Manipulating Metabolic Plasticity By Targeting Pyruvate Kinase M2 in Platelets Inhibits Arterial Thrombosis
Abstract Background: Most of the cellular responses initiated upon platelet activation are energy consuming. Like normal cells, resting platelets rely primarily on oxidative phosphorylation (OXPHOS) to generate ATP, whereas activated platelets exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen, a phenomenon referred to as the Warburg effect in tumor cells) suggesting that metabolic plasticity exists in activated platelets. Although aerobic glycolysis yields less total ATP when compared to OXPHOS, the rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which is well suited for high-energy demands during platelet activation. Pyruvate kinases (PKs) catalyzes the final step of glycolysis, the formation of pyruvate and ATP from phosphoenolpyruvate and ADP. Four PK isoforms exist in mammals: L and R isoforms are expressed in the liver and red blood cells; the M1 isoform is expressed in most adult tissues that have high catabolic demands including muscle and brain; M2 is expressed in cells including activated platelets and leukocytes. While PKM1 and tetrameric PKM2 favor ATP production from OXPHOS through the TCA cycle, dimeric PKM2 drives aerobic glycolysis. Objective: We tested an innovative concept that by manipulating the energy demand of activated platelets (metabolic plasticity), by targeting PKM2, will inhibit platelet function and thrombosis. Methods: Using a specific inhibitor of PKM2 (inhibits PKM2 dimerization and stabilizes tetramers) and standardized platelet in vitro assays, we determined the mechanistic role of PKM2 in modulating platelet function in human and mice. To provide definitive evidence, we generated a megakaryocyte or platelet-specific PKM2-/- mouse (PKM2fl/flPF4Cre). Lactate assay was performed in WT and PKM2 null platelets. Susceptibility to thrombosis was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid artery thrombosis and laser injury models) by utilizing intravital microscopy. Results: We found that PKM2 is relatively highly expressed compared to PKM1 in human and murine platelets. Transmission electron microscopy (immunogold staining) revealed that PKM2 is found in the cytoplasm and a- granule in resting platelets, whereas most of PKM2 translocated to cytoplasm upon activation. Human and mouse platelets pretreated with PKM2 inhibitor exhibited decreased platelet aggregation to sub-optimal doses of collagen and convulxin but not to thrombin. In microfluidics flow chamber assay, human and whole mouse blood pretreated with PKM2 inhibitor formed small thrombi when perfused over collagen for 5 min at an arterial shear rate of 1500s-1 (P<0.05 vs. vehicle control). Platelets from PKM2fl/flPF4Cre mice exhibited decreased platelet aggregation to sub-optimal doses of collagen and convulxin, but not to thrombin, compared to PKM2fl/fl mice concomitant with decrease lactate production. In microfluidics flow chamber assay, whole blood from PKM2fl/flPF4Cre mice formed smaller thrombi when perfused over collagen for 5 min at an arterial shear rate of 1500s-1, compared to PKM2fl/fl mice. PKM2fl/flPF4Cre mice were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury-induced mesenteric artery thrombosis models (P<0.05 vs. vehicle control, N=10 mice/group), without altering hemostasis. PKM2 regulates the phosphorylation signal transducer and activator of transcription 3 (STAT3) and p-STAT3 act as a protein scaffold that facilitates the catalytic process of activating PLCg by kinase Syk in response to low-doses of collagen and CRP, but not TRAP or ADP in human and murine platelets. Interestingly, we found that PKM2 and STAT3 colocalized in the convulixn- stimulated control platelets and less phosphorylation of STAT-3 was observed in activated PKM2 null platelets (P<0.05 vs. WT), suggesting a non-glycolytic role of the PKM2 in regulating collagen signaling. Conclusions: Our results suggest that dimeric PKM2 regulates platelet function and arterial thrombosis most likely via GPVI signaling pathway. We suggest that manipulating metabolic plasticity by targeting dimeric PKM2 may be explored as a novel strategy to inhibit platelet function and arterial thrombosis. Disclosures No relevant conflicts of interest to declare.
