| Summary: | Background: The proline-rich tyrosine kinase Pyk2 is a focal adhesion kinase expressed in blood platelets, and is activated downstream of G-protein coupled receptors as well as integrin a2ß1. Objective: In this study we have investigated the involvement of Pyk2 in integrin aIIbß3 outside-in signaling in human and murine platelets. Methods: We analyzed the stimulation of intracellular signaling pathways in platelets from Pyk2 knockout mice adherent to immobilized fibrinogen. Results: Pyk2 was rapidly phosphorylated and activated in human and murine platelets adherent to fibrinogen through integrin aIIbß3. Activation of Pyk2 was Src-dependent, but did not require phospholipase C?2 activity. Platelets from Pyk2 knockout mice showed a defective ability to adhere and spread on fibrinogen, in association with a dramatic reduction of phosphatidylinositol 3-kinase (PI3K) activation and Akt phosphorylation. Pharmacological and genetic analysis demonstrated that integrin aIIbß3 engagement selectively stimulated the ß-isoform of PI3K (PI3Kß), and that, as for Pyk2, PI3Kß activation required Src family kinases activity, but not phospholipase C?2. In fibrinogen-adherent platelets, both Pyk2 and PI3Kß were necessary for stimulation of the small GTPase Rap1b, a regulator of cell adhesion and spreading. Integrin aIIbß3 engagement triggered the association of the PI3Kß regulatory subunit p85 with the adaptor protein c-Cbl, which was mediated by the p85 SH3 domain, and was independent of c-Cbl tyrosine phosphorylation. However, p85-associated c-Cbl was tyrosine phosphorylated by activated Pyk2 in fibrinogen adherent platelets. Conclusions: These results identify a novel pathway of integrin aIIbß3 outside-in signaling and recognize the tyrosine kinase Pyk2 as a major regulator of platelet adhesion and spreading on fibrinogen. © 2012 International Society on Thrombosis and Haemostasis.
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