Figure 3: GPIbα and platelet function are unperturbed by 14-3-3ζ deficiency.

(a) Hirudin-anticoagulated blood from 14-3-3ζ-wt and 14-3-3ζ-deficient (14-3-3ζ-null) mice was perfused through collagen-coated (250 μg ml⁻¹ Type I) microslides at 1,800 s⁻¹ for 5 min. (i) DIC images are taken from 1 representative of four independent experiments (scale bar, 20 μm). (ii) Thrombi surface coverage was quantified using ImageJ software (4–5 fields analysed per flow). The histogram depicts the mean±s.e.m. (n=5; analysed using two-way ANOVA with Bonferroni’s post hoc testing). (b) VWF binding was measured as described under ‘Methods’. The graph represents one of three independent experiments. (c) Aggregation of washed mouse platelets in the presence of human VWF (10 μg ml⁻¹) and botrocetin (10 μg ml⁻¹), with stirring. The graph depicts aggregation traces from one representative of three independent experiments. (d) Comparative aggregation of washed 14-3-3ζ-wt and 14-3-3ζ-null platelets in response to CRP or ADP ((i) ADP 1 μM; (ii) CRP 20 ng ml⁻¹). Results are taken from one representative experiment. A histogram depicting the mean±s.e.m. (n=3; analysed using a two-way ANOVA with Bonferroni’s post hoc testing), is presented in Supplementary Fig. 2b. (e,f) Diluted whole blood samples from 14-3-3ζ-wt (black bars) or 14-3-3ζ-null (white bars) mice were incubated with PE-JON/A (e) or FITC-anti P-selectin antibody (f), as described under ‘Methods’, to examine integrin α_IIbβ₃ and degranulation of platelet α-granules, respectively, and analysed by flow cytometry, following incubation with the indicated agonist/concentration for 15 min. Results depict the % of gated platelets positive for antibody binding and are expressed as the mean±s.e.m. ((e) PAR4P: n=10; CRP: n=3; ADP: n=4; (f) PAR4P: n=10; CRP: n=5), where ^NSP>0.05. Note: no significant difference was observed in the geometric mean of fluorescence intensity for the same experiments. (g–i) Resting whole cell lysates were prepared from 14-3-3ζ-wt (wt) and 14-3-3ζ-null (null) washed platelets. (g) Expression levels of 14-3-3 isoforms were compared using SDS–PAGE and immunoblot analysis, as described under ‘Methods’, with immunoblots probed with the indicated 14-3-3 isoform-selective or pan-14-3-3 antibodies. These studies confirm deletion of 14-3-3ζ protein in the 14-3-3ζ-null mice, with some upregulation of 14-3-3γ. (h,i) Association of 14-3-3 proteins with the GPIbα cytoplasmic tail in the absence of 14-3-3ζ—GPIbα was immunoprecipitated from 14-3-3ζ-wt and 14-3-3ζ-null platelet lysates as described under ‘Methods’, and analysed via immunoblotting, using (h) anti-14-3-3ζ or (i) anti-pan-14-3-3. Immunoblots are taken from one representative of three independent experiments.