Figure 6: 14-3-3ζ-deficient platelets demonstrate sustained metabolic ATP following potent activation.

(a) Washed platelets (1 × 10⁸ ml⁻¹) were isolated from 14-3-3ζ-wt (black symbols) or 14-3-3ζ-deficient (14-3-3ζ-null) (white symbols). Where indicated (squares), platelets were pre-incubated with oligomycin (1 μM), prior to stimulation. Metabolic ATP was extracted and quantified in unstimulated (Resting) and stimulated platelets with a mix of CRP (0.25 μg ml⁻¹) and thrombin (0.5 U ml⁻¹) for up to 1,800 s as described in ‘Methods’ section. Results are expressed as the percentage (%) change in ATP compared with resting control levels over the indicated times, and represent the mean±s.e.m. (n=4). A two-way ANOVA was used to determine statistical difference between 14-3-3ζ-wt and 14-3-3ζ-null platelets (^NSP>0.05; *P<0.05). (b,c) Western blot analysis of phospho-AMPK in 14-3-3ζ-wt and 14-3-3ζ-null platelets: Washed platelets (3 × 10⁸ ml⁻¹) were isolated from 14-3-3ζ-wt and 14-3-3ζ-null mice and activated with CRP/thrombin (0.25 μg ml⁻¹, 1 U ml⁻¹). After activation, platelets were lysed using RIPA buffer. Platelet lysates were analysed by western blot analysis of phospho-AMPK protein using a phosphospecific antibody (40H9). The western blot in b is taken from one representative of three independent experiments. Total β-actin was used as a loading control, and quantification of phospho-AMPK signal was expressed as a ratio of protein loading (c, mean±s.e.m., n=3), using LICOR technology. A Student’s t-test was used to determine statistical difference between 14-3-3ζ-wt and 14-3-3ζ-null platelets (*P<0.05). Where appropriate, results were analysed using either unpaired Student’s t-test, two-way ANOVA with Tukey’s post hoc testing.