Figure 6.

Effects of pyruvate on antiplatelet activity of CORM-A1 in platelets. Human washed platelets (WP) were suspended in assay medium (DMEM) supplemented with glucose (1 g/L) and glutamine (2 mmol/L) with or without pyruvate (1 mmol/L). A, Aggregation of WP treated with CORM-A1 (300 µmol/L) and activated with thrombin (0.1 U/mL) as compared with control; data represent the means±SD of 4 independent experiments; n=1–2 replicates in each experiment. B, Concentration of lactate extruded from platelets (WP) treated with CORM-A1 (0, 100, 300, 1000 µmol/L); data represent the means±SD of 3 independent experiments. C, Extracellular acidification rate (ECAR) measurements of WP treated with PBS (control) or CORM-A1 (A1; 100, 300, 1000 μmol/L) in DMEM supplemented with glucose (1 g/L), glutamine (2 mmol/L), and pyruvate (1 mmol/L); Seahorse XFe96 Analyzer. Data represent means±SD from a representative experiment, n=3–6 technical replicates. D–O, Human WP suspended in PBS containing glucose (1 g/L) and glutamine (2 mmol/L) without (C, A1) or with pyruvate (1 mmol/L; P, A1/P) were untreated or treated with CORM-A1 (300 µmol/L), followed by activation with thrombin (0.1 U/mL; 6 min); data demonstrate concentrations of selected metabolic by-products of glycolysis, PPP or mitochondrial respiration are presented as means±SD (nmol/10⁹ platelets) of 3 independent experiments, n=2 replicates in each experiment. P, Aggregation of WP treated with pyruvate (100 µmol/L), CORM-A1 (300 µmol/L) and GSK2837808A (GSK; 5 µmol/L). Q, Aggregation of WP control or preincubated with dimethyl malonate (DMM; 15 min) followed by treatment with antimycin A (AA; 10 µmol/L) and GSK2837808A (GSK; 3 µmol/L). Data represent the means±SD of 4 independent experiments; n=1–3 replicates in each experiment. DHAP indicates dihydroxyacetone phosphate; F-1,6-BP, fructose-1,6-bis-phosphate; and NAD⁺, nicotinamide adenine dinucleotide. *P<0.05, #P<0.01, $P<0.001, &P<0.0001.