The selective effects of quercitrin on GPVI-induced ROS generation in human platelets
Quercitrin is already known to demonstrate free radical scavenging activity;(Li, Jiang, Wang, Liu & Chen, 2016) however, the precise effects of quercitrin on platelet activation are unclear. Therefore, we studied the anti-oxidant effects of quercitrin during the recruitment of platelets induced by CRP stimulation. To determine whether platelet agonists induce ROS (H2O2) generation, we first measured intracellular ROS levels via flow cytometry using fluorescent DCFH-DA as a probe. Because quercitrin-treated platelets were selectively defective for CRP- and U46619-induced platelet aggregation and ATP secretion (Figure 1), human platelets were stimulated with CRP (0.1 µg/ml), U46619 (3 µM), or thrombin (0.025 U/ml). Consistent with the aforementioned results, quercitrin treatment selectively inhibited CRP- or U46619-induced intracellular ROS generation (Figure 4A and 4B) relative to the findings in the vehicle controls, whereas the compound exhibited no effects on thrombin-induced ROS generation (Figure S4). These results suggest that the inhibition of intracellular ROS generation is selectively important for the effects of quercitrin on platelet activation. To further examine whether quercitrin alters H2O2 accumulation in the extracellular space, we measured extracellular ROS levels using the Amplex Red assay.(Wojtala, Bonora, Malinska, Pinton, Duszynski & Wieckowski, 2014) Compared with the findings for the vehicle control, quercitrin treatment decreased extracellular H2O2 levels in response to CRP stimulation in a concentration-dependent manner (Figure 4C). These results suggest that quercitrin regulates ROS generation, which could be important for its inhibitory effects on platelet activation.