Limiting PKM2 dimerization inhibits NETosis in vitro and reduces α-granule exocytosis in platelets from human blood. (A) NETosis assay was performed by stimulating human neutrophils with releasates collected from thrombin (0.1 U/mL)-activated human platelets in the presence or absence of ML265 (100 μM); representative microphotographs (left) of NET stained with Sytox green (stains extracellular DNA; green) and counterstained with Hoechst (stains nuclei; blue) are shown in lower (20×) and higher magnification (100×); higher magnification images showing 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 (n = 3 per group; right). Values are mean ± SEM; unpaired Student t test. (B) Human platelets pretreated with vehicle or ML265 (50 and 100 μM; 10 minutes) were stimulated by thrombin (0.1U/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. Rest, resting platelets; Veh, vehicle.