Summary. Type 2B von Willebrand disease (VWD2B) is caused by gain-of-function amino acid substitutions in the von Willebrand factor (VWF) A1 domain. These allow facilitated binding of mutated VWF to platelet GPIbα with prolonged lifetimes of VWF bonds and enhanced ADAMTS-13 cleavage of large VWF multimers. A bleeding rather than prothrombotic syndrome is due to: (i) decreased large VWF multimers in plasma; (ii) limited thrombus formation; and (iii) thrombocytopenia affecting some but not all patients. Accumulating evidence points to an altered megakaryocytopoiesis in VWD2B with the production of enlarged or giant platelets showing an abnormal ultrastructure and, in a cohort of patients, the presence of circulating platelet agglutinates. In fact, evidence from in vitro cultures and marrow aspirates suggests that the upregulated VWF function can lead to abnormal VWF trafficking in megakaryocytes, a modified platelet production with interacting proplatelets, and the presence or even release of platelet agglutinates in the bone marrow.
Type 2B von Willebrand disease (VWD2B) is caused by an abnormal von Willebrand factor (VWF) with an increased affinity for its platelet receptor GPIbα and constitutes a unique variant form of VWD [1–3]. Transmitted with autosomal dominant inheritance, VWD2B is caused by heterozygous mutations in exon 28 of the VWF gene that give rise to gain-of-function amino acid substitutions within the VWF A1 domain. While the VWF is normally synthesized, multimerized and secreted from endothelial cells, the mutations transform the protein into its GPIbα-binding conformation [4,5]. The result is spontaneous VWF binding to GPIbα, and a lowering of the ristocetin dose required to induce platelet agglutination in platelet-rich plasma (PRP). A consequence of this facilitated interaction is that the largest and most active VWF multimers are frequently lost from the plasma through adsorption to platelets. Recent studies show how A1 domain mutations in VWD2B may prolong lifetimes of VWF bonds with GPIbα and allow ADAMTS-13 to proteolyze large VWF multimers enhancing bleeding risk [6,7]. Diagnostically, the VWF:RCo/VWF:Ag ratios may or may not be less than 0.7 as typically observed for VWD2A and VWD2M; normal or reduced ratios will depend on the presence or absence of large VWF multimers. Recombinant VWD2B VWF supports platelet adhesion when adsorbed on collagen, but thrombus formation under flow is abnormal. Not only surface coverage is affected but also thrombus height with confocal microscopy showing that VWF poorly distributes within the thrombus mass [8,9]. The ability of normal VWF to restore thrombus formation has therapeutic implications and suggests that the key element in bleeding is the lack of large multimers. To this should be added the macrothrombocytopenia that affects a significant proportion of patients .
Most of the 23 VWD2B mutations cited on the ISTH VWF database (http://www.vwf.group.shef.ac.uk) cluster in the C509–C695 disulfide loop of the VWF A1 domain although some border this region. Frequent mutations repeated in unrelated families are R1306W, R1308C, V1316M and R1341Q. Early studies confirmed that recombinant forms of VWF carrying VWD2B mutations retain a spontaneous capacity to bind to GPIbα and induce platelet aggregation; nevertheless, the response was variable suggested to be related to the nature and/or location of the causative mutation [11,12]. The crystal structure of the GPIbα-A1 interface showed structural differences for the mutant A1 domain that accounts for its tighter association [5,13]. This led to the hypothesis that the α1–β2 loop of the A1 domain serves as a conformational switch that in VWD2B favors the open conformation. Gain-of-function mutations with increased RIPA but with the presence of all multimers are observed in VWD2B variants such as New York or Malmo with a P1266L mutation . In contrast, recombinant VWF possessing the P1337L mutation bound to GPIbα with high affinity resulting in the elimination of both the high and medium sized plasma multimers . Recombinant forms of R1308L and R1308C with gain-of-function binding to GPIbα also atypically bound less well to collagen types I and III . Findings such as the above show how genotype variation may in part explain the more or less severe hemostatic defect in VWD2B.
