Severe Thrombocytopenia with Impaired Megakaryocytopoiesis in a Family with von Willebrand Disease Type 2B.

Thrombocytopenia associated with type 2B von Willebrand disease (VWD) is said to result from an increased elimination of platelets due to an abnormal binding of the higher molecular weight VWF multimers. Here we report a family (brother and sister) with severe thrombocytopenia, presence of agglutinated platelets and a very severe spontaneous bleeding syndrome since childhood. Their mutation concerns the VWF A1 domain, with a R1308P substitution which corresponds to an interactive site for GPIb (French VWD network). Electron microscopy showed agglutinates composed of adjacent discoid platelets with plasma membrane domains either in very close contact or showing signs of being fusioned. By flow cytometry, increased amounts of VWF and sometimes also fibrinogen were found at the platelet surface, but not P-selectin. Binding of antibodies to the GP Ibalpha N-terminal domain was decreased whereas GPIX and GPV were normally detected. In Western Blotting (WB), GPIbalpha was normally present but there were signs of filamin degradation. Basal Ca2+ levels were elevated. Platelet proteins involved in Ca2+ regulation were analysed by quantitating mRNA or by WB. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-type 3b was found to be increased but not the isoforms 2b or 3a. Furthermore, plasma membrane Ca2+ ATPase (PMCA)-4b as well as Inositol triphosphate receptor (InsP3-R3) were also increased in concentration. Interestingly, this platelet profile, mimics that observed in normal immature MK before proplatelet formation. As PMCA4b is a target of caspase-3 in MK, another substrate the polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) was evaluated. For both patients, platelets contained increased PARP and a degradation product indicating an abnormal persistance of caspase-3 activity. In vitro, megakaryocyte (MK) culture from peripheral blood CD34+ cells in the presence of SCF and TPO showed impaired MK maturation and the production of self-associated proplatelets. All these results are in favour of a role for abnormal platelet production with abnormal apoptosis that can explain at least in part the marked thrombocytopenia in this family with VWD type 2B.