Platelets from PDK2/4 double-deficient mice exhibit impaired GPVI-mediated signaling. (A) Total tyrosine phosphorylation and (B) phospho-PLCγ2 phosphorylation (Tyr 1217) were measured in resting and collagen (25 μg/mL)–stimulated WT and PDK2/4^−/− platelets. Representative western blots for total tyrosine phosphorylation and phospho-PLCγ2 (Tyr 1217) are shown. GAPDH or total PLCγ2 were used as loading controls. The bar graphs show densitometry analysis of immunoblots. Values are mean ± SEM, n = 4 mice per group. Statistical analysis: 2-way ANOVA followed by Tukey's multiple comparisons test; ∗P < .05 and ∗∗∗∗P < .0001. (C) Calcium mobilization was evaluated in Fura-2 LR loaded WT or PDK2/4^−/− platelets washed platelets that were stimulated with CRP-XL (0.7 μg/mL) in the presence of 1.3 mM CaCl₂. The left panel shows representative calcium mobilization after stimulation with CRP-XL. The middle and right panel shows the peak calcium level and the area under the curve (AUC), respectively. Values are mean ± SEM, n = 5 mice per group. Statistical analysis: Mann-Whitney U test; ∗P < .05.