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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL093231-04
Application #
8281478
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Sarkar, Rita
Project Start
2009-05-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2014-04-30
Support Year
4
Fiscal Year
2012
Total Cost
$432,347
Indirect Cost
$144,116
Name
Temple University
Department
Physiology
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Liverani, Elisabetta; Rico, Mario C; Tsygankov, Alexander Y et al. (2016) P2Y12 Receptor Modulates Sepsis-Induced Inflammation. Arterioscler Thromb Vasc Biol 36:961-71
Buitrago, Lorena; Manne, Bhanu Kanth; Andre, Pierrette et al. (2016) Identification of novel Syk-independent functional roles of FcγRIIa in platelet outside-in signaling using transgenic mice expressing human FcγRIIa. Platelets 27:488-90
Reppschläger, Kevin; Gosselin, Jeanne; Dangelmaier, Carol A et al. (2016) TULA-2 Protein Phosphatase Suppresses Activation of Syk through the GPVI Platelet Receptor for Collagen by Dephosphorylating Tyr(P)346, a Regulatory Site of Syk. J Biol Chem 291:22427-22441
Inamdar, Vaishali; Patel, Akruti; Manne, Bhanu Kanth et al. (2015) Characterization of UBO-QIC as a Gαq inhibitor in platelets. Platelets 26:771-8
Kosoff, Rachelle E; Aslan, Joseph E; Kostyak, John C et al. (2015) Pak2 restrains endomitosis during megakaryopoiesis and alters cytoskeleton organization. Blood 125:2995-3005
Badolia, Rachit; Manne, Bhanu Kanth; Dangelmaier, Carol et al. (2015) Gq-mediated Akt translocation to the membrane: a novel PIP3-independent mechanism in platelets. Blood 125:175-84
Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol A et al. (2015) C-type lectin like receptor 2 (CLEC-2) signals independently of lipid raft microdomains in platelets. Biochem Pharmacol 93:163-70
Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol et al. (2015) Distinct pathways regulate Syk protein activation downstream of immune tyrosine activation motif (ITAM) and hemITAM receptors in platelets. J Biol Chem 290:11557-68
Zhang, Si; Zhang, Shenghui; Hu, Liang et al. (2015) Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis. Circulation 131:1160-70
Bhavanasi, Dheeraj; Kostyak, John C; Swindle, John et al. (2015) CGX1037 is a novel PKC isoform delta selective inhibitor in platelets. Platelets 26:2-9

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