PDK4 is the major determinant contributing to thrombosis in the FeCl₃ injury–induced carotid thrombosis model. (A) Platelet-rich plasma (PRP) from WT or PDK2^−/− or PDK4^−/− mice was stimulated with collagen (0.4 μg/mL), PAR4 peptide (40 μM), or ADP (0.13 μM). Results are expressed as the percent change in light transmission with respect to the blank (buffer without platelets), set at 100%. The representative aggregation curves are shown. Values are mean ± SEM, n = 4 mice per group. Statistical analysis: 1-way ANOVA followed by Tukey's multiple comparisons test ∗P < .05; ∗∗P < .01 and ∗∗∗P < .001 (B) The extent of α-granule secretion (using P-selectin exposure) in WT, PDK2^−/− and PDK4^−/− platelets (in PRP) stimulated with CRP-XL (0.1 μg/mL) or PAR4 peptide(100 μM) was determined using flow cytometry. Values are mean ± SEM, n = 4 mice per group. Statistical analysis: 2-way ANOVA followed by Tukey's multiple comparisons test; ∗∗∗P < .001 and ∗∗∗∗P < .0001. (C) ATP secretion from the dense granules of WT, PDK2^−/−, and PDK4^−/− platelets (in PRP) after stimulation with collagen (0.8 μg/mL) or PAR4 peptide (70 μM) was measured using Lumi-aggregometry. Values are mean ± SE n = 4 mice per group. Statistical analysis: 1-way ANOVA followed by Tukey's multiple comparisons test ∗P < .05; ∗∗P < .01 and ∗∗∗∗P < .0001. (D) A representative image of carotid artery thrombus (5% FeCl₃ injury for 2 minutes) as visualized by intravital microscopy in WT, PDK2^−/−, and PDK4^−/− male mice. Platelets were labeled ex vivo with calcein green. The time to occlusion is shown. These experiments were performed in the same setup with the same WT mice serving as a control group for both PDK2^−/− and PDK4^−/− mice. Values are mean ± SEM, n = 8 mice per group. Statistical analysis: Mann-Whitney U test. ∗P < .05 (E) The tail-bleeding time in WT, PDK2^−/−, and PDK4^−/− male mice was determined by the time taken for the initial cessation of bleeding after the tail transection. Values are mean ± SEM, n = 6 mice per group. Statistical analysis: Mann-Whitney U test. (F) A schematic depicting the role of PDK/PDH axis in regulating platelet activation.