Platelets become exposed to collagen during rupture of the atherosclerotic plaque and during normal vascular injury. The exposed collagen serves not only as a direct platelet agonist but also provides an adhesive surface to platelets, thus contributing to thrombosis. Two of the major collagen receptors on platelets are glycoprotein (GP) VI and the Alpha-2 Beta-1 integrin. GPVI and Alpha-2 Beta-1 are both required for full collagen mediated platelet adhesion and activation, likely involving a GPVI-mediated activation of Alpha?2 Beta-1 as demonstrated by our lab and others. However, signals leading to Alpha-2 Beta-1 activation by GPVI or other agonist receptors are not well understood. Related to this, the small GTPases Rap1 and R-Ras have each been proposed as positive regulators of integrin activation whereas Ras has been proposed as a negative regulator. We recently demonstrated that GPVI signaling in platelets leads to Rap1 activation in a manner dependent upon secreted ADP activation of the P2Y12 receptor as well as a P2Y12-independent pathway. We also provide preliminary evidence using a transducible primary mouse megakaryocyte system, that Rap1 may promote Alpha-2 Beta-1 activation. In separate studies we found that R-Ras promotes several Alpha-2 Beta-1 mediated events, and that Ras is present in platelets and activated by agonist stimulation. However, the interrelationship of Rap1 to R-Ras or Ras is not understood. In the present proposal, we aim to further define the communication between GPVI and Alpha-2 Beta-1 by first, clarifying the roles of Rap1 and R-Ras in GPVI-induced Alpha-2 Beta-1 activation, second, mapping upstream pathways leading to Rap1 activation with regard to the potential role of R-Ras and other molecules in this event, third, mapping signaling pathways downstream of activated Rap1 leading to Alpha-2 Beta-1 integrin activation, and finally, determining the role of Ras in regulating Alpha-2 Beta-1 integrin activation. Results from these studies will provide fundamental information on how these important collagen receptors communicate with one another via these small G-proteins in platelets and megakaryocytes.
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