Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation
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BackgroundWe have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.AimsTo evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.Methods and ResultsHuman and mouse washed platelets (from WT or Pyk2 KO mice) were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively) and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, a-granule secretion (P-selectin (CD62P) surface expression) and integrin aIIbß3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk), PI3-K and Bruton's tyrosine kinase (Btk) and upstream of Rac1, PLC?2, Ca2+ release, PKC, Hic-5, NOX1 and aIIbß3 activation.ConclusionOverall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.
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