The Reactive Oxygen Species Scavenger N-Acetyl-L-Cysteine Reduces Storage-Dependent Decline in Integrin α IIb β 3-Mediated Platelet Function, Inhibiting Pre-Activation of Integrin and Its β 3 Subunit Cleavage.

Abstract

Background: Premature activation of integrin α _IIb β ₃ plays a central role in the induction and development of the platelet storage lesion (PSL) characterized by an exhausted platelet phenotype that affects adhesion and spreading on fibrinogen. Given the role of reactive oxygen species (ROS) in regulating platelet activation per se, we investigated the effects of a ROS scavenger on reducing the functional decline of platelet integrin α _IIb β ₃ during storage. Methods: Platelet-rich plasma-platelet concentrates (PRP-PCs) were either treated with ROS-reducing agents (1 mM N-acetyl-L-cysteine [NAC] or 30 μM NADPH oxidase [NOX] inhibitor, VAS2870) or kept untreated during storage. CD41/CD61 (total integrin α _IIb β ₃) expression and PAC-1 binding (specific to active integrin α _IIb β ₃ conformation) were analyzed by flow cytometry over a 5 day storage period. Molecular changes in integrin β ₃ subunit were evaluated by western blotting. Platelet adhesion/spreading to fibrinogen in the presence of ROS inhibitors was also investigated during storage using fluorescence microscopy. Results: A decrease in the molecular weight of integrin β ₃ subunit was observed during platelet storage, and was significantly reduced by NAC but not VAS2870, suggesting proteolytic cleavage of β ₃ during storage. Further to this, ROS inhibitors decreased integrin activation and increased platelet adhesion to fibrinogen from day 3 of storage, while NAC but not VAS2870 improved platelet spreading. Conclusion: This is the first report of increasing β ₃ cleavage of integrin during storage that was inversely correlated with integrin α _IIb β ₃-mediated platelet function. In this regard, as a generic ROS scavenger, NAC was shown to reduce defects in platelet spreading through inhibition of β ₃ cleavage. This is in contrast to VAS2870 which selectively inhibits cytosolic NOX alone, suggesting that the reduced platelet function observed during storage may be due to cumulative effects of mitochondrial ROS. Taken together, these studies suggest that adding NAC to platelets may significantly preserve optimal integrin α _IIb β ₃ and platelet function during storage. Moreover, as a reversible scavenger, its inhibitory effect can be readily compensated after transfusion.