An update on type 2B von Willebrand disease Expert Review of Hematology Downloaded from by Nyu Medical Center on 06/19/15 For personal use only.

Expert Rev. Hematol. 7(2), 217–231 (2014)

Sameh Mikhail*1, Ehab Saad Aldin2, Michael Streiff3 and Amer Zeidan3,4 1 Department of Hematology, Ohio State University Medical Center, Columbus, OH, USA 2 Department of Internal Medicine, MedStar Good Samaritan Hospital, Baltimore, MD, USA 3 Division of Hematology, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA 4 Department of Oncology, Johns Hopkins University, Baltimore, MD, USA *Author for correspondence: Tel.: +1 410 614 4459 Fax: +1 410 955 0185 [email protected]

Type 2B von Willebrand disease (VWD) accounts for fewer than 5% of all VWD patients. In this disease, mutations in the A1 domain result in increased von Willebrand factor (VWF) binding to platelet GPIba receptors, causing increased platelet clearance and preferential loss of high molecular weight VWF multimers. Diagnosis is complicated because of significant clinical variations even among patients with identical mutations. Platelet transfusion often provides suboptimal results since transfused platelets may be aggregated by the patients’ abnormal VWF. Desmopressin may cause a transient decrease in platelet count that could lead to an increased risk of bleeding. Replacement therapy with factor VIII/VWF concentrates is the most effective approach to prevention and treatment of bleeding in type 2B VWD. KEYWORDS: bleeding disorder • deamino arginine vasopressin • inherited thrombocytopenia • type 2B von Willebrand disease • von Willebrand factor

von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, was first described by Erik von Willebrand in 1926 in several members of a family from the A¨land islands in the Baltic Sea off Finland [1]. The understanding of VWD did not evolve, however, until the immunological characterization of von Willebrand factor (VWF) and factor VIII (FVIII) in the early 1970s. This discovery led to the cloning of FVIII in 1984 [2,3] and VWF in 1985 [4–7]. Molecular defects responsible for VWD were first detected by Southern blot in 1987 [8]. The development of polymerase chain reaction in 1985 and the application of Taq polymerase in 1987 facilitated the identification of point mutations in patients with VWD [9,10]. VWD is classified into three types [11]. Type 1 is a mild-moderate quantitative deficiency of VWF that is mostly inherited in an autosomal dominant fashion. It is the most common type of VWD, accounting for approximately 75% of patients. Conversely, type 3, the least common type of VWD (with a prevalence of 0.5–6 per million people), is an autosomal recessive disease notable for a profound quantitative deficiency of VWF that is associated with a severe phenotype. Type 2 disease is characterized by a variety of qualitative defects in VWF function, and generally, with the exception of type 2N VWD, follows an autosomal dominant inheritance pattern. 10.1586/17474086.2014.868771

Type 2 VWD has been subdivided into types 2A, 2B, 2M and 2N based upon the different defects in VWF function resulting from mutations in different regions of the VWF molecule (FIGURE 1) [12]. Among type 2 variants, type 2B VWD is a rare subtype, accounting for fewer than 5% of all patients with VWD [11]. Type 2B VWD was first identified in 1977 but it took several years to begin to understand its pathophysiology [13,14]. Synthesis & structure of VWF

VWF is synthesized as a pre-pro-protein (comprising 2813 amino acids) by endothelial cells and megakaryocytes [15,16], and subsequently undergoes extensive post-translational processing in the endoplasmic reticulum and the Golgi apparatus [17]. The pro-protein of 2791 is highly repetitive and contains several structural domains, initially designated as follows: D1-D2-D´-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2 [16,18]. Homology studies later on revealed that the VWF contains a cysteine knot (CK) domain, comprising 6 cysteine residues organized into a knot-like conformation, at its C-terminal end [19]. Thus, the VWF domains have been changed to D1-D2-D´-D3-A1-A2A3-D4-B1-B2-B3-C1-C2-CK [20]. Dimerization of VWF multimers occurs in the endoplasmic reticulum via formation of disulfide bonds between their CK domains (tail-to-tail), a

 2014 Informa UK Ltd

ISSN 1747-4086



Mikhail, Aldin, Streiff & Zeidan


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FVIII Expert Review of Hematology Downloaded from by Nyu Medical Center on 06/19/15 For personal use only.




