Following vascular injury, adhesive ligands such as fibrinogen and von Willebrand factor engage integrin allbbeta3 to effect platelet aggregation and spreading during hemostasis and thrombosis. These responses are triggered by ligand-mediated allbbeta3 clustering, which initiates """"""""outside-in"""""""" signals to reorganize the actin cytoskeleton. Recent work from this laboratory has established that outside-in signaling in platelets requires Src family tyrosine kinases (SFKs), c-Src in particular, which bind to beta3 and are activated by allbbeta3 clustering in a manner dependent on PTP-1B, a protein tyrosine kinase. Here, three major unresolved questions will be asked concerning the molecular basis of outside-in signaling in platelets and its biological consequences. First, do direct interactions between integrins and SFKs represent a general mechanism for spatio-temporal initiation of outside-in signaling in platelets? Since platelets contain five different integrins and at least six different SFKs, this possibility will be evaluated by co-immunoprecipitation techniques, by bimolecular fluorescence complementation imaging in live cells, and by localization of Src activation in live cells using a FRET-based reporter. In addition, integrin/SFK interactions will be assessed in Drosophila cells to determine the extent to which direct activation of SFKs by integrins is an evolutionary conserved process. Second, how does PTP-1B activate c-Src downstream of integrins? The mechanism by which PTP-1B is recruited to the allbbeta3/c-Src complex, and possibly to other integrin/SFK complexes, will be evaluated in platelets and model cell systems, focusing on the possible role of adapter proteins. In addition, the effect of integrin clustering on PTP-1B catalytic activity will be determined. Third, does selective disruption of outside- in signaling affect thrombus formation in vivo? Here arterial thrombosis will be studied in novel gene-targeted mice predicted to have selective defects in the interaction of allbbeta with c-Src or other SFKs, or defects in downstream events required for actin reorganization. Altogether, these studies will provide molecular insights into how outside-in integrin signaling is initiated and establish the extent to which this process regulates platelet function in vivo. Thus, this line of investigation may lead to identification of new anti-thrombotic drug targets and serve as a paradigm for integrin signaling in other blood cells. ? ? ?
