratory has shown that increasing amounts of ADAMTS13 will continue to cleave recombinant human VWF multimers into smaller and smaller molecules, eventually leaving only dimers [14]. Shear stress VWF multimers are the largest molecules in circulation and can exceed 20,000 kDa [3,15]. Due to their size, VWF multimers maintain a flexible conformation that is uniquely responsive to shear stress in circulation, and shear stress has been shown to regulate many VWF interactions [10,12,15,16]. High shear stress changes the shape of the molecule from a collapsed, globular form to an elongated or stretched form (Figure 2) [10]. Stretching the molecule exposes key binding sites, thereby enhancing its activity. In this way, VWF is activated in a high shear stress environment. Notably, the cleavage site for ADAMTS13 is also exposed when VWF is stretched [16]. Thus, breakdown of VWF multimers typically only occurs after the molecule is activated; this mechanism allows ADAMTS13 to cleave VWF and avoid pathologic platelet aggregation and thrombotic thrombocytopenic purpura. The dependency of VWF activity on shear stress is important because VWF fulfills its main function in a high shear stress environment only. Understanding how shear stress influences the activity of ADAMTS13 on VWF may also help design drugs that correct aberrant proteolysis, leading to new potential treatment options for patients with type 2 VWD [16]. Aberrant proteolysis Given the complex structural and functional properties of VWF, it is not surprising that there are multiple pathomechanisms that lead to deficient or non-functional VWF [8]. For example, mutations that affect binding sites for collagen or platelets can interfere with VWF function, and mutations that affect the FVIII binding site can lead to low levels of FVIII, similar to what is seen in hemophilia A. Proteolysis is an important regulator of VWF activity, and certain conditions are associated with aberrant proteolysis. The VWD type 2A subtype IIA is characterized by constitutively increased susceptibility to proteolysis [8,14]. In this situation, VWF is cleavable by ADAMTS13 in its condensed
Fig. 2. Impact of shear stress on von Willebrand factor (VWF). In a low shear environment, VWF has a collapsed, globular conformation with few binding sites exposed. As shear forces increase, VWF is stretched and binding sites are exposed. Reproduced with permission from Schneider SW, Nuschele S, Wixforth A, Gorzelanny C, Alexander-Katz A, Netz RR, Schneider MF. Shearinduced unfolding triggers adhesion of von Willebrand factor fibers. Proc Natl Acad Sci USA 2007;104:7899-903 [10]. Copyright 2007 National Academy of Sciences, U.S.A.

form in a low shear stress environment; this prevents maintenance of large multimers in circulation, thereby decreasing platelet-dependent hemostasis. The subtype 2B is characterized by spontaneous GPIb binding. This allows spontaneous platelet binding to VWF in the circulation, thereby magnifying the shear exposed area with subsequent stretching of the molecule which is making it susceptible to ADAMTS13 cleavage. Similar to type 2A/IIA, this scenario results in a lack of large multimers necessary for hemostasis. The characteristic multimeric pattern can be determined by multimer analysis. However, the multimer pattern is indistinguishable from the 2A/IIA subtype and the final diagnosis can only be made by applying either a ristocetininduced platelet agglutination (RIPA) assay using platelet rich patient plasma or by molecular testing. Other conditions are associated with insufficient proteolysis. Mutations that result in defective platelet binding can result in reduced (subtype IIE) or even absent proteolysis (subtype IIC, IID) [17,18]. Diagnosis and classification Diagnosis of VWD is based on three components: a history of mucocutaneous bleeding; a family history of bleeding; and laboratory results that are consistent with a quantitative or qualitative defect in VWF [19]. For patients suspected of having a bleeding disorder, a number of laboratory tests may be necessary for proper diagnosis. Initial standard tests include activated partial thromboplastin time (aPTT), FVIII activity (FVIII:C), bleeding time, platelet function analyzer 100 (PFA100) assay, VWF antigen (VWF:Ag), VWF:ristocetin

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Fig. 3. Classification of von Willebrand disease [1].