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GPIb (Collagen) Botrocetin Heparin Sulphatide






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RGD αIIbβ3







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2B Propeptide


Mature VWF

All mutations are in exon 28 of the A1 domain of the VWF gene on chromosome 12 DNA change







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Figure 1. Schematic diagram showing the von Willebr and factor gene on chromosome 12 as well as the different von Willebrand factor functional domains involved in different subtypes of type 2 von Willebrand disease. Type 2A VWD refers to a disease with decreased VWF-dependent platelet adhesion with selective deficiency of high molecular weight multimers. Mutations in the D2 domain of the VWF pro-peptide, or less commonly in the D3 domain, A2 domain or the ‘cysteine knot’ domain, are responsible for type 2A VWD. Type 2B VWD refers to a disease subtype with enhanced affinity of VWF to the platelet GPIba receptors, most commonly due to a gain of function mutation of the A1 domain of the VWF. Mutations in type 2M VWD have been identified in the A1 domain of the VWF. Type 2N VWD causes an autosomal recessive hemophilia A picture, with mutations in type 2N VWD identified within the FVIII binding site of VWF (spanning the D´ domain and part of the D3 domain). The table lists the DNA mutations and associated protein changes in patients with VWD type 2B. VWD: von Willebrand disease; VWF: von Willebrand factor. Adapted from [12,20,203].

process facilitated by the addition of N-terminal carbohydrate residues that also allow for transit to the Golgi apparatus. In the Golgi apparatus, large VWF multimers (up to 20 million Daltons) are formed by the formation of ‘head-to-head’ interchain disulfide bonds between the D3 domains of VWF dimers, and further glycosylation of VWF occurs that influences processing after secretion. In the Golgi, the VWF pro-peptide sequence is cleaved producing mature VWF multimers. Multimerization occurs in the Golgi apparatus despite 218

the acidic environment and the lack of enzymes necessary for disulfide bond formation, and the fact that multimerization of VWF can be reproduced in vitro at low pH indicates that all the required factors are contained within VWF itself [16,21]. Noteworthy is that the pro-peptide does form transient intrachain disulfide bonds in the ER, and in the Golgi apparatus these are substituted by new interchain bonds between the D3 domains [22]. The pro-peptide contains two constrained CXXC motifs (Cys159-Gly-Leu-Cys162 and Cys521-Gly-Leu-Cys524) that are Expert Rev. Hematol. 7(2), (2014)

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Type 2B von Willebrand disease

crucial for the formation of VWF multimers, as are two of the 52 cysteine residues in the D´-D3 domain [16,23,24]. Although the three-dimensional structure of VWF is not fully understood, it is known that VWF polymers assemble into tubules, starting in the trans-Golgi network [25,26]. These tubules get packed into vesicles that bud off, forming immature WeibelPalade bodies that later mature and wait in the endothelial cells for a signal to undergo exocytosis and release of VWF [16,27]. Since the cleaved pro-peptide fragment is retained in Weibel-Palade body storage granules in endothelial cells, measurement of its plasma level can be useful in the diagnosis of acquired VWD. Synthesis of VWF is regulated in part by humoral factors such as estrogen and thyroid hormones [28,29]. In addition to the Weibel-Palade bodies in endothelial cells, VWF is also stored in the alpha granules of platelets [30]. VWF secretion is stimulated by epinephrine, thrombin, fibrin and histamine. Its secretion is also stimulated after exogenous administration of the vasopressin analog, desmopressin (DDAVP) [31,32]. VWF, primarily the low molecular weight multimers, is also secreted constitutively from megakaryocytes and endothelial cells [33]. Platelets contain 15% of the quantity of VWF present in an equal volume of plasma [34]. When first released from its storage sites, VWF is composed of ultra-large multimers that are rapidly cleaved into smaller multimers in plasma by the metalloproteinase, ADAMTS13 [35–40]. The A1 domain of VWF contains binding sites for platelet glycoprotein Ib (GPIb), heparin, collagen and ristocetin (FIGURE 1) [41–43]. The A2 domain contains the cleavage site for ADAMTS13 and the A3 domain contains the main binding site for collagen. Platelet GPIIb/IIIa attaches to the C1 domain while the D´ and the adjacent part of the D3 domains contain the binding site for FVIII [44–46]. The half-life of VWF in the circulation is approximately 8–12 h [47]. Pathophysiology of type 2B VWD

