Proplatelet formation in heterozygous Bernard-Soulier syndrome type Bolzano.

BACKGROUND: Although mutations of GPIb alpha 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 GPIb alpha (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, alpha-tubulin distribution within proplatelets was severely deranged. CONCLUSIONS: Megakaryocytes from patients carrying a Bolzano allele of GPIb alpha 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 GPIb alpha in proplatelet formation.

Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes.

BACKGROUND: Megakaryocytes release platelets from the tips of cytoplasmic extensions, called proplatelets. In humans, the regulation of this process is still poorly characterized. OBJECTIVE: To analyse the regulation of proplatelet formation by megakaryocyte adhesion to extracellular adhesive proteins through different membrane receptors. METHODS: Human megakaryocytes were obtained by differentiation of cord blood-derived CD34(+) cells, and proplatelet formation was evaluated by phase contrast and fluorescence microscopy. RESULTS: We found that human megakaryocytes extended proplatelets in a time-dependent manner. Adhesion to fibrinogen, fibronectin or von Willebrand factor (VWF) anticipated the development of proplatelets, but dramatically limited both amplitude and duration of the process. Type I, but not type III or type IV, collagen totally suppressed proplatelet extension, and this effect was overcome by the myosin IIA antagonist blebbistatin. Integrin alphaIIbbeta3 was essential for megakaryocyte spreading on fibrinogen or VWF, but was not required for proplatelet formation. In contrast, proplatelet formation was prevented by blockade of GPIb-IX-V, or upon cleavage of GPIbalpha by the metalloproteinase mocarhagin. Membrane-associated VWF was detected exclusively on proplatelet-forming megakaryocytes, but not on round mature cells that do not extend proplatelets. CONCLUSIONS: Our findings show that proplatelet formation in human megakaryocytes undergoes a complex spatio-temporal regulation orchestrated by adhesive proteins, GPIb-IX-V and myosin IIA.