The Use of Triphenyl Phosphonium Cation Enhances the Mitochondrial Antiplatelet Effect of the Compound Magnolol

Figure 1. Platelet cytotoxicity and apoptosis. (A) LDH release from platelets. (B) Platelet viability by Calcein-AM. (C) Platelet apoptosis by Annexin-V. The results shown were obtained from at least five independent donors and expressed as the mean ± SEM. Vehicle: DMSO 0.4%. The statistical analysis was performed using a one-way analysis of variance (ANOVA) and the Bonferroni post hoc test. ** p < 0.01 and *** p < 0.001 vs. vehicle.

Figure 2. Platelet aggregation results. Platelet aggregation was induced by (A) collagen or (B) TRAP-6, as explained in the Methods. The aggregation assays were performed on washed platelets and the results are expressed as the percentage of aggregation after 5 min of reaction. Compounds were only tested at concentrations free of cytotoxicity. The results were obtained from at least four different donors and expressed as the mean ± SEM. Vehicle: DMSO 0.4%. The statistical analysis was performed using a one-way analysis of variance (ANOVA) and the Bonferroni post hoc test. *** p < 0.001 vs. vehicle.

Figure 3. Effect of magnolol and MGN4 on platelet activation markers. (A) P-selectin expression. (B) CD63 expression. (C) GP IIb/IIIa activated (PAC-1) expression. Platelets were identified as the CD61 positive population and these CD61-expressing cells were analyzed in terms of change in mean fluorescence intensity than the vehicle. The results were obtained from at least four independent donors and bars represent the mean ± SEM. Vehicle: DMSO 0.4%. The statistical analysis was performed using a one-way analysis of variance (ANOVA) and the Bonferroni post hoc test. * p < 0.05 ** p < 0.01 and *** p < 0.001 vs vehicle; # p < 0.05 and ### p < 0.001 vs. activated control.

Figure 4. Effect of magnolol and MGN4 on platelet mitochondria. (A) Mitochondrial membrane potential (∆Ψm). (B) Intraplatelet calcium levels. (C) Intraplatelet ROS levels. The results were obtained by flow cytometry and platelets were identified as the CD61 positive population; these CD61-expressing cells were analyzed in terms of change in mean fluorescence intensity compared to the vehicle condition. FCCP was used as a positive control of mitochondrial depolarization and increased intraplatelet calcium levels; antimycin A (AA) was used as a positive control of ROS production. The results were obtained from at least six independent donors and expressed as the mean+ SEM. Vehicle: DMSO 0.4%. The statistical analysis was performed using a one-way analysis of variance (ANOVA) and the Bonferroni post hoc test. Non-statistical difference: ns, ** p < 0.01 and *** p < 0.001 vs. vehicle.

Figure 5. Effect of MGN4 on platelet mitochondrial function. (A) Representative profile of oxygen consumption rate (OCR) of platelets (25 × 106 cells/well). OCR was measured in a Seahorse XFe24 extracellular flux analyzer (Agilent, Santa Clara, CA, US) before and after the sequential addition of 3 µg/mL collagen, 2.5 µM oligomycin, 1.4 µM FCCP, and 2 µM/2 µM rotenone/antimycin A. (B) Representative profile of extracellular acidification rate (ECAR) of platelets (25 × 106 cells/well). ECAR was measured in a Seahorse XFe24 extracellular flux analyzer before and after the sequential addition of 3 µg/mL collagen, 10 mM glucose, 2.5 µM oligomycin, and 100 mM 2-DG. Data are the means ± SD (n ≥ 3). The experiment was repeated for two different donors and the results were similar (not shown).

Figure 6. Chemical structure of compounds. MGN4 and MGN10 refer to a TPP+ moiety conjugated to MGN (Magnolol) via a 4- and 10-carbon alkyl side chain, respectively [30].