Platelet aggregation and ATP secretion are inhibited by
treatment with quercitrin
To investigate the effects of quercitrin on platelet function, we first
examined platelet aggregation. Stimulation with various agonists
including CRP (0.1 µg/ml), U46619 (3 µM), collagen (1 µg/ml), thrombin
(0.025 U/ml), and ADP (2.5 µM) led to significant increases in platelet
aggregation (Figure 1). Compared with the findings for the vehicle
control, quercitrin blocked platelet aggregation induced by intermediate
concentrations of CRP (≤ 0.1 µg/ml), U46619 (≤ 3 µM), and collagen (≤ 1
µg/ml) (Figure 1A-1C), whereas such findings were not found for thrombin
(≤ 0.025 U/ml) and ADP stimulation (≤ 2.5 µM) (Figure 1D and 1E).
Although platelet aggregation was inhibited in the presence of
quercitrin upon CRP, U46619, and collagen stimulation, it is possible
that the residual activity can overcome when high concentration of
agonists (≥ 0.5 µg/ml CRP, ≥ 2.5 µg/ml Collagen, and ≥ 10 µM U46619,
Figure S1). Similar findings were observed in mouse platelets (Figure
S2). These results imply that quercitrin exhibits an important effect on
the activation of GPVI signaling, although it does not completely block
this pathway. A previous study revealed that flavonoids could bind to
plasma proteins.(Wright, Spencer, Lovegrove & Gibbins, 2013) Therefore,
the effect of quercitrin on platelet activation using PRP was also
investigated in comparison with that in washed platelets. We observed
that collagen (≤ 1 µg/ml)-stimulated platelet aggregation in PRP was
diminished by quercitrin in a concentration-dependent manner similar to
the effects of washing platelets (Figure 1F). Consistent with the
previous report, CRP stimulation did not induce platelet aggregation in
PRP compared with the findings in washed platelets (data not shown).
These results suggest that plasma proteins do not influence the effects
of quercitrin on platelet aggregation. To further confirm our findings,
we examined ADP secretion. In these experiments, CRP, U46619, and
collagen also induced ≥ 3-fold increases in ADP secretion compared with
the control findings (Figure 1A(ii)–1D(ii)). Compared with the results
for vehicle-treated platelets, quercitrin-treated platelets exhibited
significant defects in ATP secretion induced by CRP (0.1 µg/ml), U46619
(3 µM), and collagen (1 µg/ml) but not by thrombin (Figure
1A(ii)–1D(ii)). These results indicate that quercitrin could regulate
platelet aggregation and ATP secretion through a signaling pathway
induced by both CRP and U46619, but its effects are distinct from those
of thrombin.