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Aerobic glycolysis fuels platelet activation
ities, both early and late, which are categorically energy- intensive.5,6 These activities lead to the generation of macroscopic cell-cell aggregates and fibrin-rich thrombi that can stop bleeding or potentially occlude blood vessels with disastrous consequences to human health. Evidently, platelet energy metabolism sustains a thrombus until it is lysed by the fibrinolytic system. Hence, in our search for an effective anti-thrombotic strategy, we focused on meta- bolic adaptations of platelets to agonist stimulation. We reasoned that disruption of key metabolic steps would prevent platelet activation, which could be developed into a potent anti-thrombotic measure.
When we exposed platelets to physiological agonists, there was sharp rise in cellular oxygen consumption apparently driven by energy-demanding early platelet responses such as shape change, cytoskeletal reorganiza- tion, aggregation and granule secretion.4,7,33,34 Intriguingly, this rise was short-lived and was followed by an abrupt drop at a time when platelets continued to aggregate and would possibly be required to discharge resource-inten- sive late responses such as retraction of fibrin clot, shed- ding of extracellular vesicles and protein synthesis. We
sought the reason behind this rapid plunge in oxygen con- sumption despite enhanced ATP requirement. Aggregate formation could restrict access of oxygen to cells persist- ing within the core of the aggregate mass. However, this possibility was ruled out as oxygen flux in cells pre-treated with RGDS, a tetrapeptide that blocks platelet aggrega- tion, was identical to that in aggregated platelets. Possible mitochondrial dysfunction in stimulated cells was also unlikely as leak respiration and non-mitochondrial oxygen consumption in oligomycin- and antimycin A-treated acti- vated platelets, respectively, were suggestive of viable and well-coupled mitochondria. This prompted us to hypoth- esize a Warburg effect-like phenomenon in stimulated platelets whereby pyruvate is prevented from oxidation in the TCA cycle leading to a decline in mitochondrial respi- ration. Consistent with this possibility, the drop in oxygen flux was obviated when platelets were exposed to agents such as DCA (which enhances flux through the TCA cycle), or alternative electron carriers (methylene blue and toluidine blue O).
The Warburg effect or aerobic glycolysis would require enhanced uptake of glucose by the cells in order to sustain
Figure 6. Scheme for metabolic flux in stimulated platelets and sites of action of small-molecule modulators. Stimulated platelets switch to aerobic glycolysis through negative regulation of pyruvate kinase M2 and pyruvate dehydrogenase enzyme activities. The consequent increase in flux through the pentose phosphate pathway generates NADPH that fuels ROS generation by NADPH oxidase. ROS signaling in turn mediates platelet activation, thrombosis and hemostasis. Small-mol- ecule modulators that reverse this metabolic adaptation inhibit platelet activation and impair thrombus formation. DASA: diarylsulfonamide; DCA: dichloroacetate; DHEA: dehydroepiandrosterone sulphate; NOX: NADPH oxidase; PDH: pyruvate dehydrogenase; PDK: pyruvate dehydrogenase kinase PKM2: pyruvate kinase M2; PPP: pentose phosphate pathway; ROS: reactive oxygen species.
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