TNF receptor-associated factor 4 (TRAF4) is a novel binding partner of glycoprotein Ib and glycoprotein VI in human platelets

Background: Reactive oxygen species generation is one consequence of ligand engagement of platelet glycoprotein (GP) receptors GPIb-IX-V and GPVI, which bind VWF/collagen and initiate thrombosis at arterial shear; however, the precise molecular mechanism coupling redox pathway activation to engagement of these receptors is unknown. Objective: The objective of this study was to identify novel binding partners for GPIb-IX-V and GPVI that could provide a potential link between redox pathways and early platelet signaling events. Methods and Results: Using protein array analysis and affinity-binding assays, we demonstrated that the orphan TNF receptor-associated factor (TRAF) family member, TRAF4, selectively binds cytoplasmic sequences of GPIbβ and GPVI. TRAF4, p47^phox [of the NADPH oxidase (Nox2) enzyme complex] and other redox relevant signaling proteins such as Hic-5, co-immunoprecipitate with GPIb/GPVI from human platelet lysates whilst MBP-TRAF4 or MBP-p47^phox fusion proteins specifically pull-down GPIb/GPVI. GPIb- or GPVI-selective agonists induce phosphorylation of the TRAF4-associated proteins, Hic-5 and Pyk2, with phosphorylation attenuated by Nox2 inhibition. Conclusion: These results describe the first direct association of TRAF4 with a receptor, and identify a novel binding partner for GPIb-IX-V and GPVI, providing a potential link between these platelet receptors and downstream TRAF4/Nox2-dependent redox pathways.