A recent analysis of the prevalence, clinical and molecular predictors of thrombocytopenia in a cohort of 67 VWD2B patients from 38 unrelated families showed that thrombocytopenia was found in 20% of patients at baseline and rose to 57% during stress conditions including pregnancy, infections and surgery . A low platelet count was associated with a higher bleeding risk. Thrombocytopenic patients had a wide range of VWF mutations, but these did not include P1266L/Q or R1308L. Studies using a llama-derived antibody fragment (nanobody) reactive only with VWF in its GPIbα-binding conformation showed that levels of the active conformation were up to twelvefold increased compared to controls, values that increased further when the patients were thrombocytopenic . Early reports signaled that thrombocytopenia accentuates in VWD2B patients following the administration of 1-desamino-8-d-arginine vasopressin (DDAVP), a drug that increases the circulating levels of endogenous VWF . However, Federici et al.  reported a VWD2B patient with an I546V mutation, a near normal plasma VWF multimer distribution and chronic thrombocytopenia while we followed up earlier isolated reports by showing the presence of giant platelets in three patients with VWD2B with mild-to-severe thrombocytopenia . As these patients possessed different VWF structural modifications (1304insM, V1316M and P1337L), the giant platelets were not linked to a recurrent mutation. Immunogold labeling and electron microscopy (EM) showed abundant VWF on the platelet surface and within the platelet surface-connected canalicular system (SCCS) as well as within the α-granules. Typical giant platelet morphology, as well as circulating platelet agglutinates in thrombocytopenic VWD2B patients, is shown in Fig. 1.
Circulating platelet agglutinates and an altered platelet production
Type 2B Tampa was the first full report describing the presence of circulating platelet agglutinates as a variant form of VWD2B . Associated with severe thrombocytopenia, the four affected members of this family only presented with a moderate bleeding diathesis. RIPA was enhanced for all members and spontaneous platelet aggregation was in the 21%–31% range. VWFR:Co was reduced and large multimeric forms of VWF absent. We described a French family with a R1308P substitution and a constant severe thrombocytopenia with circulating platelet agglutinates . EM showed grouped platelets in close contact. An increased platelet presence of plasma membrane Ca2+ATPase-4b and inositol receptor-3 together with signs of ongoing caspase-3 activity suggested that abnormalities in the apoptotic phase of megakaryocytopoiesis are part of the phenotype of this family. Culture of peripheral blood CD34+ cells of both patients resulted in megakaryocytes (MK) showing self-associated proplatelets with VWF already bound to the cell surface (Fig. 2). More recently, a bone marrow aspirate from an unrelated patient also with a R1308P mutation revealed large platelet clumps and MK nuclei surrounded by halos of clumped platelets . Over 30 years ago, Frojmovic and his coworkers  described a Canadian kindred in which an autosomal dominant form of inherited thrombocytopenia was associated with lifelong mucocutaneous bleeding, giant platelets and spontaneous platelet agglutination. The disorder was termed Montreal platelet syndrome (MPS). The authors described that isolated MPS platelets showed a decreased aggregation to thrombin, a finding rarely reported for VWD2B. Recently, a heterozygous VWD2B V1316M mutation has been found in affected members of the MPS kindred, but not in unaffected members . This finding suggests that MPS is VWD2B with macrothrombocytopenia. As shown in Fig. 1, platelet agglutinates are present in an unrelated French family with the V1316M mutation; notwithstanding, phenotype variability is a feature of V1316M in VWD2B.
Haplotypes and modifier genes
Kunicki et al.  analyzed the association of bleeding severity with candidate gene haplotypes within pedigrees of 11 index cases of VWD type 2 (two type 2A, three type 2B and six type 2M). An increased bleeding severity score was seen with the ITGA2 promoter haplotype -52T, suggesting that variations in platelet collagen receptor expression can influence the clinical severity of the disease. Ginsburg and his coworkers  have studied the genetic regulation of plasma von Willebrand factor levels in a mouse model and identified a series of quantitative trait loci (Mvwf1-4) that explained a significant proportion of the variation of VWF plasma levels in the mouse strains used. These represent prototype studies allowing the understanding of VWF levels in humans where VWF levels vary within 50%–200% of the population average and for which genetic factors other than the VWF gene have a large contribution (with the ABO blood group a known example). A question that is yet to be resolved is whether the presence of severe thrombocytopenia and circulating platelet agglutinates in some patients is a manifestation of the hyper-reactivity of the mutated VWF or to the presence of associated gene defects. VWD2B would seem an ideal disease for a genome-wide study to determine how such phenotype variability occurs in what is assumed to be a monogenic disease.
VWD2B should always be considered in the differential diagnosis of inherited thrombocytopenia with giant platelets and especially so when there are signs of spontaneous platelet aggregation or clumping. While the primary roles of VWF as a mediator of hemostasis and as a carrier of FVIII remain unchallenged, studies on VWD2B suggest that this adhesive protein can also regulate a late stage of megakaryocytopoiesis by controlling MK maturation (or migration) and proplatelet formation perhaps via GPIbα signaling. This has important implications for the management of the disease , where platelet transfusions should accompany the administration of VWF concentrates in bleeding episodes of thrombocytopenic VWD2B patients.
We would acknowledge research support from GIS Maladies Rares and for the CRPP by the French Health Ministry.
Disclosure of Conflict of Interests
The authors state that they have no conflicts of interest.