cofactor (VWF:RCo), and VWF:collagen binding (VWF:CB). A novel latex immunoassay for VWF:GPIb binding (VWF:GPIb), which more adequately measures the platelet dependent function of VWF, is also commercially available now. If the results from these tests are normal, patients with a high suspicion of bleeding disorders may undergo additional platelet testing. Abnormal or unclear results from any standard test warrant further investigation with specialized assays related to VWD, such as multimer analysis, RIPA, VWF:FVIIIB assay, desmopressin (DDAVP) test, and if indicated, molecular testing. Classification of VWD is based on the nature of the VWF deficiency (Figure 3) [1,20]. Types 1 and 3 are characterized by quantitative deficiencies in VWF. This can be caused by defects that produce relative (type 1) or absolute (type 3) deficiency in VWF due to impaired synthesis, secretion, or half-life of the molecule. A VWF level less than 3040 IU/dL by VWF:RCo or VWF:Ag assays indicates type 1 VWF [21]. Type 2 VWD is characterized by qualitative VWF deficiency: levels of VWF:Ag may be decreased, normal or even elevated, but structural or functional defects lead to impaired activity. Many type 2 VWD phenotypes can be distinguished from VWD type 1 by their decreased ratios of functional parameters, i.e. VWF:RCo, VWFCB, VWF:GPIbB, VWF:FVIIIB, respectively, to VWF:Ag [21]. However, reliable classification of patients with VWD types 2A, 2B and 2M, additionally always requires multimer analysis. By this approach, the different subtypes of VWD type 2A (IIA, IIC, IID, IIE), and rare variants, can also be identified. Structural defects leading to impaired dimerization and/ or multimerization lead to type 2A/IID, 2A/IIC, and 2A/IIE VWD, respectively; defects that involve aberrant proteolysis can lead to type 2A/IIA and 2B disease. Functional defects that impair platelet binding, FVIII binding, and collagen binding produce type 2M, 2N, and 2CB VWD, respectively. Impaired VWF survival time can be detected in patients with type 2 VWD by the VWF propeptide assay and its ratio to VWF:Ag [21] or by DDAVP testing, which monitors the rise but also the decrease in VWF parameters after DDAVP infusion over time. However, this requires at least a time point of 4 hours after the start of DDAVP infusion [22]. Genetic analysis can help further categorize VWD, and numerous mutations in the gene encoding VWF have been identified that correlate with specific subtypes of VWD (Figure 4) [5,8,18,23]. In our laboratory, for example, we identified a distinct VWD phenotype among patients with type 2 VWD that closely resembles the previously described subtype 2A/IIE

[18]. A total of 22 different mutations were identified that correlated with this phenotype, which was characterized by a relative reduction in large multimers. Although these patients shared a common multimeric pattern, levels of VWF and functional parameters varied markedly, underscoring the complexity of diagnosing VWF disorders. Diagnosis and classification of VWD remains a challenge. No single test is diagnostic for VWD, and diagnosis is therefore based on a combination of patients medical history and the results of the battery of laboratory tests mentioned above. VWF multimer analysis is considered the gold standard for classifying VWD, but this method is technically challenging and difficult to standardize [20]. Molecular genetic analysis of patients with known or suspected VWD is often useful, and is likely to play an increasingly important role in the diagnosis and classification in the future but is a prerequisite for genetic counseling. Management Establishing the correct subtype of VWD is important because options for treatment and prophylaxis vary depending on the subtype. Situationdependent prophylaxis is warranted for most cases of type 3 VWD [24], and long-term prophylaxis is recommended for any patient with lifethreatening bleeding, severe menorrhagia not responding to hormonal regulation, and repeated bleeding in muscles, joints, or the gastrointestinal tract [25,26]. Treatment options include replacement with VWF-containing concentrates or release of VWF from endogenous storage organelles by DDAVP; antifibrinolytic agents can also be effective, particularly in patients with mucosal tissue bleeding. For female patients, hormonal regulation of menorrhagia is an important treatment option [27]. Replacement therapy involves administration of plasma-derived, VWFcontaining FVIII concentrates (several types are commercially available) or a recombinant VWF product, which is in preclinical testing and may be available in the future [19]. Given the lack of comparative trials, the relative efficacy and safety of available VWF products is difficult to determine, although it is known that these products contain varying amounts of large VWF multimers [20], which may influence their clinical effects [28]. VWF/FVIII replacement concentrates are necessary for patients who are unresponsive to DDAVP, experience major bleedings, have a decreased VWF half-life, or have type 3 VWD.

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Fig. 4. Genotypephenotype correlation in von Willebrand disease. Triangles indicate mutation clusters within individual domains of von Willebrand factor that are associated with characteristic patterns of multimerization. Arabic numerals refer to current classification; Roman numerals refer to previous classification systems. Reproduced with permission from Schneppenheim R, Budde U. von Willebrand factor: the complex molecular genetics of a multidomain and multifunctional protein. J Thromb Haemost 2011;9:209-15 [8]. 2011 International Society on Thrombosis and Haemostasis. CGLC, amino acid consensus sequence of disulfide isomerases; Collagen, collagen binding site; FVIIIB, factor VIII binding site; GPIb, glycoprotein Ib binding site; NP, normal plasma; RGD, arginineglycine-aspartic acid sequence; SP, signal peptide.

