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.