Following vascular injury, adhesive ligands such as fibrinogen and von Willebrand factor engage integrinallb/?3 to effect platelet aggregation and spreading during hemostasis and thrombosis. These responses aretriggered by ligand-mediated allb/?3 clustering, which initiates 'outside-in' signals to reorganize the actincytoskeleton. Recent work from this project has established that outside-in signaling in platelets requires Srcfamily tyrosine kinases (SFKs), c-Src in particular, which bind to /?3 and are activated by allb/?3 clustering ina manner dependent on PTP-1B, a protein tyrosine phosphatase. Here, three major unresolved questionswill be asked concerning the molecular basis of outside-in signaling in platelets and its biologicalconsequences. First, do direct interactions between integrins and SFKs represent a general mechanism forspatio-temporal initiation of outside-in signaling in platelets? Since platelets contain five different integrinsand at least six different SFKs, this possibility will be evaluated by co-immunoprecipitation techniques, bybimolecular fluorescence complementation imaging in live cells, and by localization of Src activation in livecells using a FRET-based reporter. In addition, integrin/SFK interactions will be assessed in Drosophila cellsto determine the extent to which direct activation of SFKs by integrins is an evolutionarily conserved process.Second, how does PTP-1B activate c-Src downstream of integrins? The mechanism by which PTP-1B isrecruited to the allb/?3/c-Src complex, and possibly to other integrin/SFK complexes, will be evaluated inplatelets and model cell systems, focusing on the possible role of adapter proteins. In addition, the effect ofintegrin clustering on PTP-1B catalytic activity will be determined. Third, does selective disruption of outsideinsignaling affect thrombus formation in vivo? Here arterial thrombosis will be studied in novel gene-targetedmice predicted to have selective defects in the interaction of c/llb/83 with c-Src or other SFKs, or defects indownstream events required for actin reorganization. Altogether, these studies will provide molecular insightsinto how outside-in integrin signaling is initiated and establish the extent to which this process regulatesplatelet function in vivo. Thus, this line of investigation may lead to identification of new anti-thrombotic drugtargets and serve as a paradigm for integrin signaling in other blood cells.
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