Page 180 - 2019_04-Haematologica-web

P.P. Kulkarni et al.
platelets to thrombin (0.5 U/mL), a strong physiological agonist, there was a rapid surge in oxygen consumption rate over the basal respiration (within the first 90 s of stim- ulation) (Figure 1A,B), apparently driven by energy- demanding early platelet responses. This upsurge in oxy- gen consumption rate was, however, followed by an abrupt fall (Figure 1A,B) that, intriguingly, coincided with the exponentially rising phase of the platelet aggregatory response (Online Supplementary Figure S1A). We recorded identical peaking and plunging of oxygen consumption in platelets upon stimulation with collagen, another physio- logical agonist (Online Supplementary Figure S1B).
We sought to identify the reason underlying this rapid plunge in oxygen consumption of stimulated platelets, despite continued ATP requirement for energy-intensive processes such as cytoskeletal reorganization, shedding of extracellular vesicles, and protein synthesis, which are all associated with platelet activation. As aggregate forma- tion could restrict access of oxygen to cells persisting within the core of the aggregate mass, we performed respirometry experiments after pre-treatment with Arg- Gly-Asp-Ser (RGDS), a tetrapeptide that prevents platelet aggregation. Strikingly, we noticed no difference in polarogram profiles (Online Supplementary Figure S1C), which ruled out any contribution of cell-cell aggregate for- mation to the observed drop in oxygen consumption. A reduced rate of cellular respiration could reflect dysfunc- tional mitochondria in agonist-treated platelets.18 We found that oxygen flux following sequential treatment of platelets with oligomycin (an inhibitor of ATPase), car- bonyl cyanide m-chlorophenyl hydrazine (an uncoupler), and antimycin A (an inhibitor of respiratory complex III) was consistent with the presence of well-coupled and viable mitochondria in stimulated platelets (Online Supplementary Figure S2A). This prompted us to hypothe- size a Warburg-like phenomenon19 in stimulated platelets. Consistent with this proposition, the decline in oxygen flux was obviated when platelets were exposed to DCA (Figure 1A,B), which enhances flux through the tricar- boxylic acid (TCA) cycle by promoting pyruvate dehydro- genase activity, or to methylene blue, an alternative elec- tron carrier (Online Supplementary Figure S2B).
The Warburg effect, or aerobic glycolysis, entails aug- mented uptake of glucose from external medium by the cells. We detected 1.8- and 2-fold increases in glucose uptake and lactate generation, respectively, by platelets upon exposure to thrombin (Figure 1C). Enhanced glucose uptake by stimulated platelets is mediated through cell membrane translocation of cytosolic preformed GLUT3.20 As expected, we observed a nearly 41% rise in surface expression of GLUT3 upon stimulation of platelets with thrombin (0.5 U/mL) (Figure 1D,E). The surface mobiliza- tion of GLUT3 in neuronal cells is regulated by the activity of AMP-activated protein kinase (AMPK).21 In line with this, pre-treatment of platelets with compound C, which is a specific inhibitor of AMPK, resulted in a significant drop (by ̴31%) of GLUT3 expression on thrombin-stim- ulated platelets (Figure 1D,E).
Stimulated platelets switch to aerobic glycolysis through negative regulation of pyruvate dehydrogenase and pyruvate kinase M2
We next sought to elucidate the mechanism underlying the observed switch to aerobic glycolysis from oxidative phosphorylation in stimulated platelets. Pyruvate dehy-
drogenase (PDH), the ‘gate-keeper’ enzyme, determines the relative fluxes through glycolysis and oxidative phos- phorylation. PDH activity is regulated through inhibitory phosphorylation (at S293) by pyruvate dehydrogenase kinase (PDK).22 We examined the expression of phospho- rylated PDH in thrombin-stimulated platelets by using a phospho-specific antibody. Thrombin (0.5 U/mL) induced a rise in the level of phosphorylated PDH in platelets (Figure 2A,B), which diverts flux away from the TCA cycle. DCA (20 mM), a pharmacological inhibitor of PDK, almost completely abolished phosphorylation of PDH (Figure 2A,B) and partially restored the drop in oxygen consumption in thrombin-treated platelets (Figure 1A lower panel, 1B). Pre-exposure to DCA also led to signifi- cant inhibition of the thrombin-induced rises in the rates of glucose uptake and lactate generation (by 33% and 28%, respectively) (Figure 1C), suggesting PDK-mediated inactivation of PDH and decreased flux through the TCA cycle in stimulated platelets. Interestingly, DCA also trig- gered a 20% drop in GLUT3 externalization (Figure 1D,E) in thrombin-stimulated platelets. AMPK is known to induce phosphorylation of PDH under conditions of nutri- ent-deprivation leading to inhibition of PDH activity.23 Exposure to compound C reversed the increases in PDH phosphorylation (Figure 2A,B) as well as glucose uptake/lactate secretion rates (Figure 2E) that were observed in thrombin-stimulated platelets. As AMPK activity in platelets is upregulated by thrombin,24 this kinase is positioned in the thrombin signaling pathway, upstream of PDH.
Neoplastic transformation of cells is associated with expression of PKM2, a splice variant of pyruvate kinase, which, unlike PKM1, facilitates aerobic glycolysis through a low-activity dimer state.25 We report that PKM2 is signif- icantly expressed in human platelets (Online Supplementary Figure S3A). Expression of the PKM2 mRNA splice form was remarkably higher than that of its PKM1 counterpart (Online Supplementary Figure S3B). Phosphorylation of PKM2 at Y105 is associated with sustenance of the dimer state with attenuated catalytic activity.26 Using a phospho- specific antibody we observed that exposure to thrombin (0.5 U/mL) evoked appreciably higher phosphorylation (Y105) of PKM2, suggesting low enzymatic activity in stimulated platelets. This phosphorylation was reversed by PP2, an inhibitor of Src family tyrosine kinases, which are activated in stimulated platelets (Figure 2C). PP2 also regressed the thrombin-induced increases in glucose uptake/lactate secretion rates in platelets (Figure 2E). Pre- treatment of cells with DASA (200 mM), an activator of PKM2, decreased thrombin-induced GLUT3 externaliza- tion by 36% (Figure 1D,E). Thus, our findings suggest that stimulated platelets make a metabolic switch to aerobic glycolysis through post-translational regulation of PDH and PKM2 enzyme activities.
Enhanced flux through the pentose phosphate pathway supports reactive oxygen species-dependent integrin activation
Aerobic glycolysis and low PKM2 activity would lead to pooling of glycolytic intermediates upstream of pyruvate, which include glucose-6-phosphate, a substrate for the PPP.25 Hence, we studied metabolic flux through the PPP in thrombin-stimulated platelets, as reflected by the ratio of NADPH to total NADP(H) levels. Thrombin (0.5 U/mL) induced a significant rise (by 50%) in the ratio of NADPH
808
haematologica | 2019; 104(4)