Platelet activation plays a major role in hemostasis and thrombosis. Platelet agonists activate complex signaling cascades resulting in shape change, aIIbb3 integrin activation, and dense granule release and thromboxane A2 (TXA2) generation, amplifying the initial signal. The signaling cascades and the intracellular interaction of these signaling molecules regulating platelet physiological events have not been completely understood. In this application, we propose to test the overall hypothesis that PDK1-Akt-Pyk2 axis regulates platelet functional responses. We propose to understand the interaction of these signaling molecules and the downstream events in the agonist-induced platelet activation using complimentary biochemical, pharmacological, and gene knockout approaches. PDK1 is known to phosphorylate a number of kinases, but its function in platelets has not been elucidated.
Aim 1) We hypothesize that PDK1 plays an important positive regulatory role in platelets through selective phosphorylation and activation of Akt. We propose to test this hypothesis through selective pharmacological inhibitors of PDK1 and using conditional PDK1 null mice. Our preliminary data shows that PDK1 phosphorylation of Thr308 is crucial for the activity of Akt and downstream signaling events. Furthermore our preliminary data shows that PDK1 inhibition affects agonist-induced platelet integrin activation and TXA2 generation.
Aim 2) We hypothesize that intracellular association of PAK with Akt is crucial for its phosphorylation by PDK1. We propose to evaluate the role of PAK in translocation of Akt to the membrane using pharmacological approaches and PAK1 and 2 knockout mice. In preliminary data, we show that Akt translocation to the membrane and phosphorylation follows different kinetics. We postulate that PYK2, through tyrosine phosphorylation of PI3 kinases and PDK1, regulates platelet functional responses and Akt phosphorylation. We also postulate that Pyk2 is activated by G12/13 pathways and regulates thromboxane generation. We will evaluate the function of Pyk2 in aIIbb3 integrin activation, TXA2 generation, clot retraction, and spreading on immobilized fibrinogen, using knockout mice and pharmacological inhibitors. Our preliminary data shows that Pyk2 plays an important role in these platelet functional responses. These studies will enhance our understanding of the intracellular interactions of signaling molecules and their role in platelet activation, and might identify potential newer targets for the treatmentof thrombosis.
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