title: ITAM receptor-mediated generation of reactive oxygen species in human platelets occurs via Syk-dependent and Syk-independent pathways.
abstract: BACKGROUND
  Ligation of the platelet-specific collagen receptor, GPVI/FcRγ, causes rapid, transient disulfide-dependent homodimerization, and the production of intracellular reactive oxygen species (ROS) generated by the NADPH oxidase, linked to GPVI via TRAF4.
  
  
  OBJECTIVE
  The aim of this study was to evaluate the role of early signaling events in ROS generation following engagement of either GPVI/FcRγ or a second immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor on platelets, FcγRIIa.
  
  
  RESULTS
  Using an H(2) DCF-DA-based flow cytometric assay to measure intracellular ROS, we show that treatment of platelets with either the GPVI agonists, collagen-related peptide (CRP) or convulxin (Cvx), or the FcγRIIa agonist 14A2, increased intraplatelet ROS; other platelet agonists such as ADP and TRAP did not. Basal ROS in platelet-rich plasma from 14 healthy donors displayed little inter-individual variability. CRP, Cvx or 14A2 induced an initial burst of ROS within 2 min followed by additional ROS reaching a plateau after 15-20 min. The Syk inhibitor BAY61-3606, which blocks ITAM-dependent signaling, had no effect on the initial ROS burst, but completely inhibited the second phase.
  
  
  CONCLUSIONS
  Together, these results show for the first time that ROS generation downstream of GPVI or FcγRIIa consists of two distinct phases: an initial Syk-independent burst followed by additional Syk-dependent generation.
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria, Australia. jane.arthur@monash.edu
authors.forename: J F
authors.initials: JF
authors.lastname: Arthur
authors.forename: J
authors.initials: J
authors.lastname: Qiao
authors.forename: Y
authors.initials: Y
authors.lastname: Shen
authors.forename: A K
authors.initials: AK
authors.lastname: Davis
authors.forename: E
authors.initials: E
authors.lastname: Dunne
authors.forename: M C
authors.initials: MC
authors.lastname: Berndt
authors.forename: E E
authors.initials: EE
authors.lastname: Gardiner
authors.forename: R K
authors.initials: RK
authors.lastname: Andrews
mesh_terms: D020816:Amino Acid Motifs; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002352:Carrier Proteins / Q000494:pharmacology; D003435:Crotalid Venoms / Q000494:pharmacology; D004789:Enzyme Activation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D037181:Lectins, C-Type; D008297:Male; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000819:agonists / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D017452:Receptors, IgG / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000072377:Syk Kinase; D013997:Time Factors
chemical_list: C477642:2-(7-(3,4-dimethoxyphenyl)imidazo(1,2-c)pyrimidin-5-ylamino)nicotinamide; D002352:Carrier Proteins; D003435:Crotalid Venoms; C080568:Fc gamma receptor IIA; D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D017382:Reactive Oxygen Species; D017452:Receptors, IgG; C435908:collagen-related peptide; C098341:platelet membrane glycoprotein VI; D009536:Niacinamide; C028890:convulxin; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase