In vitro and ex vivo effects of intra- and extra-platelet NO on human platelet aggregation and of NO/NO⁺ donors on recombinant bovine cyclooxygenase-1 activity. (A) Effects of SNP, glycerol trinitrate (GTN), CSNO and S-nitrosoglutathione (GSNO) on the activity of recombinant bovine COX-1 (5 U, 1.2nM; Cayman Chemicals) measured as prostaglandin E₂ (PGE₂) formation rate (100% corresponds to 1.8 ng of PGE₂ per ng COX-1 and per minute). Concentrations used were each 100μM for GSNO, CSNO, SNP and GTN, 1μM for the COX-1 inhibitor diclofenac (Diclo) serving as a positive control, and 10μM for arachidonic acid as the COX-1 substrate. Incubations were performed at 37°C in 100mM phosphate buffer, pH 8, containing 5mM EDTA, 2mM phenol and 1μM hematin. PGE₂ was measured by GC-MS/MS. Data are shown as mean ± SD, n = 3. (B) Effect of GSNO on the activity of recombinant bovine COX-1. Data are shown as mean ± SD, n = 6 for each GSNO concentration. See panel A for more details. (C) Aggregation (y axis) and NOS activity (x axis) represented by the [¹⁵N]nitrate enrichment measured in platelet-rich plasma (PRP) from citrated blood donated by an apparently healthy female volunteer (aged 49 years). PRP was incubated with L-[guanidine-¹⁵N₂]-arginine (98% at both ¹⁵N atoms; Cambridge Isotope Labs) at a final concentration of 40μM in the absence or in the presence of externally added recombinant human endothelial NOS (heNOS, 50 μg/mL; ALEXIS) and with all cofactors (5μM FAD, 5μM FMN, 800μM NADPH, 10μM tetrahydrobiopterin, 500nM calmodulin, 500μM calcium). Incubations were performed at 37°C. Data are shown as mean ± SD. Platelet aggregation was induced by ADP (2μM) 3 minutes before starting aggregometric measurements. Reaction was stopped by acetone and [¹⁵N]nitrate was measurement by GC-MS.⁹  Without heNOS, aggregation was 69%, 68% and 69% and [¹⁵N]nitrate enrichment was 7%, 6% and 7%.