Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca2+-dependent tyrosine kinase Pyk2

Abstract

Key Points• The tyrosine kinase Pyk2 regulates a p38MAPK-cPLA2 pathway required for thrombin-induced thromboxane A2 production and platelet aggregation.• Pyk2 deletion in mice confers protection against pulmonary thromboembolism and arterial thrombosis.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 ␣ IIb ␤ 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 IntroductionPyk2, also known as RAFTK or CAD␤, is a nonreceptor tyrosine kinase that is highly homologous to the focal adhesion kinase FAK and is predominantly expressed in the CNS and hematopoietic cells. 1-3 Like FAK, Pyk2 does not possess SH2 or SH3 domains, but has a centrally located catalytic domain flanked by an N-terminal FERM domain and a C-terminal focal adhesion targeting FAT domain. Pyk2 can be tyrosine phosphorylated and activated by a variety of different cellular stimuli, including cytokines, growth factors, agonists of G-protein-coupled receptors (GPCRs), integrin ligands, and stress stimuli. [3][4][5][6] Typically, Pyk2 activation is mediated by both Src kinase-and cytosolic Ca 2ϩ -dependent pathways. Src kinases phosphorylate Pyk2 at Tyr579, Tyr580, and Tyr881, increasing the catalytic activity of the kinase and promoting autophosphorylation on Tyr402 in the FERM domain. 5,6 Even in the absence of Src-mediated phosphorylation, Pyk2 can be activated on increase of the cytosolic Ca 2ϩ concentration. [4][5][6][7] It has been shown that Ca 2ϩ and calmodulin bind to the N-terminal FERM domain of Pyk2, triggering Pyk2 dimerization, activation, and autophosphorylation at Tyr402. 8 Phosphorylated Tyr402 is a binding site for the SH2 domain of Src. 5,6 Therefore, Pyk2 is a kinase that can link Ca 2ϩ -based signaling pathways to protein tyrosine phosphorylation.Some observations reported over the past 2 decades have implicated Pyk2 in platelet activation. Pyk2 is highly expressed in megakaryocytes and platelets and is rapidly phosphorylated on stimulation with se...

Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis

“…There are ample published examples in which deletion of a signaling cascade protein causes only modest defects in platelet activation in ex vivo assays, yet the protein contributes significantly to in vivo hemostasis and thrombosis. 56,[76][77][78][79] In a recent example, STXBP5 deletion yielded modest defects on dense granule release but was absolutely required for hemostasis in the tail transection model and for thrombosis in the FeCl 3 -induced carotid injury model. 80 Therefore, it is possible that the sensitivity of the ex vivo assays we employed are insufficient to fully delineate the effects of deleting the autophagy machinery.…”

Distinct Role of Pyk2 in Mediating Thromboxane Generation Downstream of Both G12/13 and Integrin αIIbβ3 in Platelets

“…In addition, we found that Pyk2 inhibition had no effect on ADP-induced platelet aggregation in aspirin-treated platelets, which is consistent with a previous study (41) showing that there was no difference in ADP-induced platelet aggregation from WT or TxA 2 receptor null mice. Recent study (42) has characterized the platelets from Pyk2 knock-out mice and no significant differences are observed between WT and Pyk2 knock-out mice. In addition, arachidonic acid induces normal platelet aggregation in Pyk2-deficient platelets, and Pyk2 is linked to cPLA2 activation (42).…”

“…Recent study (42) has characterized the platelets from Pyk2 knock-out mice and no significant differences are observed between WT and Pyk2 knock-out mice. In addition, arachidonic acid induces normal platelet aggregation in Pyk2-deficient platelets, and Pyk2 is linked to cPLA2 activation (42). In the experimental model adopted from a previous study (21) to confirm the role of Pyk2 downstream of G 12/13 pathways in TxA 2 generation, we found that G 12/13 pathways failed to substitute for integrin-mediated signaling for TxA 2 generation in the presence of the Pyk2 inhibitor confirming the contribution of Pyk2 downstream of G 12/13 pathways.…”

Platelet PI3Kβ and GSK3 regulate thrombus stability at a high shear rate

“…11 Among them, Rap1b and the Ser/Thr kinase Akt have been implicated in a IIb b 3 inside-out and/or outside-in signaling in a PI3Kb-dependent manner. 6,19,[31][32][33][34][35][36][37] Using PDK1-deficient mice, it was proposed that the PDK1/Akt axis modulates platelet aggregation and clot retraction. 38 Although the b3 integrin can be phosphorylated on Thr 753 by PDK1 and Akt in vitro 39 , the exact regulatory mechanism involving these kinases remains to be established.…”