title: Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.
abstract: BACKGROUND
  We 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.
  
  
  OBJECTIVE
  To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.
  
  
  RESULTS
  Human 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.
  
  
  CONCLUSIONS
  Overall, 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.
authors.affiliation: Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.
authors.forename: Naadiya
authors.initials: N
authors.lastname: Carrim
authors.affiliation: Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.
authors.forename: Tony G
authors.initials: TG
authors.lastname: Walsh
authors.affiliation: Laboratories of Biochemistry, Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Consonni
authors.affiliation: Laboratories of Biochemistry, Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Torti
authors.affiliation: Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Curtin Health Innovation Research Institute, Faculty of Health Sciences, Curtin University, Perth, Australia.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Berndt
authors.affiliation: Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Curtin Health Innovation Research Institute, Faculty of Health Sciences, Curtin University, Perth, Australia.
authors.forename: Pat
authors.initials: P
authors.lastname: Metharom
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D060588:LIM Domain Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D015539:Platelet Activation / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D053449:TNF Receptor-Associated Factor 4 / Q000235:genetics / Q000378:metabolism
chemical_list: D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D010980:Platelet Membrane Glycoproteins; D017382:Reactive Oxygen Species; C490351:TGFB1I1 protein, human; D053449:TNF Receptor-Associated Factor 4; C506367:TRAF4 protein, human; C090090:Tgfb1i1 protein, mouse; C506368:Traf4 protein, mouse; C098341:platelet membrane glycoprotein VI; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; C496050:PTK2 protein, human; C103547:PTK2B protein, human; C496051:Ptk2 protein, mouse; C496053:Ptk2b protein, mouse