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P.P. Kulkarni et al.
adequate ATP generation by glycolysis. In keeping with this and an earlier report,20 we observed surface transloca- tion of cytosolic GLUT3, the major glucose transporter, in thrombin-stimulated platelets. As already observed for neuronal cells,21 GLUT3 externalization in platelets was facilitated by AMPK, whose activity is known to be upregulated by agonist stimulation.24 Enhanced surface expression of GLUT3 was corroborated by a nearly 2-fold rise in the uptake of glucose in thrombin-activated platelets. In parallel there was enhanced secretion of lac- tate into medium, which was in line with earlier reports of extracellular acidification induced by agonists.34-37
The metabolic checkpoint at the level of pyruvate, which is generated by the enzymatic activity of pyruvate kinase, determines whether glucose will be catabolized to lactate (by glycolysis) or through mitochondrial pathways. The enzyme PDH, part of a larger complex, catalyzes con- version of pyruvate to acetyl-CoA, which is then chan- neled into the TCA cycle for further oxidation and gener- ation of ATP. Reactions catalyzed by PDH and pyruvate kinase are thus two critical hubs in the landscape of cellu- lar glucose metabolism. PDH activity is regulated through inhibitory serine-293 phosphorylation by the enzyme PDK.22 An increase in the activity of PDK and/or reduced PDH activity restricts flux of pyruvate into the TCA cycle and favors aerobic glycolysis. Inhibitory phosphorylation of PDH is known to be induced by AMPK under condi- tions of nutrient-deprivation.23 We observed an increase in phosphorylated PDH in agonist-stimulated platelets, which was reversed by inhibition of AMPK activity. Notably, we also discovered that platelets express PKM2, a tissue-specific isoform of pyruvate kinase, which, in low-activity state, facilitates aerobic glycolysis.38 Phosphorylation of PKM2 at Y105 is associated with sus- tenance of a dimer state with attenuated catalytic activi- ty.26 Interestingly, agonist stimulation of platelets also led to increased expression of phosphorylated PKM2, which could be reduced to a basal level by inhibition of Src fam- ily kinases. Thus, we show here that metabolism in stim- ulated platelets switches to aerobic glycolysis through active regulation of PDH and PKM2; this switch leads to accumulation of glycolytic intermediates upstream of pyruvate and facilitates flux through the PPP.25 Consistent with this we observed significant enrichment in the pool of NADPH, a direct readout of the PPP, in thrombin-stim- ulated platelets.
Increased levels of NADPH in most cells lead to a reduc- tive state through generation of reduced glutathione.25 Yet, in an apparent paradox, NADPH can also serve as a sub- strate for the enzyme NADPH oxidase generating super- oxide free radicals in the phagocytic cells, such as neu- trophils, of the innate immune system.39 Platelets are now widely recognized to be key players in immune responses.40,41 Possibly owing to genealogical and now increasingly recognized functional relationships of platelets with innate immunity, these cells, too, express significant levels of NOX activity,28 which is in fact the pri- mary source of ROS in activated platelets.27,28 Our findings were consistent with a rise in the levels of NADPH in stimulated platelets serving to increase the generation of ROS through activity of NOX. There is now compelling evidence indicating that NOX-generated ROS are impor- tant mediators of platelet activation signaling,29,42 which culminates in the expression of a conformationally active form of integrins αIIbβIIIa.27,43 Remarkably, pre-treatment of
platelets with small-molecule inhibitors of either aerobic glycolysis (DCA or DASA) or the PPP (DHEA) brought about a significant drop in agonist-induced ROS produc- tion as well as integrin activation, and was associated with profound impairment in platelet responses to agonists (platelet aggregation, fibrinogen binding and secretion of contents of dense and α granules). The metabolic inhibitors also attenuated phosphatidylserine exposure and extracellular vesicle release, both measures of a pro- coagulant phenotype31,32 in platelets, thus underlining the significance of aerobic glycolysis and the PPP-NOX axis in platelet activation and thrombosis. In agreement with our findings, genetic deficiency of glucose 6-phosphate dehy- drogenase, the key regulatory enzyme in the PPP, is known to be associated with a remarkably lower risk of cardiovascular mortality.44,45 Strikingly, intravenous admin- istration of metabolic modulators significantly delayed thrombus formation in mesenteric arterioles, retarded thrombus growth and prolonged time needed for com- plete vascular occlusion in murine models. Thus, our find- ings suggest that aerobic glycolysis and associated flux through the PPP in stimulated platelets play critical roles in arterial thrombosis and underscore the therapeutic poten- tial of targeting metabolic pathways as a novel anti- thrombotic approach (Figure 6). These results are also con- sistent with two recent articles by Abel’s group, which establish the role of glucose metabolism in platelet activa- tion using transgenic mice with platelet-specific ablation of glucose transporters.36,37
Currently available anti-platelet agents are plagued by limited efficacy2 which could be attributed to remarkable redundancy in agonist stimuli and downstream signaling pathways that lead to platelet activation. Thus, conven- tional anti-platelet therapeutic regimens that target spe- cific agonists/activation pathways may not confer absolute protection against thrombotic episodes, as platelets can still be stimulated by other potential triggers and parallel signaling inputs. It is, therefore, vital to dis- cover novel anti-platelet strategies to address these chal- lenges and cater to this largely unmet medical need. The small-molecule metabolic modulators employed in our study block essential metabolic checkpoints in stimulat- ed platelets and effectively prevent platelet activation irrespective of the nature of the agonists and ensuing sig- naling pathways.
We validated our findings in a murine model of throm- bosis in which deep vein thrombosis and pulmonary embolism were induced by intravenous administration of collagen and epinephrine. Notably, mice pre-treated with small-molecule metabolic modulators were protected from pulmonary thromboembolism. However, lung sec- tions in these animals revealed extravasated erythrocytes, which was in keeping with prolonged tail-bleeding fol- lowing administration of metabolic modulators. Thus, these small molecules can impair hemostasis despite con- ferring significant protection against thrombosis. Therefore, targeting platelet metabolism, much like cur- rently available anti-platelet regimens, would involve a tightrope-walk of dose adjustment for effective preven- tion of thrombotic events without raising the risk of bleeding complications. Remarkably, metabolic modula- tors employed in our study have some differential effects on tail bleeding and thrombus stability, which raises hope for preferential targeting of thrombosis over hemostasis in the future by tweaking specific metabolic pathways.
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haematologica | 2019; 104(4)