Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca(2+)-dependent tyrosine kinase Pyk2.

In the present study, we used a knockout murine model to analyze the contribution of the Ca(2+)-dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2-deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin α(IIb)β(3) and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A(2) production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA(2) phosphorylation through p38 MAPK. The results of the present study show the importance of the focal adhesion kinase Pyk2 downstream of G-protein-coupled receptors in supporting platelet aggregation and thrombus formation.

[1]  E. Ongini,et al.  Nitric oxide enhances the anti-inflammatory and anti-atherogenic activity of atorvastatin in a mouse model of accelerated atherosclerosis. , 2012, Cardiovascular research.

[2]  E. Hirsch,et al.  Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling. , 2012, Blood.

[3]  Koji Ikeda,et al.  Early Inflammatory Reactions in Atherosclerosis Are Induced by Proline-Rich Tyrosine Kinase/Reactive Oxygen Species–Mediated Release of Tumor Necrosis Factor-&agr; and Subsequent Activation of the p21Cip1/Ets-1/p300 System , 2011, Arteriosclerosis, thrombosis, and vascular biology.

[4]  Michael D Schaller,et al.  Cellular functions of FAK kinases: insight into molecular mechanisms and novel functions , 2010, Journal of Cell Science.

[5]  C. Lipinski,et al.  Targeting Pyk2 for therapeutic intervention , 2010, Expert opinion on therapeutic targets.

[6]  R. Ganju,et al.  HIV-1 gp120-induced migration of dendritic cells is regulated by a novel kinase cascade involving Pyk2, p38 MAP kinase, and LSP1. , 2009, Blood.

[7]  C. Libert,et al.  Loss of matrix metalloproteinase 2 in platelets reduces arterial thrombosis in vivo , 2009, The Journal of experimental medicine.

[8]  P. Gresele,et al.  Potentiation and priming of platelet activation: a potential target for antiplatelet therapy. , 2008, Trends in pharmacological sciences.

[9]  Terukatsu Sasaki,et al.  Protein-tyrosine kinase CAKβ/PYK2 is activated by binding Ca2+/calmodulin to FERM F2 α2 helix and thus forming its dimer , 2008 .

[10]  F. Mancini,et al.  The low-molecular-weight phosphotyrosine phosphatase is a negative regulator of FcgammaRIIA-mediated cell activation. , 2007, Blood.

[11]  A. Monopoli,et al.  NCX 6560, a nitric oxide-releasing derivative of atorvastatin, inhibits cholesterol biosynthesis and shows anti-inflammatory and anti-thrombotic properties. , 2007, European journal of pharmacology.

[12]  H. Matsubara,et al.  Central Role of Calcium-Dependent Tyrosine Kinase PYK2 in Endothelial Nitric Oxide Synthase–Mediated Angiogenic Response and Vascular Function , 2007, Circulation.

[13]  G. Leoncini,et al.  Activation of p38 MAPKinase/cPLA2 pathway in homocysteine‐treated platelets , 2006, Journal of thrombosis and haemostasis : JTH.

[14]  P. Gresele,et al.  Nitroaspirin plus clopidogrel versus aspirin plus clopidogrel against platelet thromboembolism and intimal thickening in mice , 2005, Thrombosis and Haemostasis.

[15]  R. Mohanlal,et al.  Effect of selective inhibition of the p38 MAP kinase pathway on platelet aggregation , 2004, Thrombosis and Haemostasis.

[16]  W. Durante,et al.  Role of the Pyk2–MAP Kinase–cPLA2 Signaling Pathway in Shear-Dependent Platelet Aggregation , 2004, Annals of Biomedical Engineering.

[17]  Takao Shimizu,et al.  Regulatory mechanism and physiological role of cytosolic phospholipase A2. , 2004, Biological & pharmaceutical bulletin.

[18]  F. Sinigaglia,et al.  Contribution of Protease-activated Receptors 1 and 4 and Glycoprotein Ib-IX-V in the Gi-independent Activation of Platelet Rap1B by Thrombin* , 2004, Journal of Biological Chemistry.

[19]  M. Sheetz,et al.  Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[20]  F. Sinigaglia,et al.  Proline-rich Tyrosine Kinase 2 and Focal Adhesion Kinase Are Involved in Different Phases of Platelet Activation by vWF , 2002, Thrombosis and Haemostasis.

[21]  Y. Fu,et al.  RAFTK/Pyk2 involvement in platelet activation is mediated by phosphoinositide 3‐kinase , 2001, British journal of haematology.

[22]  T. Ohmori,et al.  Involvement of Hic-5 in platelet activation: integrin alphaIIbbeta3-dependent tyrosine phosphorylation and association with proline-rich tyrosine kinase 2. , 2001, The Biochemical journal.

[23]  V. Duronio,et al.  Thrombin‐stimulated phosphatidylinositol 3‐kinase activity in platelets is associated with activation of PYK2 tyrosine kinase: activation of both enzymes is aggregation independent , 2000, Journal of cellular physiology.

