Platelet activation plays a major role in hemostasis and thrombosis. Platelet agonists activate complex signaling cascades resulting in shape change, aIIbb3 integrin activation, and dense granule release and thromboxane A2 (TXA2) generation, amplifying the initial signal. The signaling cascades and the intracellular interaction of these signaling molecules regulating platelet physiological events have not been completely understood. In this application, we propose to test the overall hypothesis that PDK1-Akt-Pyk2 axis regulates platelet functional responses. We propose to understand the interaction of these signaling molecules and the downstream events in the agonist-induced platelet activation using complimentary biochemical, pharmacological, and gene knockout approaches. PDK1 is known to phosphorylate a number of kinases, but its function in platelets has not been elucidated.
Aim 1) We hypothesize that PDK1 plays an important positive regulatory role in platelets through selective phosphorylation and activation of Akt. We propose to test this hypothesis through selective pharmacological inhibitors of PDK1 and using conditional PDK1 null mice. Our preliminary data shows that PDK1 phosphorylation of Thr308 is crucial for the activity of Akt and downstream signaling events. Furthermore our preliminary data shows that PDK1 inhibition affects agonist-induced platelet integrin activation and TXA2 generation.
Aim 2) We hypothesize that intracellular association of PAK with Akt is crucial for its phosphorylation by PDK1. We propose to evaluate the role of PAK in translocation of Akt to the membrane using pharmacological approaches and PAK1 and 2 knockout mice. In preliminary data, we show that Akt translocation to the membrane and phosphorylation follows different kinetics. We postulate that PYK2, through tyrosine phosphorylation of PI3 kinases and PDK1, regulates platelet functional responses and Akt phosphorylation. We also postulate that Pyk2 is activated by G12/13 pathways and regulates thromboxane generation. We will evaluate the function of Pyk2 in aIIbb3 integrin activation, TXA2 generation, clot retraction, and spreading on immobilized fibrinogen, using knockout mice and pharmacological inhibitors. Our preliminary data shows that Pyk2 plays an important role in these platelet functional responses. These studies will enhance our understanding of the intracellular interactions of signaling molecules and their role in platelet activation, and might identify potential newer targets for the treatmentof thrombosis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL118593-01A1
Application #
8580272
Study Section
Special Emphasis Panel (ZRG1-VH-J (02))
Program Officer
Sarkar, Rita
Project Start
2013-08-01
Project End
2017-06-30
Budget Start
2013-08-01
Budget End
2014-06-30
Support Year
1
Fiscal Year
2013
Total Cost
$448,406
Indirect Cost
$159,112
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
Zhou, Yuhang; Abraham, Shaji; Andre, Pierrette et al. (2015) Anti-miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice. Blood 126:2871-81
Bhavanasi, Dheeraj; Badolia, Rachit; Manne, Bhanu Kanth et al. (2015) Cross talk between serine/threonine and tyrosine kinases regulates ADP-induced thromboxane generation in platelets. Thromb Haemost 114:558-68
Badolia, Rachit; Manne, Bhanu Kanth; Dangelmaier, Carol et al. (2015) IPA3 non-specifically enhances phosphorylation of several proteins in human platelets. Platelets 26:501-3
Inamdar, Vaishali; Patel, Akruti; Manne, Bhanu Kanth et al. (2015) Characterization of UBO-QIC as a Gαq inhibitor in platelets. Platelets 26:771-8
Kostyak, John C; Kunapuli, Satya P (2015) PKCθ is dispensable for megakaryopoiesis. Platelets 26:610-1
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

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