Protein kinases are critical regulators of cellular functions. Work from this and other laboratories established that protein kinase C (PKC)-pathways modulate agonist-induced fibrinogen receptor activation and secretion in platelets. However, the identity of PKC isoforms and the underlying mechanisms are incompletely understood. For example, previous studies from this lab have shown that PKCd plays a negative regulatory role in GPVI- mediated dense granule release whereas it promotes secretion downstream of thrombin receptors. Similarly, the physiological significance of tyrosine phosphorylation of platelet novel class PKC (nPKC) isoforms requires further elucidation. Our overall hypothesis is that different nPKC isoforms play different roles in platelet functions and differentially tyrosine phosphorylated PKCd isoforms trigger distinct signaling cascades leading to diverse functional responses. We will test this overall hypothesis using complimentary cell biological, pharmacological, biochemical, and molecular genetic approaches.
Our specific aim 1 is to evaluate the functional role of different PKC isoforms in platelet fibrinogen receptor activation and secretion. We will test the hypothesis that "thromboxane A2 and thrombin activate specific PKC isoforms that regulate dense granule release;ADP, however, fails to activate these isoforms". In support of this idea, we have recently demonstrated the PKCd isoform, which is not activated by ADP, plays an important role in dense granule release.
Aim 2 is to delineate the molecular basis for differential regulation of dense granule release by PKCd in platelets. We hypothesize that differential regulation of PKCd, downstream of GPVI and PARS, occurs due to its differential association with SHIP1. Preliminary studies that show selectively association of SHIP1 with PKCd, downstream of GPVI but not PARs, supports this hypothesis.
The aim 3 is to investigate the molecular mechanism of differential interaction of PKCd and SHIP1 in platelets. We hypothesize that tyrosine phosphorylated PKCd triggers different signaling cascades. PKC isoforms have several tyrosine residues that can be phosphorylated. We hypothesize that diverse signaling pathways downstream of G protein-coupled receptors and tyrosine kinase-linked receptors phosphorylate different tyrosine residues on PKCd and these differential phosphorylations modify the functional implications of these isoforms. Our preliminary studies indicate that G protein-coupled PARs and tyrosine kinase-linked collagen receptor GPVI differentially phosphorylate Y-311 and Y-155 residues, respectively. We propose to test the role of these phospho-tyrosine residues in the interaction with SHIP1 by molecular cell biological approaches. Finally, we will identify additional signaling molecules associated with differentially phosphorylated PKCd by biochemical and proteomic approaches. The studies proposed in this application will identify novel therapeutic targets towards treatment of thrombosis.
Project Relevance: Platelet activation is critical for hemostasis and can lead to thrombotic events. The signal transduction mechanisms downstream of agonist receptors are important to understand the molecular basis of platelet activation. The proposed research examines the regulation and function of signaling events downstream of platelet surface receptors through a combination of biochemical, pharmacological and genetic approaches. An in-depth understanding of these mechanisms will aid in identifying novel targets of antithrombotic therapeutics.
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|Bhavanasi, Dheeraj; Kostyak, John C; Swindle, John et al. (2015) CGX1037 is a novel PKC isoform delta selective inhibitor in platelets. Platelets 26:9-Feb|
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|Liverani, Elisabetta; Rico, Mario C; Yaratha, Laxmikausthubha et al. (2014) LPS-induced systemic inflammation is more severe in P2Y12 null mice. J Leukoc Biol 95:313-23|
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|Kostyak, John C; Bhavanasi, Dheeraj; Liverani, Elisabeta et al. (2014) Protein kinase C ? deficiency enhances megakaryopoiesis and recovery from thrombocytopenia. Arterioscler Thromb Vasc Biol 34:2579-85|
|Liverani, Elisabetta; Rico, Mario C; Garcia, Analia E et al. (2013) Prasugrel metabolites inhibit neutrophil functions. J Pharmacol Exp Ther 344:231-43|
|Buitrago, Lorena; Bhavanasi, Dheeraj; Dangelmaier, Carol et al. (2013) Tyrosine phosphorylation on spleen tyrosine kinase (Syk) is differentially regulated in human and murine platelets by protein kinase C isoforms. J Biol Chem 288:29160-9|
|Kim, Soochong; Cipolla, Lina; Guidetti, Gianni et al. (2013) Distinct role of Pyk2 in mediating thromboxane generation downstream of both G12/13 and integrin *IIb*3 in platelets. J Biol Chem 288:18194-203|
|Manne, Bhanu Kanth; Getz, Todd M; Hughes, Craig E et al. (2013) Fucoidan is a novel platelet agonist for the C-type lectin-like receptor 2 (CLEC-2). J Biol Chem 288:7717-26|
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