Cytoskeleton modulation of platelets and megakaryocytes

Platelets perform a critical role in both physiologic hemostasis and pathologic thrombosis, and the production and activation of platelets involves intricate regulation of cytoskeletal processes within these cells. A greater understanding of these cytoskeletal processes within platelets will offer the ability to manipulate them in hope of mitigating pathologies. In this dissertation I present a continuum of work progressing from the study of neural guidance cues in immune cells to neural guidance cues in platelet cytoskeletal function and finally to cytoskeletal alterations required for platelet production in thrombopoiesis. This work begins with the finding that, contrary to published reports, Slit2 does not regulate neutrophils directly, but instead acts through Robo4 to blunt endothelial hyper-response to cytokine storm. These studies led naturally into studies of guidance cues in other hematopoietic lineages, specifically platelets. I present data suggesting that Semaphorin 3E (Sema3E) inhibits all stages of platelet activation. Additionally, Sema3E reversibly inhibits platelet function by inactivating Rap1b, which is required for activation of 􀀁llb􀀂3 integrin. In attempts to determine the platelet receptor for Sema3E, using mice with a platelet-specific conditional deletion of the Sema3E receptor PlexinD1, I determined that PlexinD1 is not necessary for the platelet inhibitory activities of Sema3E. Simultaneously, in an attempt to produce Sema3E protein, I determined that effects originally attributed to Sema3E were in fact a result of Tween-20 contamination from commercially produced Sema3E formulation, thus terminating my studies of Semaphorins in platelets and suggesting the need for another look at the previously published reports of Sema3A in platelets. The study of cytoskeletal regulation in platelets lent naturally to the study of cytoskeletal functions in thrombopoiesis. Here I show that the proteasome inhibitor bortezomib causes thrombocytopenia by blunting platelet production in mice, consistent with clinical studies in humans. Furthermore, bortezomib prevents the production of proplatelets from megakaryocytes. I demonstrate that bortezomib treatment elevates both RhoA protein levels and RhoA activation in megakaryocytes and that inhibition of RhoA or its downstream kinase ROCK restores proplatelet production. As currently-approved drugs exist that inhibit both RhoA and ROCK, I suggest that these pathways are primary targets for treating bortezomib-induced thrombocytopenia.