OC 49.5 - Mitochondrial ATP generation in stimulated platelets is essential for granule secretion but dispensable for aggregation and procoagulant activity

Background: Platelets exhibit considerable plasticity in energy metabolism. Yet, we recently demonstrated that diverting the metabolic flux from aerobic glycolysis towards OXPHOS prevents platelet activation. In addition, several studies have established that inhibitors of OXPHOS alone do not compromise thrombin-induced platelet aggregation.

Aims: We aim to determine if mitochondrial respiration is redundant for platelet bioenergetics and function in response to agonist-stimulation.

Methods: We studied the effect of antimycin (complex III inhibitor), oligomycin (FoF1 ATP synthase inhibitor) and CCCP (uncoupler), on platelet responses to agonist-stimulation by lumi-aggregometry, flow cytometry, and shear-based thrombosis studies. The underlying mechanism mediating the observed effects was explored by flow cytometry and biochemical assays. Significance in difference of means was determined by repeated measures ANOVA and Dunnett's multiple comparison test.

Results: Thrombin-induced platelet IIb3 integrin activation, aggregation and procoagulant activity were spared in presence of either oligomycin or antimycin, but were significantly retarded by CCCP. Cellular ATP level, however, remained unchanged under above conditions through a compensatory rise in glycolysis. CCCP, but neither oligomycin nor antimycin, profoundly abrogated thrombin-induced mitochondrial ROS generation and calcium transients that are essential for platelet aggregation and procoagulant activity, respectively. Strikingly, both oligomycin and CCCP restrained platelet granule secretion, as evidenced from compromised ATP release and P-selectin exposure, which was associated with diminished platelet-neutrophil interactions and platelet thrombus formation on immobilized collagen under arterial shear. The effect of oligomycin and CCCP on thrombus growth was abolished in the presence of ticagrelor, an ADP receptor antagonist.

Conclusion(s): Mitochondrial ATP generation is dispensable for platelet aggregation and procoagulant activity, which are fueled by glycolytic ATP. However, maintenance of proton gradient across inner mitochondrial membrane plays a vital role in these processes by supporting ROS generation and mitochondrial calcium influx, respectively. Mitochondrial ATP is critical for platelet granule secretion, platelet-neutrophil interaction and thrombus growth, especially when inadequately compensated by glycolysis.