Elucidation of the mechanisms whereby integrin mediates platelet activation.

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Specific ligand binding to integrins initiates 'outside-in' signaling that coordinates with signaling cascades initiated through growth factor-, cytokine-, and G protein-coupled receptors to regulate actin reorganization, cell survival, and proliferation. In platelets, the binding of fibrinogen or von Willebrand factor(VWF) to integrin αIIbβ3 triggers signals that promote cytoskeletal changes, spreading and formation of stable platelet thrombi. Physical interactions between αIIbβ3 and non-receptor protein kinases, such as Src and Syk, have been demonstrated, and fibrinogen binding to the integrin results in activation of these tyrosine kinases. Studies with human platelets, platelets from gene-targeted mice and heterologous expression systems have suggested a model in which the integrin β3 cytoplasmic domain interacts directly and constitutively with the SH3 domain of c-Src. Upon integrin ligation and clustering, it is proposed that integrin-associated c-Src is activated rapidly, leadi … More ng to tyrosine phoshorylation of c-Src substrates and assembly of a nascent αIIbβ3-based signaling complex known to date that includes Syk, SLP-76, Vav, and ADAP(adhesion and degranulation promoting adaptor protein ; previously called Fyn-binding protein, Fyb or SLAP-130). Lnk is an SH2 domain-containing adapter protein that inhibits cytokine signaling. Lnk^<-/-> mice exhibit a marked thrombocytosis, as evidenced by 5-fold increase in platelet count, due to proliferation and maturation of precursor cells in response to cytokines. We show that Lnk is expressed in human and mouse platelets, and that Lnk plays an unanticipated role in platelet integrin αIIbβ3 outside-in signaling. Lnk^<-/-> platelets exhibit defects in spreading on fibrinogen, β3 subunit tyrosine phosphorylation, clot retraction and formation of thrombi on collagen under flow conditions. In contrast, inside-out signaling is normal in Lnk^<-/-> platelets, as evidenced by agonist-induced fibrinogen binding. In platelets, Lnk forms a complex with c-Src and ADAP, Fyn-binding adaptor, in a manner dependent on αIIbβ3 ligation and Src activation, and may be required for Fyn recruitment to αIIbβ3 together with tyrosine phosphorylation of the β3 subunit. These results demonstrate for the first time that Lnk adaptor plays a pivotal role in the adhesion responses of platelets and thrombus formation through regulation of outside-in integrin αIIbβ3 signaling. In addition, we addressed the role of WASP/WAVE family on actin cytoskeletal changes in megakaryocytes and platelets. In this part of the projects, we demonstrated that the WAVE2/Abil signaling complex mediates integrin-dependent lamellipodia formation and maturation of megakaryocytopoiesis. As a final result, we established a novel method to generate human ES cell-derived platelets in vitro. Less