Figure 2.

Effects of the mixture of KKO and PF4 on platelets pre-treated with a monoclonal Ab IV.3 against the FcγRII-receptor. Platelets were incubated for 60 min at 37 °C under the following conditions: untreated platelets (negative control), treated with KKO/PF4 (positive control) and treated with KKO/PF4 after pre-treatment for 10 min at 37 °C with a monoclonal Ab IV.3 against the FcγRII-receptor. Final concentrations: 10 µg/mL PF4, 50 µg/mL KKO, 50 µg/mL IV.3. (A) Fraction of platelets expressing P-selectin (CD62P-positive). (B) Fraction of platelets expressing phosphatidylserine (Annexin V-positive). (C) Fraction of live platelets with normal mitochondrial membrane potential determined with a ΔΨm-sensitive (mitochondrial membrane potential) dye MitoTrackerDeepRed. (D) Subpopulations of platelets double-stained with a ΔΨm-sensitive dye MitoTrackerDeepRed and FITC-Annexin V normalized by the total number of gated platelets taken as 100%. Platelets were segregated into 2 subpopulations: MitoTracker-negative/Annexin V-positive or “dead platelets” (dark boxes) and MitoTracker-positive/Annexin V-positive or “live activated platelets” (light boxes). Each experiment was performed with platelets isolated from three independent donors. * p < 0.05, **** p < 0.0001 for the corresponding data without and with the addition of Ab IV.3 (A-C, Kruskal–Wallis test; D, 2-way ANOVA test with Dunn’s post-hoc test).