[24]  T. Ohmori,et al.  Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on alphaIIbbeta3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2. , 2000, The Biochemical journal.

[25]  Shin-Young Park,et al.  RAFTK/Pyk2-mediated cellular signalling. , 2000, Cellular signalling.

[26]  M. Gelb,et al.  Stress stimuli increase calcium-induced arachidonic acid release through phosphorylation of cytosolic phospholipase A2. , 1999, The Biochemical journal.

[27]  G. Tokiwa,et al.  Adaptor Proteins Grb2 and Crk Couple Pyk2 with Activation of Specific Mitogen-activated Protein Kinase Cascades* , 1999, The Journal of Biological Chemistry.

[28]  P. Majumder,et al.  Activation of p38 Mitogen-activated Protein Kinase by PYK2/Related Adhesion Focal Tyrosine Kinase-dependent Mechanism* , 1999, The Journal of Biological Chemistry.

[29]  T. Ohmori,et al.  Suppression of protein kinase C is associated with inhibition of PYK2 tyrosine phosphorylation and enhancement of PYK2 interaction with Src in thrombin-activated platelets. , 1999, Thrombosis research.

[30]  C. Leslie.  Properties and Regulation of Cytosolic Phospholipase A2 * , 1997, The Journal of Biological Chemistry.

[31]  S. Watson,et al.  Phosphorylation and activation of cytosolic phospholipase A2 by 38-kDa mitogen-activated protein kinase in collagen-stimulated human platelets. , 1997, European journal of biochemistry.

[32]  H. Avraham,et al.  Tyrosine Phosphorylation of the Novel Protein-tyrosine Kinase RAFTK during an Early Phase of Platelet Activation by an Integrin Glycoprotein IIb-IIIa-independent Mechanism* , 1997, The Journal of Biological Chemistry.

[33]  O. Mandelboim,et al.  Protection from Natural Killer Cell-Mediated Lysis by HLA-G Expression on Target Cells , 1996, Science.

[34]  S. Lev,et al.  A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation , 1996, Nature.

[35]  S. Lev,et al.  Activation of Pyk2 by Stress Signals and Coupling with JNK Signaling Pathway , 1996, Science.

[36]  H. Avraham,et al.  Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes. , 1996, Blood.

[37]  Robert A. H. White,et al.  Identification and Characterization of a Novel Related Adhesion Focal Tyrosine Kinase (RAFTK) from Megakaryocytes and Brain (*) , 1995, The Journal of Biological Chemistry.

[38]  S. Watson,et al.  Cytosolic Phospholipase A2 Is Phosphorylated in Collagen- and Thrombin-stimulated Human Platelets Independent of Protein Kinase C and Mitogen-activated Protein Kinase (*) , 1995, The Journal of Biological Chemistry.

[39]  Terukatsu Sasaki,et al.  Cloning and Characterization of Cell Adhesion Kinase β, a Novel Protein-tyrosine Kinase of the Focal Adhesion Kinase Subfamily (*) , 1995, The Journal of Biological Chemistry.

[40]  E. Peles,et al.  Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions , 1995, Nature.

[41]  Y. Kiyota,et al.  Beneficial effect of CV-4151 (Isbogrel), a thromboxane A2 synthase inhibitor, in a rat middle cerebral artery thrombosis model. , 1995, Thrombosis research.

[42]  M. Nakashima,et al.  Arterial thrombosis model with photochemical reaction in guinea-pig and its property. , 1992, Thrombosis research.

[43]  J. Bruno,et al.  3,4-Dihydroquinolin-2(1H)-ones as combined inhibitors of thromboxane A2 synthase and cAMP phosphodiesterase. , 1992, Journal of medicinal chemistry.

[44]  G. Nenci,et al.  Picotamide Protects Mice from Death in a Pulmonary Embolism Model by a Mechanism Independent from Thromboxane Suppression , 1990, Thrombosis and Haemostasis.

[45]  E. Cronkite,et al.  Morphology and enumeration of human blood platelets. , 1950, Journal of applied physiology.

[46]  松井 朗裕.  Central role of calcium-dependent tyrosine kinase PYK2 in endothelial nitric oxide synthase mediated angiogenic response and vascular function , 2008 .

[47]  H. Ostergaard,et al.  Focal adhesion kinase-related protein tyrosine kinase Pyk2 in T-cell activation and function , 2005, Immunologic research.

[48]  L. Moro,et al.  Nongenomic effects of 17β-estradiol in human platelets: potentiation of thrombin-induced aggregation through estrogen receptor β and Src kinase , 2005 .

[49]  L. Moro,et al.  Nongenomic effects of 17beta-estradiol in human platelets: potentiation of thrombin-induced aggregation through estrogen receptor beta and Src kinase. , 2005, Blood.

[50]  P. Gresele,et al.  Thromboxane synthase inhibitors, thromboxane receptor antagonists and dual blockers in thrombotic disorders. , 1991, Trends in pharmacological sciences.