DISCUSSION
A better understanding of the relationship between flavonoids and
platelet function could lead to improved strategies for treating
cardiovascular diseases. In this study, we characterized quercitrin, a
dietary flavonoid with various pharmacological activities that is widely
distributed in nature.(Ma, Luo, Jiang & Liu, 2016; Muzitano et al.,
2006; Sanchez de Medina, Vera, Galvez & Zarzuelo, 2002; Zhi et al.,
2016) Although quercitrin exhibits weaker effects on the inflammatory
response and platelet activation in vitro than quercetin, the
beneficial effects of quercitrin have not been reproduced in
vivo .(Comalada et al., 2005) Indeed, quercetin is not well absorbed
through the intestines, and thus, its plasma levels are not sufficient
for inhibiting platelet function.(Crespy, Morand, Manach, Besson,
Demigne & Remesy, 1999; Hubbard, Wolffram, Lovegrove & Gibbins, 2004)
Further, previous studies did not explore the mechanism by which
quercitrin inhibits platelet activation. In this study, we demonstrated
that quercitrin can inhibit platelet aggregation and prevent platelet
thrombus formation by suppressing GPVI-mediated signaling and ROS
generation. However, platelet activation induced by the GPCR agonist
thrombin was not inhibited by quercitrin, indicating that quercitrin
inhibits platelet function via GPVI-dependent signaling pathways.
Although a variety of agonists can induce strong platelet aggregation,
collagen only substantially stimulated ROS generation in platelets, in
accordance with other observations.(Caccese et al., 2000; Krotz et al.,
2002) The binding of collagen to GPVI induced the activation and
phosphorylation of PLCγ via numerous tyrosine phosphorylation steps. The
inhibition of tyrosine kinases prevented collagen-dependent ROS
generation. However, this finding cannot fully explain the observation
that only collagen induced an increase of ROS generation, as thrombin is
also known to induce the phosphorylation of tyrosine kinases.(Coughlin,
2001; Zielinski, Wachowicz, Saluk-Juszczak & Kaca, 2001) Recent
findings demonstrated that TRAF4 directly binds to the cytoplasmic tail
of GPVI, thereby providing a mechanism of NADPH oxidase
activation(Arthur et al., 2011). Further, p47phox, the
cellular subunit of NADPH, binds to TRAF4, which constitutive interacts
with Syk and Lyn upon GPVI engagement, thereby activating redox
signaling pathways.(Arthur et al., 2011) In our experiments, quercitrin
substantially abolished the phosphorylation of members of the
TRAF4/p47phox/Hic5 complex without affecting the
interaction between TRAF and GPVI (Figure 6G-6J), suggesting that
quercitrin may be a negative regulator of activation of the
TRAF4/p47phox/Hic5 complex during GPVI-mediated
platelet activation. Thus, this finding could explain the differences in
ROS generation between the distinct platelet activation pathways
mediated by GPVI and PARs. However, we could not rule out the
possibility that a low concentration of thrombin can induce
phosphorylation of kinases but may not be reached the increase of
platelet ROS generation.
Additionally, we observed that quercitrin markedly diminished platelet
aggregation and ROS generation not only evoked by CRP stimulation but
also by U46619 stimulation in a concentration-dependent manner (Figure 1
and 4). These results are consistent with the effects of the
TxA2-induced thromboxane receptor (TP)-ROS signaling
pathway,(Muzaffar, Shukla, Lobo, Angelini & Jeremy, 2004) and the
binding of TxA2 to TP on platelets induces PLC
activation and stimulates inositol 1,4,5-triphosphate and diacylglycerol
production, resulting in increased intracellular Ca2+accumulation and ROS generation.(Moers et al., 2003; Offermanns, 2006)
Therefore, it appears that quercitrin inhibits
TxA2-mediated NADPH oxidase activation and that
TxA2-mediated platelet aggregation is also influenced by
ROS generation, which is crucial for positive feedback related to
platelet activation. However, the relevance of TP-mediated ROS signaling
to the pathophysiological actions of TxA2 requires
further investigation.
A number of flavonoids have been demonstrated to inhibit various active
pathways, likely through the inhibition of tyrosine and lipid kinases
involved in platelet responses.(Benavente-Garcia & Castillo, 2008;
Wright, Spencer, Lovegrove & Gibbins, 2013) Since quercitrin inhibited
CRP-induced platelet aggregation and activation, we also investigated
the activation of GPVI signaling proteins. CRP-mediated GPVI signaling
induces tyrosine phosphorylation of the Fc receptor γ-chain by Fyn and
Lyn, which allows the assembly of Syk and subsequent formation of a
complex among LAT, SLP-76, PI3K, PLCγ2, and Btk, leading to activation
of the signaling molecules.(Nieswandt & Watson, 2003; Varga-Szabo,
Braun & Nieswandt, 2009) In particular, PIP3 produced
by PI3K is necessary for the membrane translocation and full activation
of PLCγ2 during GPVI-mediated signaling.(Bobe et al., 2001; Watanabe et
al., 2003) We found that the phosphorylation of GPVI-activated signaling
proteins such as Syk, PLCγ2, PI3K, and AKT was dramatically diminished
in a concentration-dependent manner by quercitrin (Figure 6A and 6B).
However, their phosphorylation was not changed by other agonists such as
thrombin and ADP (data not shown). These data suggest that quercitrin
can influence the immediate effectors within the GPVI signaling pathway,
which do not participate in activation mechanisms stimulated by other
agonists.
Protein tyrosine phosphorylation has been widely accepted to exhibit a
central role in the GPVI signaling pathway, and tyrosine phosphorylation
has been considered to be controlled by the reconciled action of protein
tyrosine kinases and PTPs including SHP1, SHP2, and PTEN.(Senis, 2013)
Thus, PTPs are considered important regulators of GPVI-mediated signal
transduction. Recent studies indicated that ROS are important for the
conformational change-mediated inactivation of PTPs, thereby leading to
the phosphorylation of immunoreceptor tyrosine-based inhibition
motif-containing receptors.(Senis, 2013) However, we found that the
phosphorylation levels of PTEN, SHP1, and SHP2 were reduced by
quercitrin, and these changes were correlated with GPVI-mediated signal
transduction (Figure 6E and 6F). Thus, consistent with a previous report
demonstrating the importance of ROS to PTP activation during platelet
activation,(Jang et al., 2014) these findings provide evidence that
quercitrin regulates ROS-mediated PTP signaling pathways. We speculate
that quercitrin modulates an early stage of GPVI-mediated signal
transduction, whereas ROS participate in a later stage of the feedback
cycle of platelet activation by inactivating PTP. Although the detailed
mechanisms remain to be elucidated, our results suggest that ROS
generation and kinase activity are mutually regulated.
Overall, our studies provide mechanistic insight into the novel effects
of quercitrin on platelet function and ROS generation upon GPVI-mediated
platelet activation. Thus, our results provide evidence that quercitrin
may be a novel therapeutic agent for the treatment of thrombotic
diseases.