Reducing state attenuates ectodomain shedding of GPVI while restoring adhesion capacities of stored platelets: evidence addressing the controversy around the effects of redox condition on thrombosis.

Thrombosis involves different stages including platelet adhesion to the site of injury, aggregatory events governed by integrin activation, pro-inflammatory responses recruiting leukocytes and finally, pro-coagulant activity which results in fibrin generation and clot formation. As important signaling agents, reactive oxygen species (ROS) reduce thrombus volume and growth, however given such a multistage mechanism, it is not well-elucidated how ROS inhibition modulates thrombosis. PRP-platelet concentrates (PCs) were either treated with ROS-reducing agents (1 mM NAC or 30 µM NOX inhibitor, VAS2870) or kept untreated during storage. Shedding and expression of platelet adhesion receptors in presence of inhibitors, agonists and CCCP (as controls) were analyzed by flow cytometery and western blot respectively. Besides above parameters, platelet adhesion to collagen in stored platelets was examined in presence of ROS inhibitors using fluorescence-microscopy. Highest levels of adhesion receptors shedding were achieved by ionophore and CCCP while collagen induces much more GPVI shedding than that of GPIbα. ROS inhibition reduced receptors shedding from day 3 of storage while enhanced their expressions. ROS inhibition not only did not reduce platelet adhesion capacity but it also enhanced platelets adhesion (in presence of NAC) or spreading (in presence of VAS2870) in 5 days-stored PCs. While reducing state significantly inhibits platelet aggregation and thrombus growth, our results indicated that as a first stage of thrombosis, platelet adhesion is resistance to such inhibitory effects. These findings highlight the fact that integrin-dependent platelet activation is much more vulnerable to the inhibition of ROS generation than GPVI-dependent platelet adhesion. Presumably, inhibition of platelet activating signals by ROS inhibitors preserves platelet adhesiveness to collagen due to lessening GPVI shedding.

Intraplatelet reactive oxygen species (ROS) correlate with the shedding of adhesive receptors, microvesiculation and platelet adhesion to collagen during storage: Does endogenous ROS generation downregulate platelet adhesive function?

Platelets storage lesion is mainly orchestrated by platelet activating signals during storage. Reactive oxygen species (ROS) are being considered as important signaling molecules modulating platelet function while their production has also been shown to be augmented by platelet activation. This study investigated to what extent endogenous ROS generation during platelet storage could be correlated with platelet receptor shedding, microvesiculation and adhesive function. 10 PRP-platelet concentrates were subjected to flow cytometry analysis to examine the generation of intraplatelet ROS on days 1, 5 and 7 after storage. In 5 day-stored platelets considering 40% of ROS generation as a cutoff point, samples were divided into two groups of those with higher or lower levels of ROS. The expression of adhesion receptors (GPVI, GPIbα), the amount of microparticles and phosphatidylserine exposure in each group were then examined by flow cytometry. Platelet receptor shedding and adhesion to collagen matrix were respectively measured by western blotting and microscopic assays. Our data showed lowered expression of GPIbα (p < 0.05) and GPVI in samples with ROS > 40% than those with ROS ≤ 40%, whereas receptors shedding and microvesiculation were (p < 0.05) elevated in platelets with higher levels of ROS. Functionally, we observed significantly (p < 0.05) lower levels of platelet adhesion to collagen matrix in samples with ROS generation more than 40%. Taken together, we showed correlations between intraplatelet ROS generation and either platelet receptors or microparticle shedding as well as platelet adhesive capacity to collagen. These findings suggest that augmented ROS generation during storage might be relevant to down-regulation of platelet adhesive function.