Formation of platelet thrombi is dependent on the agonist-induced inside-out signaling to integrin ?IIb?3 that regulates soluble fibrinogen binding, and outside-in signaling through aIIbb3 that controls the platelet cytoskeletal rearrangement. Inside-out signaling is generated by several agonists that engage the G protein coupled receptors (GPCR). Intrinsic to inside-out and outside-in signaling, is the reversible tyrosine (Tyr and serine/threonine (Ser/Thr) phosphorylation-dependent assembly of multiple effectors. The phosphorylation and the activity of several effectors are regulated by protein kinases and phosphatases. While kinase mediated phosphorylation events during inside-out and outside-in signaling has been intensely investigated, the contribution of the catalytic subunits of Ser/Thr protein phosphatase 1 (PP1c) and protein phosphatase 2A (PP2Ac) is relatively unexplored. In the current funding period, we noticed decreased thrombin-induced inside- out signaling and delayed in vivo thrombus formation in mice lacking the catalytic subunit of protein phosphatase 1 ? (PP1c?). Outside-in signaling was unaffected by the loss of PP1c?, but increased in the absence of PP2Ac. Our overarching hypothesis is that the specific subtypes of Ser/Thr phosphatases orchestrate a spatial regulation of inside-out and outside-in signaling. Our goal is to decipher the molecular details underpinning the functional coupling of PP1c with the G protein signaling and PP2Ac with the integrin signaling, during physiological responses to injury.
Aim 1 will define the role of PP1c and its interacting protein G?1 during inside-out signaling. G?1, a component of the heterotrimeric G proteins that couple to GPCR, interacted with PP1c in resting platelets, while agonist treatment dissociated this complex. Depletion of Gb1 in murine megakaryocytes or blockade of G?? signaling in platelets, decreased thrombin receptor activating peptide induced fibrinogen binding and aggregation. Using platelets from human and mice deficient in PP1c?, PP1ca and G?1, our goal is to test if G?1 targets PP1c to the GPCR complex and positively regulates inside- out signaling.
Aim 2 will define the role of PP2Ac during aIIbb3 mediated outside-in signaling. Src activation is critical for outside-in signaling and we showed that PP2Ac depletion activates Src. CIN85 is an adaptor protein that associated with PP2Ac and outside-in signaling dissociated this complex in platelets. CIN85 depletion reduced Src activation and ?IIb?3 adhesiveness. Using platelets and aIIbb3 model cells, we will test if PP2Ac negatively regulates outside-in signaling via CIN85. The proposed research is innovative because it represents a departure from the kinase-mediated phosphorylation events to a phosphatase mediated dephosporylation events during platelet activation. The proposed research is significant because it will advance our understanding of the molecular mechanisms of platelet activation, and lay the basic groundwork for identifying phosphatase-interacting proteins as the new therapeutic targets for future anti-thrombotic therapy.
Platelet activation events initiate heart attack and stroke. This proposal will examine new concepts of platelet activation, with the expectation that the studies will identify new targets for anti-thrombotic therapy.
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