PDK2/4-double-deficient mice exhibit reduced platelet functions. (A)The deletion of PDK2 and PDK4 was confirmed using western blotting. (B) Platelet-rich plasma (PRP) from WT or PDK2/4^−/− mice was stimulated with collagen (0.4 μg/mL), CRP-XL (0.05 μg/mL), PAR4 peptide (40 μM), thrombin (0.013 U/mL; washed platelelets were used), 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 = 5 to 6 mice per group. Statistical analysis: Mann-Whitney U test; ∗P < .05 and ∗∗P < .01. (C) The extent of integrin αIIbβ3 activation (using JonA binding), and (D) α-granule secretion (using P-selectin exposure) in WT and PDK2/4^−/− 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 = 3 to 4 mice per group. Statistical analysis: 2-way ANOVA followed by Tukey multiple comparisons test; ∗P < .05; ∗∗P < .01 and ∗∗∗∗P < .0001. (E) ATP secretion from the dense granules of WT and PDK2/4^−/− 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 = 6 mice per group. Statistical analysis: Mann-Whitney U test; ∗∗P < .01.