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

Background: Upon vascular damage, the exposed subendothelial matrix recruits circulating platelets to site of injury while inducing their firm adhesion mainly via GPVI-collagen interaction. GPVI also supports aggregatory and pro-coagulant functions in arterial shear rate even on the matrix other than collagen. Reactive oxygen species (ROS) modulate these stages of thrombosis; however 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. Methods: Fresh PRP-PCs (platelet concentrates) were either stimulated with potent ROS-inducers PMA and CCCP or stored for 5 days. Intra-platelet superoxide (O2-) or mitochondrial-ROS and GPVI expression were detected by flowcytometery. GPVI shedding, platelet aggregation and spreading/adhesion to collagen were analyzed by western blot, aggregometry and fluorescence-microscopy, respectively. Results: 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 higher than those induced by these potent stimuli. GPVI expressions were reduced comparably in CCCP treated and 5 days-stored PCs. Platelet adhesion was also diminished during storage while demonstrating significant reverse correlation with GPVI shedding. However, only firm adhesion (indicated by platelets spreading or adhesion surface area) was relevant to GPVI expression. Platelet adhesion and aggregation also showed reverse correlations with both O2- and mitochondrial-ROS formations; nonetheless mitochondrial-ROS was only relevant to firm adhesion. Conclusion: As a sensitive indicator of platelet activation, GPVI shedding was correlated with either simple adhesion or spreading to collagen, while GPVI expression was only relevant to platelet spreading. Thereby, if the aim of GPVI evaluation is to examine platelet firm adhesion, expression seems to be a more specific choice. Furthermore, the comparable levels of ROS generation in 5 days-stored PCs and CCCP treated platelets, indicated that these products are significantly affected by oxidative stress. Reverse correlation of accumulating ROS with collagen-dependent platelet dysfunction is also a striking sign of an oxidant-induced lesion that may raise serious question about the post-transfusion quality and competence of longer-stored platelet products.