Figure 5.
Thrombin-Stimulated Generation of FAs, Eicosanoids, OxPLs, and Mitochondrial Oxygen Consumption Is Sensitive to cPLA2 Inhibition, while sPLA2 Inhibition Suppresses OxPLs Only
Heatmaps were generated using the pheatmap package in R and lipids color-coded according to lipid group. Levels of treatment response are represented by a color gradient ranging from blue to red (increase in response). Lipids are color-coded by group and clustered by similarity in overall response to the treatments
(A–C) Effect of PLA2 inhibitors on generation of FAs and oxPLs by platelets. Washed platelets were incubated with inhibitors or vehicle for 15 min, then activated using 0.2 U/mL thrombin for 30 min and generation of lipids determined using LC/MS/MS as described in Supplemental Information. Inhibitors were as follows: (A) cPLA2, 50–100 nM cPLA2i; (B) iPLA2, 50 nM BEL; and sPLA2, 2 μM OOEPC. (C) Inhibition of oxPLs by PLA2 inhibitors. oxPLs are grouped depending on the sn2 fatty acid to aid data visualization. Results are shown as heatmaps, with full data in Figures S3–S5.
(D–G) cPLA2 inhibition suppresses mitochondrial oxygen consumption, but not glycolysis. Platelets were plated on Cell-Tak-coated XF96 plates, and pre-treated with cPLA2i (<200 nM) for 15 min prior to bioenergetic measurements. (D) Basal OCR of platelets was measured prior to injection of thrombin (0.5 U/mL), followed by 1 μg/mL oligomycin (O), 0.6 μM FCCP (F), and 10 μM antimycin A (Anti). (E) cPLA2i (0–200 nM) dose response of thrombin-linked OCR presented as a percentage of control. (F) Indices of mitochondrial function, basal, thrombin responsive, ATP-linked, proton leak, maximal, reserve capacity, and non-mitochondrial OCR were calculated. (G) Basal and thrombin-responsive ECARs were calculated from parallel ECAR measurements. Data expressed as mean ± SEM from one representative donor, n = 3–5 replicates per sample. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005 different from control (ANOVA and Bonferroni post hoc test).