Deletion of PDK2 and PDK4 inhibits PDH phosphorylation and aerobic glycolysis in activated platelets. PDH phosphorylation at Ser293 and Ser300 was measured in (A) collagen- (25 μg/mL) or (B) PAR4 peptide (70μM)–stimulated WT or PDK2/4^−/− platelets. Representative western blots are shown. Total PDH was used as a loading control. The bar graphs show densitometry analysis of immunoblots. Values are mean ± SEM, n = 5 to 6 mice per group. Statistical analysis: Mann-Whitney U test; ∗P < .05 and ∗∗P < .01. The glycolytic proton efflux rate (glycoPER) was measured in WT or PDK2/4^−/− platelets stimulated with (C) collagen (25 μg/mL) or (D) PAR4 peptide (50μM) using a Seahorse extracellular flux analyzer. The bar graph shows the quantified data (for the line graph values marked with a box). Values are mean ± SEM, with n = 4 to 5 mice per group. Statistical analysis: 2-way ANOVA followed by Tukey multiple comparisons test; ∗P < .05; ∗∗P < .01 and ∗∗∗∗P < .0001.