Platelet thrombus formation is dependent on inside-out signaling to integrin allbb3 that regulates ligand binding and outside-in signaling through allbbS that controls the platelet cytoskeletal rearrangment. Inside- out and outside-in signaling events involve reversible phosphorylation of tyrosine (Tyr) or serine/threonine (Ser/Thr) residues on multiple proteins. The net Tyr or Ser/Thr phosphorylation of a protein substrate is regulated by the activities of both protein kinases and phosphatases. Historically, kinases and phosphorylation events have held the center stage during integrin-mediated signaling, while a role for the phosphatases is poorly understood. Our preliminary studies demonstrate for the first time that the catalytic subunits of protein phosphatase 1 (PP1c) and protein phosphatase 2A (PP2Ac) but not protein phosphatase 2c (PP2Cc) associate constitutively with the integrin allbbS and regulates allbbS adhesive function. We hypothesize that PP1c and PP2Ac orchestrate a temporal and spatial regulation of integrin allbbS signaling and participate in platelet function. Our goal is to study the mechanisms by which Ser/Thr phosphatases regulate integrin allbbS activation, signaling and function using human platelets, platelets from PP1c null mice, murine megakaryocytes and a cell line with thrombin activatable allbbS (DT40 PAR1/allbb3).
Aim 1 will test the hypothesis that PP1c a) dephosphorylates Ser/Thr residues on integrin bS in resting platelets, b) dephosphorylates cofilin thereby activating the cytoskeletal reorganization, and c) is involved in inside-out and outside-in signaling through allbbS.
Aim 2 will examine if a) the PP1c-allb interaction modulates the binding of other allb binding proteins, b) the platelet lipid rafts have a role in the spatial regulation of the integrin-phosphatase association. The possibility that PP1c interacts with other integrins bearing the PP1c binding site will be evaluated.
Aim 3 will establish the role for PP2Ac in the biology of integrin allbbS. Notably, we will map the PP2Ac binding site on integrin allbbS, study the role of PP2Ac in allbbS inside-out and outside-in signaling. Thus, by elucidating the mechanisms and consequences of allbbS-phosphatase interactions, these studies should provide novel insights into the understudied aspects of platelet function and may provide potential new therapeutic targets for anti-thrombotic therapy.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Hemostasis and Thrombosis Study Section (HT)
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Kindzelski, Andrei L
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Baylor College of Medicine
Internal Medicine/Medicine
Schools of Medicine
United States
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Khatlani, Tanvir; Pradhan, Subhashree; Da, Qi et al. (2016) A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in αIIbβ3 Integrin Signaling. J Biol Chem 291:17360-8
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