Pyruvate kinase M2 promotes venous thrombosis by enhancing SNAP23-mediated platelet exocytosis and consequent NETosis

Little is known about the role of metabolic regulatory mechanisms in the pathobiology of deep vein thrombosis (DVT). Recent studies have demonstrated the involvement of the metabolic enzyme pyruvate kinase M2 (PKM2) in platelet function; however, whether platelet PKM2 contributes to DVT has not been investigated yet. Using platelet-specific PKM2−/− (PKM2Plt-KO) or wild-type (WT) mice orally administered ML265 (a small molecule that limits PKM2 dimers by stabilizing PKM2 tetramers), we found reduced thrombus burden at 48 h post-surgery in the inferior vena cava (IVC) stenosis model compared with littermate controls. This reduction was associated with lower levels of CitH3, a marker of neutrophil extracellular traps (NETs), in the harvested thrombi and improved IVC wall contraction and relaxation responses (assessed by myography). Mechanistically, thrombin-stimulated platelets from PKM2Plt-KO mice or ML265-pretreated platelets from WT mice showed reduced SNAP23 phosphorylation and diminished PF4 release (a marker of α-granule exocytosis). The releasate collected from thrombin-stimulated platelets was less effective at inducing NETosis, compared to respective controls. Utilizing ML265-pretreated human whole blood perfused over a tissue factor-coated surface at a venous shear rate, we found that the area covered by platelet-leukocyte aggregates was profoundly reduced compared to vehicle control. Consistent with murine data, human platelets pretreated with ML265 and stimulated with thrombin exhibited decreased PF4 release and generated releasates that were less potent in inducing NETosis. These findings reveal for the first time that targeting PKM2 genetically or pharmacologically reduces SNAP23-mediated α-granule exocytosis in platelets, platelet releasate-induced NETosis, and susceptibility to DVT.