ADP is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. ADP causes platelets to change their shape, to aggregate, to release contents of granules, and to produce thromboxane A2, another potent agonist for platelets. The physiological effects and intracellular responses of ADP on platelets have been well characterized and the receptor mediating these effects has been cloned. However the molecular mechanism of these ADP-mediated physiological processes remains obscure. During the previous grant period, we have demonstrated 3 ADP receptor subtypes, P2Y1, P2Y12, and P2X1, on platelets and elucidated a number of signaling mechanisms in platelets. In this grant period, we propose to enhance our understanding by elucidating the molecular mechanisms of ADP-induced platelet activation and evaluate the relative contribution of P2Y receptors to thrombus formation. We hypothesize that different tyrosine kinases are activated downstream of different P2Y receptor subtypes. Our studies in the previous grant period led to the conclusions that Src family kinases contribute to fibrinogen receptor activation, thromboxane A2 generation, and phosphorylation of PKC-5 isoform and Akt downstream of the P2Y12 receptor activation. We propose to test this hypothesis by pharmacological, biochemical, and molecular genetic approaches. We hypothesize that charge interactions between negatively charged phosphates in ADP and some of the positively charged basic amino acid residues in the extracellular domains of the P2Y12 receptor are involved in ligand binding. We will test this hypothesis using chimeric receptors and site-directed mutagenesis. We will change the conserved basic amino acid residues in the P2Y12 receptor to alanine by site-directed mutagenesis, express the mutant receptors transiently in mammalian cells, and determine the effect on binding of labeled nucleotides and ADP-induced inhibition of adenylyl cyclase. In addition, we will pharmacologically characterize a constitutively active P2Y12 receptor generated in our lab. We hypothesize that the P2Y1 and P2Y12 receptors synergistically contribute to the thrombus growth and stability in vivo. We further propose to evaluate the relative contribution of the P2Y1 and the P2Y12 receptors to thrombus formation, using P2Y1 or P2Y12 deficient mice and treatment with antagonists and models of in vivo thrombosis and thromboembolism.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL080444-03
Application #
7229003
Study Section
Special Emphasis Panel (ZRG1-HEME-D (02))
Program Officer
Sarkar, Rita
Project Start
2005-05-01
Project End
2009-04-30
Budget Start
2007-05-01
Budget End
2008-04-30
Support Year
3
Fiscal Year
2007
Total Cost
$445,591
Indirect Cost
Name
Temple University
Department
Physiology
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Bhavaraju, Kamala; Lakhani, Parth R; Dorsam, Robert T et al. (2011) G(12/13) signaling pathways substitute for integrin ?IIb?3-signaling for thromboxane generation in platelets. PLoS One 6:e16586
Bynagari-Settipalli, Yamini S; Chari, Ramya; Kilpatrick, Laurie et al. (2010) Protein kinase C - possible therapeutic target to treat cardiovascular diseases. Cardiovasc Hematol Disord Drug Targets 10:292-308
Getz, T M; Dangelmaier, C A; Jin, J et al. (2010) Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in platelets. J Thromb Haemost 8:2283-93
Mayanglambam, Azad; Dangelmaier, Carol A; Thomas, Dafydd et al. (2010) Curcumin inhibits GPVI-mediated platelet activation by interfering with the kinase activity of Syk and the subsequent activation of PLCgamma2. Platelets 21:211-20
Thomas, Dafydd H; Getz, Todd M; Newman, Tiffanny N et al. (2010) A novel histidine tyrosine phosphatase, TULA-2, associates with Syk and negatively regulates GPVI signaling in platelets. Blood 116:2570-8
Daniel, James L; Dangelmaier, Carol A; Mada, Sripal et al. (2010) Cbl-b is a novel physiologic regulator of glycoprotein VI-dependent platelet activation. J Biol Chem 285:17282-91
Mao, Yingying; Zhang, Lili; Jin, Jianguo et al. (2010) Mutational analysis of residues important for ligand interaction with the human P2Y(12) receptor. Eur J Pharmacol 644:10-6
Garcia, Analia; Kim, Soochong; Bhavaraju, Kamala et al. (2010) Role of phosphoinositide 3-kinase beta in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways. Biochem J 429:369-77
Mao, Yingying; Zhang, Ming; Tuma, Ronald F et al. (2010) Deficiency of PAR4 attenuates cerebral ischemia/reperfusion injury in mice. J Cereb Blood Flow Metab 30:1044-52
Ding, Zhongren; Bynagari, Yamini S; Mada, Sripal R et al. (2009) Studies on the role of the extracellular cysteines and oligomeric structures of the P2Y12 receptor when interacting with antagonists. J Thromb Haemost 7:232-4

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