Following vascular injury, adhesive ligands such as fibrinogen and von Willebrand factor engage integrin allb/?3 to effect platelet aggregation and spreading during hemostasis and thrombosis. These responses are triggered by ligand-mediated allb/?3 clustering, which initiates """"""""outside-in"""""""" signals to reorganize the actin cytoskeleton. Recent work from this project has established that outside-in signaling in platelets requires Src family tyrosine kinases (SFKs), c-Src in particular, which bind to /?3 and are activated by allb/?3 clustering in a manner dependent on PTP-1B, a protein tyrosine phosphatase. 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 evolutionarily conserved process. Second, how does PTP-1B activate c-Src downstream of integrins? The mechanism by which PTP-1B is recruited to the allb/?3/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 outsidein 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 c/llb/83 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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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University of California San Diego
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