Characterization of the p.A1461D Mutation Causing Von Willebrand's Disease Type 2B with Severe Thrombocytopenia, Circulating Giant Platelets and Defective α -Granule Secretion

Abstract 1074

Von Willebrand's disease type 2B (VWD2B) is a rare bleeding disorder characterized by enhanced binding of von Willebrand factor (VWF) to platelet GPIbα due to gain-of-function mutations clustering in the VWF A1 domain. Binding of mutated type-2B VWF to GPIbα on circulating platelets may result in spontaneous platelet agglutination and increased ADAMTS13-mediated proteolysis of bound VWF. Consequently, thrombocytopenia, loss of larger VWF plasma multimers, and increased agonist-induced platelet agglutination at low concentrations of ristocetin are typical findings in VWD2B. However, not all of these laboratory features must be present, and confirming a diagnosis of VWD2B can thus be a challenge. We describe the case of a first-time pregnant woman (MCMDM-1 bleeding severity score, 10) with hereditary thrombocytopenia (platelet count, 17 × 10⁹/L) and spontaneous ex-vivo platelet aggregate formation, in whom previous testing for VWD2B had been inconclusive due to absent additional platelet agglutination at low concentrations of ristocetin. Therefore, platelet-type VWD caused by mutated GPIbα remained possible. Microscopic examination of the patient's peripheral blood smear revealed both small agglutinates of moderately enlarged and scattered single giant platelets. VWF:CB was 26% (normal, 50–250%), and VWF:Ag was 56% (50–160%), resulting in an abnormal activity to antigen ratio of 0.46 (0.8–1.2). Multimer analysis revealed loss of larger and intermediate sized VWF plasma multimers, and VWD2B was eventually diagnosed by identification of the mutation p.A1461D in the VWF A1 domain. Using an in-house ELISA, we assessed binding of recombinant wild-type (WT) and mutant VWF, respectively, to an immobilized GPIbα fragment in the absence and presence of increasing concentrations of ristocetin (0.3–1.5 mg/mL). Surprisingly, when compared to WT VWF in the presence of 1.5 mg/mL ristocetin (100%), binding of the p.A1461D mutant was already increased to 177% in the absence of ristocetin with only marginal additional binding evident at 1.5 mg/mL (238%). In comparison, binding of the p.V1316M mutant causative of VWD2B formerly described as the Montreal platelet syndrome, an inherited form of thrombocytopenia characterized by mucocutaneous bleeding and circulating giant platelets, was only 73% at 0 mg/mL ristocetin with significantly increased binding at 1.5 mg/mL (171%). Interestingly, ex-vivo stimulation of the patient's platelets with ADP or TRAP-6 resulted in only minimal surface CD62P expression, as analyzed by flow cytometry, while no CD62P was present on un-stimulated platelets, suggesting defective α -granule secretion. Similar findings were obtained on platelets from the patient's father and brother who also had severe thrombocytopenia and genetically confirmed VWD2B. Successful delivery of a healthy infant was achieved by only two peripartal doses of highly purified VWF concentrate in addition to systemic antifibrinolytics. In summary, this report further highlights the importance of genetic testing for the diagnostic work-up of suspected VWD2B, because the typical VWF activity pattern may not be present in all patients. Furthermore, our ELISA data demonstrate significant ristocetin-independent binding of the p.A1461D VWF mutant to platelet GPIbα, suggesting that the assay could be useful in differentiating VWD2B from platelet-type VWD in cases with severe thrombocytopenia and/or pronounced spontaneous platelet agglutination. Finally, our flow cytometry experiments support the concept of altered megakaryocytopoiesis and dysfunctional (pro)platelet production in VWD2B, which are likely due to adhesive intracellular interactions between mutated VWF and GPIbα and may result in concomitant α -storage pool disease.