Figure 4: Reduced PS exposure and thrombin generation in 14-3-3ζ-deficient platelets. | Nature Communications

Figure 4: Reduced PS exposure and thrombin generation in 14-3-3ζ-deficient platelets.

From: 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function

Figure 4

(a–c) PS exposure was quantified in anticoagulated whole blood isolated from 14-3-3ζ-wt or 14-3-3ζ-null mice (a), or in washed platelets isolated from either 14-3-3ζ-wt and 14-3-3ζ-null mice (c) or healthy human donors (b). Where indicated, washed platelets were treated with a 14-3-3 dimer destabilizer (RB-011, 10 μM) or vehicle (sodium mesylate salt), prior to activation. In whole blood, PS exposure was quantified following stimulation of platelets with buffer (resting), CRP/Thrombin (CRP (0.25 μg ml−1)/Thr (0.5 U ml−1)) or calcium ionophore A23187 (Iono, 1 μm), for the indicated times. Washed platelets were stimulated with CRP (0.25 μg ml−1)/TRAP (1 μM) or CRP (0.25 μg ml−1)/PAR4 activating peptide (200 μM) (for human and mouse platelets, respectively) for the indicated times. PS was detected through measurement of Alexa-488-labelled Annexin V binding (ANV+ve), as described under ‘Methods’. Results are expressed as the mean±s.e.m. (n=8), where ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; NSP>0.05. (d–f) Procoagulant function of washed human (d) or mouse platelets (e,f) was examined using a modified aPTT assay with Ellagic acid as a soluble activator, as described under ‘Methods’. Resting platelets or those activated for 10 min with CRP/TRAP (d) or CRP/PAR4-activating peptide (e,f) were added as a source of phospholipid. Results depict the mean±s.e.m. (n=4; where ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; NSP>0.05). Results represent the aPTT in vehicle- and RB-011-treated activated platelets, relative to resting platelets (d), the fold difference in aPTT when comparing activated vehicle- and RB-011-treated human (d, inset) or 14-3-3ζ-wt and 14-3-3ζ-null mouse platelets (f), or the fold difference in aPTT when comparing activated 14-3-3ζ-wt and 14-3-3ζ-null mouse platelets (e). Where appropriate, results were analysed using either unpaired Student’s t-test, one-way or two-way ANOVA (with Tukey’s or Bonferroni’s post hoc testing).

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