Proplatelet formation in heterozygous Bernard-Soulier syndrome type Bolzano

Background: Although mutations of GPIbα are among the most frequent causes of inherited platelet disorders, the mechanisms for the onset of thrombocytopenia and platelet macrocytosis are still poorly defined. Objective: In this work we analyzed in vitro megakaryocyte differentiation and proplatelet formation in six subjects heterozygous for the Ala156Val mutation in the GPIbα (Bolzano mutation). Methods: Human megakaryocytes were obtained by differentiation of patient cord blood-derived CD34⁺ cells and peripheral blood-derived CD45⁺ cells. Proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: Megakaryocyte differentiation from both cord blood (one patient) and peripheral blood (five patients) was comparable to controls. However, proplatelet formation was reduced by about 50% with respect to controls. An identical defect of proplatelet formation was observed when megakaryocytes were plated on fibrinogen, von Willebrand factor or grown in suspension. Morphological evaluation of proplatelet formation revealed an increased size of proplatelet tips, which was consistent with the increased diameters of patients' blood platelets. Moreover, α-tubulin distribution within proplatelets was severely deranged. Conclusions: Megakaryocytes from patients carrying a Bolzano allele of GPIbα display both quantitative and qualitative abnormalities of proplatelet formation in vitro. These results suggest that a defect of platelet formation contributes to macrothrombocytopenia associated to the Bolzano mutation, and indicate a key role for GPIbα in proplatelet formation.