Use of DDAVP mobilizes endogenous VWF from WeibelPalade bodies in the endothelium [25,29]. Some endothelial cells contain receptors for the antidiuretic hormone vasopressin, and vasopressin and its synthetic analog DDAVP bind to these receptors and stimulate the exocytosis of VWF [30]. This can rapidly provide a 3- to 5-fold increase in VWF levels, including a high proportion of large VWF multimers, and is very effective in many patients with VWD type 1 and also in some patients with VWD type 2 [22,31]. DDAVP therefore has a significant role in the prevention and treatment of bleeding episodes in patients with milder forms of VWD. It is, however, not effective in patients with type 3 VWD because these patients cannot produce any VWF; DDAVP therefore cannot be used in patients with type 3 VWD. Similarly, DDAVP is not used in patients with type 2B disease, because it may exacerbate associated thrombocytopenia [32]. For patients with type 1 VWD (relative, quantitative VWF deficiency), testing for DDAVP responsiveness is an important step in determining the optimal treatment approach. A typical DDAVP test in a patient with type 1 VWD will reveal a rapid and sustained increase in VWF levels and activity; this indicates that DDAVP can be effectively used to manage nose bleeds or as prophylaxis for minor surgery in these patients. Response to DDAVP, however, varies among patients with type 1 VWD, depending on the type of VWF mutation [33]. The DDAVP test is therefore necessary even in patients with type 1 VWD to determine if response is sufficient. Response to DDAVP among patients with type 2 VWD is mixed. In general, most type 2 patients produce dysfunctional VWF, so increasing levels of dysfunctional endogenous VWF with DDAVP is not a viable strategy. Patients who lack large VWF multimers due to defects of dimerization or multimerization (e.g. type 2/IIA, IIE, IIC, IID), would not be expected to have an adequate increase in VWF activity following DDAVP administration. We have tested patients with various mutations associated with type 2 VWD and found that the type of mutation present correlated with DDAVP response [22]. Among patients with type 2A/IIA with enhanced proteolysis, those with mutations in the A2 domain, e.g. R1597Q, had a rapid decline in VWF levels subsequent to an initial rise of VWF:Ag and VWF:RCo on DDAVP

stimulation, indicating that DDAVP may not provide adequate prophylaxis for surgical procedures in these patients. Those with the G1503R mutation showed no change in functional parameters of VWF after DDAVP stimulation, indicating that DDAVP would not be effective in these patients. Other specific mutations in patients with type 2A/IIC (M1051T/4820delG) and 2M (Y1409F) known to impair VWF function were found to predict an insufficient DDAVP response. While the mutations mentioned above predict negative DDAVP response, others were found to predict an unexpectedly positive response. For example, among patients with a type 1/2A (smeary) profile, for whom DDAVP is not normally indicated, those with the R1315C mutation were found to have an adequate DDAVP response. Similarly, patients with type 2B VWD are not usually candidates for DDAVP due to severe thrombocytopenia [34], but those with the common R1341Q mutation do not exhibit severe thrombocytopenia, and DDAVP has been safely used in these patients (own unpublished observations). These findings provide further insight into the correlation between genotype and phenotype in VWD and highlight the importance of DDAVP testing in patients with type 1 or 2 VWD before making treatment decisions. Summary Current knowledge regarding the pathophysiology of VWD continues to expand and is already having direct implications on the management of patients with VWD. The complexity of VWF structure and function provides numerous opportunities for error, and many of the resulting pathomechanisms produce unique phenotypic characteristics. While all pathomechanisms ultimately result in deficient VWF activity, their unique characteristics can help determine the best therapeutic approach. For patients with type 3 VWD, for example, the total lack of VWF indicates that DDAVP is not beneficial, while prophylaxis with plasma-derived, VWF-containing concentrates is necessary. Since even type 1 VWD turned out to be a heterogeneous disorder, a DDAVP test is also recommended for these patients. DDAVP may not be sufficient in certain type 1 patients, such as those with mutations that lead to

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a markedly reduced half-life of high molecular-weight VWF in the circulation (i.e. subtype 1 Vicenza). Among certain patients with type 2 VWD, a DDAVP test is also recommended: DDAVP may be effective in certain patients with minor bleeding, such as those with type 2A/IIA, 2M, and patients with a mild 2N VWD mutation (R854Q). For those with major bleeding, DDAVP will not be sufficient. The ability to predict DDAVP response based on the presence of certain mutations in patients with type 1 and 2 VWD has provided further insight into genotypephenotype correlations in VWD; genetic analysis and genotypephenotype correlations are expected to play an increasingly important role in patient management, treatment decisions, and genetic counseling in the future. Acknowledgements The author wishes to acknowledge Swiss Medical Press GmbH for editorial assistance. Conflict of interest R. Schneppenheim received research funding by CSL Behring. Role of the funding source Phenotype-genotype correlation with respect to DDAVP response. References
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