VWF plays two important roles in hemostasis. It is a carrier protein for FVIII, protecting it from degradation by activated protein C. It is also an adhesive protein essential for binding platelets (via GPIb/IX) to subendothelial collagen and platelets to other platelets (via GPIIb/IIIa) [48–50]. In primary hemostasis, the N-terminus of the platelet GPIba receptor binds to exposed subendothelium via VWF’s A1 domain [51,52]. In healthy subjects, VWF multimers in plasma fail to bind to the GPIba platelet receptor spontaneously since VWF binding is controlled by a disulfide double-linked double-loop region just below the leucine-rich repeats of GPIba [53]. In vitro, the interaction between VWF and GPIba can be induced by ristocetin (a glycoprotein antibiotic derived from a fungus) or botrocetin (a platelet agglutinating protein derived from snake venom) [54]. At sites of vascular injury, the binding of VWF and GPIba is induced by physiological factors such as fluid shear stress and surface tethering of VWF [54]. In VWD type 2B, mutations leading to amino acid substitutions in the A1 domain [55–61] give rise to a gain of function of the VWF multimers, causing spontaneous binding of VWF to platelet GPIba


receptors [55,62,63]. Since larger VWF multimers have more binding sites for GPIb, they are more reactive to platelets and are selectively cleared from the circulation [64]. Thus, VWFGPIba binding typically results in loss of the largest VWF multimers from plasma, blocking GPIb function and causing thrombocytopenia [64]. In some patients with type 2B VWD and more severe thrombocytopenia, defects in megakaryocytopoiesis have been identified as a cause for reduced platelet counts [65]. Nurden et al. have demonstrated that the R1308P substitution in A1 domain of VWF results in severe thrombocytopenia due to impaired megakaryocytopoiesis. Their studies have identified that VWF interacts with megakaryocytes via GPIb and that this interaction stimulates platelet production [65]. Further support for the concept that VWF plays an important role in thrombocytopoiesis comes from the investigation by Jackson et al. of a kindred previously diagnosed as having the Montreal platelet syndrome, a hereditary form of macrothrombocytopenia associated with mucocutaneous bleeding and spontaneous platelet aggregation in vitro [66]. Noting the similarities with this disease and type 2B VWD, Jackson et al. identified a V1308M mutation in family members with this syndrome, confirming that this syndrome in fact represents type 2B VWD [67]. In addition, recent studies in a mouse model of type 2B VWD indicate that thrombocytopenia occurs as a consequence of mutant VWF binding to platelets and targeting the VWF/platelet complex for destruction in macrophages [68]. The prevalence and the clinical and molecular predictors of thrombocytopenia in a cohort of 67 type 2B VWD were explored by Federici et al. in 2009 [69]. Platelet parameters were studied at baseline as well as during physiologic (pregnancy) or pathologic (infections, surgeries) stress conditions. Thrombocytopenia was found to be present in 30% of patients at baseline and increased to 57% after stress conditions. In patients with the P1266L/Q or R1308L mutations, there was no thrombocytopenia in all stress conditions [69]. The risk of bleeding was higher in those with thrombocytopenia (adjusted hazard ratio: 4.57; 95% CI: 1.17–17.90) and in those with the highest tertile of bleeding severity score, a score originally devised to determine a bleeding severity in VWD type 1 [69,70]. Increased proteolysis of VWF by ADAMTS13 has been clearly established in patients with type 2A VWD, yet until recently the data were scarce on increased proteolysis of VWF by ADAMTS13 in those with other types of VWD. Rayes et al. evaluated the proteolytic activity of recombinant ADAMTS13 on different types of recombinant VWF (in patients with type 2A, 2B and 2M) as well as on wild-type VWF [71]. All mutations were shown to be associated with increased proteolysis of recombinant VWF compared with wild-type VWF, although the susceptibility to proteolysis was to 2A > 2B > 2M. Type 2M was shown to have similar susceptibility to proteolysis to wildtype VWF under physiologic salt concentration (150 mm NaCl). The study concludes that in type 2B VWD, the spontaneous binding to platelets by VWF as well as its degradation of high-molecular-weight multimers by ADAMTS13 may 219


