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.