INTRODUCTION
The response of platelets to vascular damage is essential for hemostasis, whereas excessive platelet activation leads to thrombotic vascular conditions such as stroke, myocardial infarction, and arterial thrombosis.(Yeung, Li & Holinstat, 2018) In response to vascular injury, platelets rapidly adhere to immobilized adhesive proteins such as von Willebrand factor and collagen, thereby triggering the activation and aggregation of platelets and subsequent thrombus formation.(Furie & Furie, 2008) Collagen-induced platelet activation is initiated by binding to the primary glycoprotein VI (GPVI) receptor. The signaling pathway is initiated by Src family kinase-mediated phosphorylation of tyrosine residues associated with immunoreceptor tyrosine-based activation motif-containing FcRγ chains.(Nieswandt & Watson, 2003) Subsequently, phospholipase Cγ2 (PLCγ2) is activated by the signaling cascades involved in the recruitment and activation of spleen tyrosine kinase (Syk), lymphocyte cytosolic protein 2 (SLP-76), Vav1, phosphatidylinositol 3-kinase (PI3K), and Bruton’s tyrosine kinase (Btk). The tyrosine phosphorylation-based activation of PLCγ2 eventually leads to intracellular Ca2+ accumulation, a marker of platelet activation and thrombus formation.(Munnix et al., 2005; Varga-Szabo, Braun & Nieswandt, 2009) In addition, TNF receptor-associated factor 4 (TRAF4), as a binding partner of p47phox in NADPH oxidase 1 and 2 complexes, interacts with the intracellular sequences of GPVI, thereby providing redox signaling pathways and a major source of reactive oxygen species (ROS) generation in platelets.(Arthur et al., 2011) Further, TRAF4 associates with Hic-5 and Pyk2, which constitutively interact with the Src family tyrosine kinase Lyn upon GPVI engagement, leading to propagation of the GPVI signalosome and the recruitment and phosphorylation of Syk.(Arthur et al., 2011; Carrim, Walsh, Consonni, Torti, Berndt & Metharom, 2014) Protein tyrosine phosphorylation has been widely accepted as a central event in the regulation of GPVI signaling in platelets.
Previous studies illustrated that ROS levels are profoundly increased by GPVI stimulation in platelets, whereas intracellular ROS levels do not actively change in response to G protein-coupled receptor (GPCR) agonists such as thrombin and ADP.(Begonja et al., 2005; Krotz et al., 2002; Pignatelli, Pulcinelli, Lenti, Gazzaniga & Violi, 1998) Upon GPVI stimulation, the production of ROS is crucial for propagation of the GPVI signalosome, including PLCγ2 activation, Ca2+mobilization, granule secretion, and αIIbβ3 integrin activation.(Begonja et al., 2005; Krotz et al., 2002; Pignatelli, Pulcinelli, Lenti, Gazzaniga & Violi, 1998) Further, GPVI-mediated ROS production triggers the oxidative inactivation of cytosolic protein tyrosine phosphatases (PTPs) including SH2 domain-containing tyrosine phosphatases 1 and 2 (SHP1 and SHP2), thereby triggering tyrosine phosphorylation-dependent signal transduction.(Jang et al., 2014; Senis, 2013) Therefore, an oxidative submembranous environment in activated platelets would potentially be controlled by the coordinated action of protein tyrosine kinases and PTPs.(Jackson, Schoenwaelder, Yuan, Salem & Cooray, 1996; Tonks & Neel, 1996) However, the underlying mechanism by which ROS regulate the GPVI signaling cascade remains elusive.
A large epidemiological study conducted in the USA regarding the dietary prevention of cardiovascular diseases indicated that thrombogenesis could be reduced by the quality and quantity of dietary fruits and vegetables.(Pallazola et al., 2019; Yamamoto, Ijiri, Ikarugi, Otsui, Inoue & Sakariassen, 2018) Thus, investigating novel natural products and defining novel targets for the prevention of thrombogenesis became important areas of cardiovascular disease research. Flavonoids are ubiquitous secondary metabolites produced under various environmental conditions in fruits and vegetables.(Hertog, Feskens, Hollman, Katan & Kromhout, 1993) Flavonoids are primarily typified by the ability to inhibit enzymes, and they exhibit a number of biological activities such as anti-oxidant and anti-inflammatory properties.(Panche, Diwan & Chandra, 2016; Serafini, Peluso & Raguzzini, 2010) These activities could explain the beneficial effects of flavonoid intake on a variety of human pathologies, including hypertension, inflammatory conditions, and cardiovascular disease.(Comalada et al., 2005) Among the flavonoids, quercetin is the most common flavonoid in nature, and it exists primarily in glycosylated forms such as quercitrin (3-rhamnoside).(Hertog, Feskens, Hollman, Katan & Kromhout, 1993) Quercitrin is known to exhibit biological effects such as anti-oxidant, anti-inflammatory, and anti-apoptotic activities.(Ma, Luo, Jiang & Liu, 2016; Zhi et al., 2016) The sugar moiety of quercitrin usually increases its solubility in polar solvents, consequently resulting in improved absorption.(Gee, DuPont, Rhodes & Johnson, 1998) Thus, quercitrin might be a more potent anti-oxidant than quercetin because of its high bioavailability in the digestive tract.(Dai, Ding, Zhang, Cai & Li, 2013; Jo et al., 2008) However, little is known about the biological properties of the glycoside form (quercitrin) compared with the aglycone form (quercetin) because of the lack of commercial standards. Although a variety of pharmacological activities of quercitrin have been studied,(Ma, Luo, Jiang & Liu, 2016; Zhi et al., 2016) the mechanism underlying its potential inhibitory effects on platelet thrombus formation has not been studied previously. Given that GPVI-mediated platelet activation is inhibited by anti-oxidant enzymes, it is suggested that flavonoids are key negative regulators of GPVI-mediated signaling pathways via regulating ROS scavenging. However, the role of flavonoids in GPVI-stimulated platelet activation is unclear. Therefore, the present study aimed to clarify the anti-platelet activity of quercitrin.
In the present study, we demonstrated that quercitrin inhibits thrombus formation in vivo and in vitro . Quercitrin specifically inhibits platelet activation and aggregation induced by collagen-related peptide (CRP) and U46619. Using biochemical approaches, we revealed that quercitrin acts as a negative regulator of the GPVI-mediated signalosome, probably dependently of GPVI-mediated ROS generation. Further, the anti-oxidant effect of quercitrin is probably derived from the decreased phosphorylation of components of the TRAF4/p47phox/Hic5 signalosome. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that quercitrin plays a crucial role in stroke-induced brain damage. The tail bleeding time was not significantly higher in quercitrin-treated mice than in control mice. These results demonstrate that quercitrin can potentially exert anti-platelet and antithrombotic effects without affecting hemostasis. Hence, quercitrin could be an effective therapeutic agent for the treatment of thrombotic diseases.