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Aerobic glycolysis fuels platelet activation
to total NADP(H), which was reversed by up to ~20% in the presence of DHEA (200 mM) (Figure 2F). DHEA, an endogenous steroid hormone, is an inhibitor of glucose 6- phosphate dehydrogenase, the rate-limiting enzyme of the PPP. Small-molecule modulators that facilitate the TCA cycle and check the rate of aerobic glycolysis would also restrict flux of metabolites through the PPP. Predictably, pre-exposure of platelets to DCA (20 mM) or DASA (200 mM) led to reductions in the ratio of NADPH to total NADP(H) of 47% and 12%, respectively (Figure 2F).
Activity of NADPH oxidase (NOX) has been widely documented as a significant source of ROS in stimulated platelets.27,28 We investigated whether NADPH accumulat- ed in thrombin-stimulated platelets acts as a substrate for NOX, and leads to enhanced generation of ROS. In keep- ing with this possibility, pre-incubation of platelets with either DHEA or apocynin (a NOX inhibitor) prior to expo- sure to thrombin resulted in 63% and 62% attenuation in ROS generation, respectively. Remarkably, pre-treatment with DCA and DASA also led to 59% and 43% decreases, respectively, in ROS production in thrombin-stimulated platelets (Figure 2G,H). These decreases were consistent with the impeded PPP flux observed in the presence of these molecules.
There is now considerable evidence to suggest that ROS are important mediators of the platelet activation signal- ing29 that culminates in the expression of conformational- ly-active integrin αIIbβIIIa.27 Consistently, DHEA as well as NOX inhibitors – DPI and apocynin – attenuated the bind- ing of PAC-1 (an antibody directed against conformation- ally-active integrin αIIbβIIIa) to thrombin-stimulated platelets by 59%, 56%, and 62%, respectively (Figure 3A,B). DCA and DASA, which limit flux through the PPP, also inhibited PAC-1 binding by 32% and 65%, respec- tively. Furthermore, the role of ROS in mediating platelet activation was underscored by near-total abrogation of thrombin-induced PAC-1 binding to platelets in the pres- ence of N-acetyl cysteine (10 mM), a ROS scavenger (Online Supplementary Figure S4). As an active conformer of αIIbβ3 is endowed with high affinity towards fibrinogen, we next studied the association of fluorescently labeled fibrinogen to thrombin-stimulated platelets in the pres- ence of these small-molecule modulators. DCA, DASA, DHEA, DPI and apocynin attenuated fibrinogen binding to stimulated platelets by ̴28%, 58%, 37%, 49%, and 63% respectively (Figure 3C,D).
Small-molecule inhibitors of aerobic glycolysis
and the pentose phosphate pathway impair platelet responsiveness
As we demonstrated that small-molecule inhibitors of aerobic glycolysis and the PPP could prevent platelet sur- face integrins αIIbβ3 from switching to an active conforma- tion and attenuate fibrinogen binding, we asked whether they could also inhibit activation-initiated platelet responses including aggregation and secretion of granule contents. Pre-treatment with DCA (20 mM), DASA (200 mM) or DHEA (200 mM) led to reductions in platelet aggre- gation evoked by thrombin (0.5 U/mL) by 61%, 58% and 19%, respectively (Figure 3E,F). A similar inhibition of col- lagen (2 mg/mL)-induced platelet aggregation, albeit to a much greater extent, was also observed (Figure 3G,H), which was consistent with a previous report that described inhibition of platelet aggregation by DHEA in
an Akt/ERK/p38 MAPK-dependent fashion.30 Surface externalization of P-selectin from α-granules and secretion of adenine nucleotides stored in dense granules dropped upon pre-treatment with DCA, DASA or DHEA (Online Supplementary Figure S5). These small molecules also inhibited phosphatidylserine exposure (measured through annexin V binding) on the surface of stimulated platelets (Figure 3I,J), which is critical for the procoagulant activity of the platelets.31 Thrombin elicited shedding of extracel- lular vesicles (size range, 25-800 nm; predominantly 100- 250 nm) bearing a phosphatidylserine-rich procoagulant surface32 from platelets, a phenomenon which was also mitigated by ~40% in the presence of DCA (Online Supplementary Figure S6). The foregoing observations strongly underscore the significance of aerobic glycolysis and the PPP-NOX axis in platelet activation and thrombo- sis. In contrast to DCA, pre-treatment with either antimycin or oligomycin did not significantly inhibit platelet aggregation induced by thrombin (0.2 U/mL) (Online Supplementary Figure S7), suggesting that mito- chondrial respiration is relatively dispensable for platelet activation responses. To rule out the possibility that the observed attenuation in platelet activity was a conse- quence of cell death induced by the small-molecule mod- ulators, we performed a lactate dehydrogenase leak assay. There was no significant release of lactate dehydrogenase activity from platelets when cells were exposed to DCA (20 mM), DASA (200 mM) or DHEA (200 mM) for up to 2 h (Online Supplementary Figure S8).
Small-molecule inhibitors of aerobic glycolysis and the pentose phosphate pathway preclude thrombosis in mice
Platelets play a pivotal role in the pathogenesis of arteri- al thrombosis which underlies occlusive vasculopathies such as acute myocardial infarction and ischemic stroke. In order to establish a link between energy metabolism in activated platelets and arterial thrombosis in vivo, we stud- ied the effect of small-molecule metabolic modulators in a murine model of mesenteric thrombosis induced by ferric chloride. We fluorescently labeled the platelets and induced intramural thrombus in exteriorized mesenteric arterioles of mice administered DCA (200 mg/kg intraperi- toneal), DHEA (50 mg/kg intraperitoneal), DASA (40 mg/kg intravenous) or vehicle (control). Intravital imaging of thrombus formation was carried out by epifluorescence video microscopy using a high-speed camera. We docu- mented the time required for initial thrombus formation, rate of thrombus growth and time to occlusion as pointers to the initiation, propagation and stabilization of throm- bus, respectively. Strikingly, mice pre-treated with DCA (Online Supplementary Video S2), DHEA (Online Supplementary Video S3) or DASA (Online Supplementary Video S4) had significantly prolonged mean times to form first thrombus compared to vehicle-treated mice (DCA, 9.6 ± 1.2 min; DHEA, 11.8 ± 1.02 min; DASA, 9.67 ± 1.2 min, versus control, 5.9 ± 0.60 min) (Figure 4A,B) and impaired thrombus growth rate (Figure 4A,C,D) com- pared to vehicle-treated mice (Online Supplementary Video S1). While the mean occlusion time for control mice was 20.4 ± 2.38 min, stable occlusion failed to occur even after 40 min from the time of injury in all DHEA-pretreated mice and most of the mice administered DCA or DASA (except one from each group) (Figure 4E). These results suggest that agents that block metabolic reprogramming
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