Regulation and function of PDK1-Akt-Pyk2 axis in platelets

Summary

Principal Investigator: Satya P Kunapuli
Abstract: DESCRIPTION (provided by applicant): 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.
Funding Period: 2013-08-01 - 2017-06-30
more information: NIH RePORT

Top Publications

  1. pmc Trypsin causes platelet activation independently of known protease-activated receptors
    Yingying Mao
    Satya P Kunapuli, PhD, Department of Physiology, Temple University, 217MRS, 3420 N Broad Street, Philadelphia, Pennsylvania 19140, USA, Tel 1 215 707 4615, Fax 1 215 707 4003, E mail
    Thromb Haemost 110:1241-9. 2013
  2. pmc RhoG protein regulates glycoprotein VI-Fc receptor γ-chain complex-mediated platelet activation and thrombus formation
    Soochong Kim
    From the Departments of Physiology
    J Biol Chem 288:34230-8. 2013
  3. pmc LPS-induced systemic inflammation is more severe in P2Y12 null mice
    Elisabetta Liverani
    1 Temple University School of Medicine, 3420 N Broad St, Philadelphia, PA 19140, USA
    J Leukoc Biol 95:313-23. 2014
  4. pmc PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses
    C Dangelmaier
    Satya P Kunapuli, PhD, Department of Physiology, Temple University, Rm 217 MRB, 3420 N Broad Street, Philadelphia, Pennsylvania 19140, USA, Tel 1 215 707 4615, Fax 1 215 707 4003, E mail
    Thromb Haemost 111:508-17. 2014

Detail Information

Publications5

  1. pmc Trypsin causes platelet activation independently of known protease-activated receptors
    Yingying Mao
    Satya P Kunapuli, PhD, Department of Physiology, Temple University, 217MRS, 3420 N Broad Street, Philadelphia, Pennsylvania 19140, USA, Tel 1 215 707 4615, Fax 1 215 707 4003, E mail
    Thromb Haemost 110:1241-9. 2013
    ..In conclusion, our study demonstrates a novel protease signalling pathway in platelets that is independent of PARs. This protease-induced novel signalling pathway regulates platelet shape change through SFKs and p160ROCK. ..
  2. pmc RhoG protein regulates glycoprotein VI-Fc receptor γ-chain complex-mediated platelet activation and thrombus formation
    Soochong Kim
    From the Departments of Physiology
    J Biol Chem 288:34230-8. 2013
    ..Our data demonstrate that RhoG is expressed and activated in platelets, plays an important role in GPVI-Fc receptor γ-chain complex-mediated platelet activation, and is critical for thrombus formation in vivo. ..
  3. pmc LPS-induced systemic inflammation is more severe in P2Y12 null mice
    Elisabetta Liverani
    1 Temple University School of Medicine, 3420 N Broad St, Philadelphia, PA 19140, USA
    J Leukoc Biol 95:313-23. 2014
    ..Furthermore, clopidogrel may have effects that are independent of P2Y12 receptor blockade. ..
  4. pmc PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses
    C Dangelmaier
    Satya P Kunapuli, PhD, Department of Physiology, Temple University, Rm 217 MRB, 3420 N Broad Street, Philadelphia, Pennsylvania 19140, USA, Tel 1 215 707 4615, Fax 1 215 707 4003, E mail
    Thromb Haemost 111:508-17. 2014
    ..These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses. ..

