Collagen-dependent platelet dysfunction and its relevance to either mitochondrial ROS or cytosolic superoxide generation: a question about the quality and functional competence of long-stored platelets

Abstract

Upon vascular damage, the exposure of sub-endothelial matrix to circulating platelets causes their firm adhesion and spreading on the site of injury mainly via GPVI-collagen interaction. Whilst reactive oxygen species (ROS) modulate this stage of thrombosis, augmented oxidant stress also disturbs platelet functions. Stored-dependent platelet lesion is associated with the increasing levels of ROS. Whether ROS accumulation is also relevant to collagen-dependent platelet dysfunction is the main interest of this study. Here, fresh PRP-PCs (platelet concentrates) were either stimulated with PMA and CCCP or stored for 5 days. Intra-platelet superoxide or mitochondrial-ROS was detected by flowcytometery. GPVI shedding, platelet aggregation and spreading/adhesion to collagen were analyzed by western blot, aggregometry and fluorescence-microscopy respectively. Mitochondrial-ROS levels in 5 days-stored PCs were comparable to those induced by mitochondrial uncoupler, CCCP while O2-- generations were higher than those achieved by PMA. Shedding levels in 5 days-stored PCs were also higher than those induced by these potent stimuli. Platelet adhesion surface area in 5 days-stored PCs was tremendously reduced compared to that of fresh ones. Whereas platelet spreading/adhesion and aggregation in response to collagen showed reverse correlations with both O2-- and mitochondrial-ROS formations, total number of adhesion was only relevant to O2-- generations. Conclusively, ROS accumulations in 5 days-stored platelets were at the levels comparable to those induced by potent stimuli. Direct correlation of accumulating ROS with collagen-dependent platelet spreading/adhesion and aggregation is a striking sign of an oxidant-induced lesion that may raise serious question about the post-transfusion quality and competence of longer stored products.