Mikhail, Aldin, Streiff & Zeidan

collectively contribute to the clearance of VWF and platelets and subsequently to the bleeding diathesis [71].

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Molecular genetics of type 2B VWD

Mutations affecting the VWF-GPIba binding domain have been localized to a peptide fragment within the VWF protein encoded by VWF exon 28 [72]. This discovery allowed several groups to analyze the DNA coding sequence for those fragments and gain a better understanding of the molecular genetics of VWD [60,64,73–81]. Mutations in type 2B VWD induce protein conformational changes that enhance the binding of the VWF A1 domain to GPIba. However, different mutations at different residues can results in variable binding affinities of VWF to platelets [69,82,83]. Several specific amino acid substitutions and insertions have been identified within a small segment of the VWF A1 domain, which stretches between amino acids 1260 and 1479 [83]. To date, over 25 mutations have been described in type 2B VWD [83]. Four mutations account for nearly 90% of type 2B mutations discovered to date [64]. Of these, V1316M is the most common and most extensively studied [83]. Several type 2B VWD variants have been reported and include patients with complete VWF multimers [84], chronic thrombocytopenia [85,86], giant platelets [83,87,88] and/or impaired megakaryopoiesis [89]. It is worth noting that mutations just outside the A1 loop can induce changes responsible for type 2B VWD phenotype [85,90], such as the H1268D/N [91,92] and the L1460V mutations [93]. Diagnosis of type 2B VWD

The enhanced binding of VWF to platelets in type 2B VWD results in accelerated clearance of high molecular weight (HMW) VWF multimers and platelets, which may manifest clinically as a bleeding disorder [14,62]. Diagnosis of type 2B VWD is complicated because significant clinical variation may exist, even among patients with identical mutations [69,94,95]. Patients may present with easy bruisability, mucocutaneous bleeding, petechiae, epistaxis, menorrhagia and/or gastrointestinal bleeding. Bleeding is commonly triggered by trauma, surgery, dental work or delivery [82]. Platelet counts may be mildly or moderately reduced or may be normal. In addition, some patients may also exhibit intermittent thrombocytopenia [54,82]. The bleeding variability has been primarily attributed to variability in platelet counts [83]. Platelet counts may be reduced by conditions that increase the endogenous release of VWF (e.g., pregnancy, stress, infection, exercise or DDAVP therapy), which may induce bleeding, [82,96–98]. Patients with VWD type 2B cannot be diagnosed based on history alone. Evaluation for this disorder comes as part of the general evaluation of a suspected bleeding disorder. Evaluation is initiated in those with a personal history of excessive bleeding and/or a family history of similar complaints or of known bleeding disorders. Factors to consider are the spontaneity, severity, duration and site of bleeding, as well as the type of insult associated with bleeding and the ease with which bleeding is stopped [99]. The use of standardized questionnaires for 220

