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.