The goal of this proposal is to better define the roles of Akt in platelet signaling and thrombosis. The Aktkinases are serine-threonine kinases that have well-described roles in cell survival, proliferation, andmetabolism. 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 densegranule secretion and that Akt2 knockout mice are resistant to arterial thrombosis. However, themechanisms by which Akt regulates platelet function are not understood. This proposal will test thehypothesis that Akt activation by G protein-coupled receptors (GPCRs) supports specific signaling pathwaysin platelets, including the regulation of GSKSbetaand integrin outside-in signaling pathways, that contributeto arterial thrombosis. We will test this hypothesis in the following 3 Specific Aims, which focus onelucidating signaling pathways upstream and downstream of Akt activation in platelets.
Aim 1 is to elucidatethe mechanisms of Akt activation by G protein-coupled receptors, focusing on the ability of arrestin-2 toserve as a scaffoldfor PIS kinase subunits and GPCRs. Our preliminary studies show that arrestins formagonist-dependent complexes with p85-PI3K subunits in human platelets. By inhibiting arrestin expressionin megakaryocytic cells and studying arrestin-2-/- mice, we propose to determine the components of arrestincomplexes, 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 functionsthat are positively regulated by Akt. Our hypothesis is that GSKSbeta acts to suppress platelet function, andthat 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 byintegrin alphallb-betaS. Our preliminary data indicate that the rate of clot retraction and spreading onfibrinogen are dependent on Akt2 in platelets. Since these functions are dependent on alphallb-betaS, wewill 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.
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