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.