Journal: Nature Communications
Article Title: 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function
Figure Lengend Snippet: 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 IIb β 3 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 NS P > 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.
Article Snippet: All aggregation studies were initiated by addition of the indicated concentrations of adenosine diphosphate (ADP) or CRP to platelet suspensions stirred at 600 r.p.m. for 10 min at 37 °C in a four-channel automated platelet analyser (AggRAM, Helena Laboratories, Tyne and Wear, UK) in the presence of 1 mM calcium and 0.5 mg ml−1 fibrinogen.
Techniques: Mouse Assay, Software, Flow Cytometry, Binding Assay, Incubation, Cytometry, Concentration Assay, Fluorescence, Expressing, SDS Page, Western Blot, Immunoprecipitation