The cell division control protein 42 /Src family kinase/Neural Wiskott-Aldrich syndrome protein pathway regulates human proplatelet formation

SummaryBackground Cytoskeleton rearrangements are essential in platelet release. The RHO small GTPase family, as regulators of the actin cytoskeleton, plays an important function in proplatelet formation. In the neuronal system, CDC42 is involved in the axone formation, a process that combined elongation and branching as proplatelet formation. Objective To analyze the role of CDC42 and its effectors of the WASP family on proplatelet formation Methods Human MKs were obtained from CD34+ cells. The inhibition of CDC42 in MK was obtained with a chemical inhibitor CASIN or of an active or a dominant-negative form of CDC42. The invalidation of N-WASP was obtained by an shRNA strategy Results Herein, we show that CDC42 activity increased during megakaryocyte (MK) differentiation. The use of a chemical inhibitor CASIN or of an active or a dominant-negative form of CDC42 demonstrated that CDC42 positively regulates proplatelet formation in vitro. We determined that N-WASP but not WASP regulated proplatelet formation. We found that N-WASP knock-down, led to a marked decrease in proplatelet formation due a defect in demarcation membrane system (DMS). This was associated with RHOA activation, and a concomitant augmentation in the phosphorylation of MLC2. N-WASP phosphorylation, a primed form of N-WASP, increased during MK differentiation. Phosphorylation inhibition by two Src family kinase inhibitors decreased proplatelet formation. Conclusions We conclude that N-WASP positively regulates the DMS development and PPF and that the Src family kinases in association with CDC42 regulate proplatelet formation through N-WASP. This article is protected by copyright. All rights reserved.