Abstract
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, α-granule secretion (P-selectin (CD62P) surface expression) and integrin αIIbβ3 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, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbβ3 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.
Highlights
Glycoprotein (GP)VI is a major platelet collagen receptor
To determine the precise location of focal adhesion kinase (FAK) within the GPVI pathway, we analyzed the effect of PF-228 inhibition in collagen related peptide (CRP)-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk), phosphoinositide 3-kinase (PI3-K) and Bruton’s tyrosine kinase (Btk) and upstream of Rac1, PLCc2, Ca2+ release, protein kinase C (PKC), Hic-5, NOX1 and aIIbb3 activation
The Role of FAK in reactive oxygen species (ROS) Formation by Platelets. Overall, these data suggest a novel role for FAK in GPVIdependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway
Summary
Glycoprotein (GP)VI is a major platelet collagen receptor. Following vascular injury, platelet binding to immobilized collagen within the extracellular matrix initiates a cascade of intra-platelet signaling pathways which are essential for platelet activation and subsequent thrombus formation [1]. We have previously shown the presence of a GPVI-associated complex involving tumor necrosis factor receptor-associated factor (TRAF), the NADPH oxidase (NOX) organizer subunit, p47phox, Hic, and proline rich tyrosine kinase 2 (Pyk2), consistent with a potential novel role of this complex in GPVIdependent ROS formation [9]. Pyk was recently shown to be a key regulator of NOX-dependent ROS formation in endothelial cells [10] Both FAK and Pyk are activated downstream of ligand binding to GPVI, but the significance of both these PTKs in GPVIdependent ROS formation and an extensive characterization of their relevance to the GPVI signaling pathway remains unclear [11, 12]. We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. 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 Rac, PLCc2, Ca2+ release, PKC, Hic-5, NOX1 and aIIbb activation
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