Quercitrin alters focal adhesion tyrosine kinase phosphorylation during GPVI-dependent platelet activation
Since the presence of TRAF4/p47phox/Hic5 complex associated with the platelet collagen receptor facilitates GPVI-dependent ROS formation,(Carrim, Walsh, Consonni, Torti, Berndt & Metharom, 2014) we determined whether quercitrin could affect the interaction of GPVI and TRAF4 during platelet activation. We found that the binding of TRAF4 to GPVI was not changed in CRP-activated platelets in the presence or absence of quercitrin, whereas TRAF4 phosphorylation was abrogated in quercitrin-treated platelets (Figure 6G and 6H). Further, because the association of TRAF4 with p47phox(NADPH oxidase organizer subunit) and Hic5 (which is constitutively bound to Syk and Lyn upon GPVI engagement) is involved in GPVI-dependent ROS generation,(Arthur et al., 2011) we also examined the phosphorylation levels of p47phox and Hic5. We found that the phosphorylation levels of p47phox and Hic5 were significantly reduced by quercitrin following CRP stimulation (Figure 6I and 6J). These results suggest that quercitrin does not affect the binding of TRAF4 to GPVI, but it impairs the phosphorylation of members of the TRAF4/p47phox/Hic5 complex associated with the platelet collagen receptor, thereby regulating ROS generation and GPVI-mediated signaling.