Research Grants30

  1. Hemostatic factors and sickle cell disease
    Punam Malik; Fiscal Year: 2013
    ..abstract_text> ..
  2. Novel strategies to prevent FcgRIIA-dependent platelet activation and thrombosis
    Wolfgang Bergmeier; Fiscal Year: 2013
    ..The successful completion of this proposal will accelerate the rational design of novel therapeutics for immune-mediated platelet disorders. ..
  3. Immune Responses To AAV-Mediated FIX Gene Transfer
    Hildegund C J Ertl; Fiscal Year: 2013
    ..HC ErtI): T Cells to AAV and AAV-Encoded Transgene Products Project 3 (RW Herzog, C Terliorst): Pathways Towards Immune Tolerance to Coagulation Factors Core A (HC ErtI): Administrative Core Core B (S Zliou): Vector Core ..
  4. Blood Systems Biology
    Scott L Diamond; Fiscal Year: 2013
    ....
  5. MULTIDISCIPLINARY STRUCTURES AT VASCULAR CELL SURFACES
    Timothy A Springer; Fiscal Year: 2013
    ..Administrative (Springer) and Protein Expression (Lu) Cores enhance efficiency of the PPG. (End of Abstract) ..
  6. Outside-in signaling mechanisms of platelet integrin alpha-llb-beta3
    Xiaoping Du; Fiscal Year: 2013
    ....
  7. Role of 12-lipoxygenase in platelet reactivity and type 2 diabetes mellitus
    MICHAEL ALLAN HOLINSTAT; Fiscal Year: 2013
    ..Finally, this study will give significant insight into alternative approaches such as 12-LOX inhibition in order to regulate platelet activity in the growing T2DM population. ..
  8. Endogenous suppression of integrin signaling
    Ulhas P Naik; Fiscal Year: 2013
    ..To test thi hypothesis, we will use Jam-A knockout mice and evaluate the molecular mechanisms through which JAM-A suppresses platelet activation and thus thrombosis. ..
  9. The platelet mitochondrial permeability transition and thrombosis
    Shawn M Jobe; Fiscal Year: 2013
    ....
  10. Novel Molecules in Calcium Signaling in Platelets
    Wolfgang Bergmeier; Fiscal Year: 2013
    ..An in-depth analysis of how CD-GEFI regulates platelet function in vitro and in vivo will aid in its establishment as a target for antiplatelet therapy. ..
  11. Regulation of Platelet Glycoprotein (GP) Ib-IX Complex Function by Membrane Lipid
    Yuandong Peng; Fiscal Year: 2013
    ..The platelet glycoprotein GP Ib-IX complex is one of the proteins regulated by membrane lipid domain. In this project, we sight to investigate this regulatory mechanism. ..
  12. Regulation of Platelet Integrin Signaling in Hemostasis and Thrombosis
    Brian G Petrich; Fiscal Year: 2013
    ..Together our proposed experiments, consistent with the mission of the NHLBI, may point to novel therapies for the prevention and treatment of heart attack and stroke. ..
  13. The role of pleckstrin &pleckstrin-2 in platelet biology.
    Charles S Abrams; Fiscal Year: 2013
    ....
  14. The Biological Roles of Phoshadylinositol Transfer Proteins in Platelets
    Charles S Abrams; Fiscal Year: 2013
    ..In Aim 2, I propose experiments designed to understand the distinct biochemical functions of the individual PITP isoforms. In the final Aim, we will identify the role of PITP isoforms in platelet activation ex vivo and in vivo. ..
  15. Serine/Threonine Phosphatases and Platelet Physiology
    K Vinod Vijayan; Fiscal Year: 2013
    ....
  16. Ask1 a novel regulator of platelet function
    Ulhas P Naik; Fiscal Year: 2013
    ..The identification and characterization of such regulatory mechanisms may define new targets for developing potential therapeutic agents toward thrombotic disorders. ..
  17. Adhesive Signaling by Platelet Integrins
    Sanford J Shattil; Fiscal Year: 2013
    ..The proposed studies will establish common and unique mechanisms of integrin activation in physiological and pathological circumstances, with diagnostic and therapeutic implications for vascular biology and beyond. ..
  18. Chemistry and Biology of Coagulation
    Sriram Krishnaswamy; Fiscal Year: 2013
    ..This program seeks to develop new information by which key reactions of blood clotting are regulated. This information will lead to new concepts and strategies for the treatment of blood clotting-related human disease. ..
  19. Platelet activation by cocaine in HIV-1 patients involves IKK
    MICHELLE MEGAN KIEBALA; Fiscal Year: 2013
    ....
  20. TRIP-PCI: PAR1 Pepducin-Based Interventions in Arterial Thrombosis
    Athan Kuliopulos; Fiscal Year: 2013
    ....
  21. B Cells in Health and Disease
    IGNACIO E SANZ; Fiscal Year: 2013
    ..Finally, and central to this PPG, our results will greatly enhance our ability to design better and safer BCDT therapies and to develop biomarkers of B cell targeted treatments efficacy and safety. ..
  22. Regulation of the early events of platelet activation
    Lawrence F Brass; Fiscal Year: 2013
    ..A better understanding of this critical, but under-explored aspect of platelet biology is key to understanding and preventing what goes wrong when people have heart and strokes. ..
  23. Functional Genetics of COPD
    Edwin K Silverman; Fiscal Year: 2013
    ..resistant Inbred strains which are tested with long-term cigarette smoke exposure. We anticipate that these studies will provide insight into the pathways and mechanisms for COPD susceptibility...
  24. Novel Signaling Pathways in Ischemic Stroke
    JAMES KUANG JAN LIAO; Fiscal Year: 2013
    ..This research application proposes to investigate the role of an emerging signaling pathway, Rho kinase (ROCK), in platelets as a potential therapeutic target for preventing and treating ischemic strokes. ..