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.