PKM2 regulates SNAP-23–mediated platelet exocytosis and consequent NETosis. (A) NETosis assay was performed by stimulating bone marrow–derived WT neutrophils with releasates collected from thrombin (0.1 U/mL)-activated platelets isolated from PKM2^WT or PKM2^Plt-KO mice. Representative microphotographs (left) of NET in lower magnification (20×) and higher magnification (100×) stained with Sytox green (stains extracellular DNA, green) and counterstained with Hoechst (stains nuclei, blue). Higher magnification images show NET from the boxed regions (scale bar for 20× image, 50 μm; scale bar for 100× image, 10 μm). Quantification of the percentage of cells releasing NET (right) calculated in lower magnification (20×; n = 4 per group). Values are mean ± SEM; Mann-Whitney U test. (B) Platelets isolated from WT and PKM2^Plt-KO mice were stimulated with thrombin (0.1 U/mL; 2 minutes), and the level of SNAP-23 phosphorylation was measured by western blot; shown are a representative western blot (upper) and a densitometry analysis (lower) of western blots (n = 3 per group). Values are mean ± SEM; 1-way analysis of variance (ANOVA) with Tukey multiple comparisons test. (C) Platelets isolated from PKM2^WT and PKM2^Plt-KO mice were stimulated with thrombin (0.1 U/mL; 2 minutes) and centrifuged for 3 minutes; the level of PF4 was measured in the supernatant by western blot. The upper panel shows a representative western blot, and the lower panel shows Ponceau S staining (n = 3 per group). Values are mean ± SEM; 1-way ANOVA with Tukey multiple comparisons test.