Mitochondrial Reactive Oxygen Species (ROS) Are Involved in Coated Platelet Formation and Platelet Procoagulant Activity.

Abstract Increased vascular oxidative stress has been demonstrated in many pathological conditions associated with an increased predisposition for thrombotic events, including atherosclerosis, diabetes mellitus, and hypertension. We hypothesized that oxidative stress may promote thrombosis through increases in platelet procoagulant activity and coated platelet formation. Upon activation with convulxin (an agonist for the collagen receptor GPVI) and thrombin, approximately 70% of murine platelets acquired the coated platelet phenotype, characterized by increased alpha granule protein retention, decreased binding of the α2bβ3 activation-dependent antibody JON/A, and increased binding of annexin V. Dual stimulation with convulxin and thrombin also resulted in a 4-fold increase in platelet procoagulant activity (measured in a prothrombinase assay) together with rapid oxidation of the redox-sensitive dye, H2DCF. Only small increases in H2DCF oxidation were observed in platelets stimulated with thrombin or convulxin alone demonstrating a unique effect of dual agonist stimulation on platelet oxidant production. Formation of coated platelets and platelet procoagulant activity following stimulation with convulxin and thrombin were inhibited with the non-specific antioxidants, Tiron or Trolox. No inhibitory effect was observed using the NADPH oxidase inhibitor diphenylene iodonium or the NO synthase inhibitor L-NNA. The mitochondrial electron transport inhibitor, rotenone, effectively inhibited ROS production, coated platelet formation, and platelet procoagulant activity (53 ± 7%) (p<0.01) following dual agonist stimulation. We also found that hydrogen peroxide (H2O2) could effectively substitute for convulxin in mediating these dual-stimulated activities. Stimulation with H2O2 (100 mM) in the presence of thrombin (0.5 U/mL) resulted in a 4-fold increase in coated platelets and annexin V binding and a 3-fold increase in platelet procoagulant activity relative to platelets stimulated with thrombin alone (p<0.05). This response was dose-dependent, and no effect was observed with H2O2 in the absence of thrombin. Together these results suggest a central role for oxidant signaling and mitochondrial ROS production in platelet procoagulant activity and coated platelet production and point to a possible mechanism for thrombosis in pathological conditions associated with increased oxidative stress.