ITAM receptor-mediated generation of reactive oxygen species in human platelets occurs via Syk-dependent and Syk-independent pathways

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. Objectives: 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. Methods and Results: Using an H₂DCF-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 2min followed by additional ROS reaching a plateau after 15-20min. 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.