Akt-regulated pathways in platelet function

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The goal of this proposal is to better define the roles of Akt in platelet signaling and thrombosis. The Akt kinases are serine-threonine kinases that have well-described roles in cell survival, proliferation, and metabolism. We and others have shown that Akt kinases also play important roles in platelet function. Specifically, our previous studies show that Akt2 plays a role in promoting fibrinogen binding and dense granule secretion and that Akt2 knockout mice are resistant to arterial thrombosis. However, the mechanisms by which Akt regulates platelet function are not understood. This proposal will test the hypothesis that Akt activation by G protein-coupled receptors (GPCRs) supports specific signaling pathways in platelets, including the regulation of GSKSbetaand integrin outside-in signaling pathways, that contribute to arterial thrombosis. We will test this hypothesis in the following 3 Specific Aims, which focus on elucidating signaling pathways upstream and downstream of Akt activation in platelets. Aim 1 is to elucidate the mechanisms of Akt activation by G protein-coupled receptors, focusing on the ability of arrestin-2 to serve as a scaffoldfor PIS kinase subunits and GPCRs. Our preliminary studies show that arrestins form agonist-dependent complexes with p85-PI3K subunits in human platelets. By inhibiting arrestin expression in megakaryocytic cells and studying arrestin-2-/- mice, we propose to determine the components of arrestin complexes, to establish whether Akt activation in platelets is dependent on arrestins, and to determine the role of arrestins in platelet activation. Aim 2 is to determine the role of the Akt substrate, GSKSbetain platelet function and thrombosis. GSKSbetais a ser/thr kinase that frequently suppresses cellular functions that are positively regulated by Akt. Our hypothesis is that GSKSbeta acts to suppress platelet function, and that the removal or inhibition of GSKSbeta should enhance platelet aggregation or thrombosis. Our preliminary data suggest that this is the case. Aim 3 is to define the impact of Akt on outside-in signaling by integrin alphallb-betaS. Our preliminary data indicate that the rate of clot retraction and spreading on fibrinogen are dependent on Akt2 in platelets. Since these functions are dependent on alphallb-betaS, we will seek to define how Akt regulates alphallb-betaS signaling by studying phosphorylation of the betaStail, actin assembly, and activation of rho family members in platelets lacking Akt or expressing activated Akt.