history collection is advisable to appreciate the severity of the bleeding tendency [72,100,101]. Further discussion on these standardized questionnaires is beyond the scope of this review, but a bleeding score (obtained from specifically designed questionnaires) above a certain cutoff suggests the need for further evaluation for bleeding disorders [72,102,103]. In patients undergoing evaluation for VWD, diagnosis and appropriate classification of VWD usually is dependent on an array of tests (TABLE 1) in presence of supporting history, including family history of known or suspected VWD. An algorithm delineating the approach to the diagnosis of VWD is presented in FIGURE 2 [99]. Type 2B VWD is characterized by reduced VWF ristocetin cofactor activity (VWF:RCo), normal or reduced VWF antigen (VWF:Ag) levels, a low ratio of VWF:RCO to VWF:Ag (VWD: RCo/VWF:Ag) and VWF collagen binding (VWF:CP) to VWF: Ag (VWF: CB/VWF:Ag), absence of HMW multimers, reduced or normal platelet counts and reduced or normal FVIII levels [104]. TABLE 2 summarizes the findings. The above phenotypic features are similar to those of type 2A VWD (with the exception of thrombocytopenia), which is characterized by a qualitative defect in VWF causing decreased platelet adhesiveness and aggregation, in addition to decreased VWF polymerization [104]. Type 2B differs, however, from type 2A by enhanced ristocetininduced platelet aggregation (RIPA). It is worth noting that infusion of DDAVP in patients with type 2B VWD will typically increase FVIII and VWF:Ag levels but induces a modest response in VWF:RCO, does not improve VWF:CB activity and causes thrombocytopenia due to an increase in numbers of HMW multimers in plasma [105]. Recently, the VWF pro-peptide/VWF antigen ratio has been proposed as another strategy to identify patients with type 1 VWD due to accelerated clearance (such as VWD type 1 Vicenza) which are unlikely to have sustained responses to DDAVP [106,107]. Casonato et al. have also demonstrated that the VWF pro-peptide/VWF Ag ratio has utility in the diagnosis of type 2B VWD [107]. In mothers with type 2B VWD, special consideration should be given to obtaining a platelet count on their newborn because the disorder is inherited in an autosomal dominant fashion [82]. Analysis of cord blood for VWF HMW multimers is the most expedient method to definitely diagnose the disease in the peripartum period [82]. DNA sequencing to pinpoint type 2B VWD can identify or confirm the diagnosis [73,74,76]. Genetic testing, however, usually requires laboratories that have experience in performing and interpreting these tests and is available in fewer than 5% of VWD-testing laboratories [54]. In general, genetic testing is not the definitive modality for diagnosis, due to the presence of many different genetic mutations causing type 2B VWD. Clinicians still require evidence of VWD type 2B via the more traditional functional assays rather than genetic testing. Genetic tests may then be used for confirmation. Differentiating type 2B VWD from similar disorders

The diagnosis of type 2B VWD is often challenging due to the varied clinical manifestations and laboratory abnormalities of Expert Rev. Hematol. 7(2), (2014)

Type 2B von Willebrand disease


Table 1. Laboratory assays for the diagnosis of von Willebrand disease. Tests

Test principle

Test commentary pros and cons

Ristocetin cofactor activity assay (VWF:RCo)

Formalin-fixed normal platelets are added to patient plasma sample with ristocetin (1 mg/ml). Ristocetininduced platelet aggregation provides a functional measure of VWF activity in the patient sample

Pro: The current gold standard for measurement of VWF function Con: Limitations include wide coefficient of variation, difficulties with standardization, presence of a common SNP results in artifactually low test results and low sensitivity for levels

An update on type 2B von Willebrand disease.

Type 2B von Willebrand disease (VWD) accounts for fewer than 5% of all VWD patients. In this disease, mutations in the A1 domain result in increased v...
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