Aspirin acetylates nitric oxide synthase type 3 in platelets thereby increasing its activity

Aims Acute administration of aspirin increases nitric oxide (NO) synthesis by platelets, an effect not shared by other non-steroidal anti-inflammatory drugs. The aim of the present study was to determine the mechanism by which aspirin acutely increases the activity of NO synthase type 3 (NOS-3), the predominant NOS isoform expressed by platelets, and specifically whether this occurs through an increase in its acetylation. Methods and results Platelets isolated from the blood of healthy human subjects were exposed in vitro to vehicle or aspirin at different concentrations (5 mu mol/L-4 mmol/L). Changes in intraplatelet Ca2+ concentration were determined from fura-2 fluorescence. Following immunoprecipitation of NOS-3 from platelet lysates, its activity was determined from L-[H-3]arginine to L-[H-3]citrulline conversion, and its serine phosphorylation quantified by western blotting. Acetylation of NOS-3 in platelets was assessed by the incorporation of radioactivity into the immunoprecipitated enzyme from [acetyl-C-14] aspirin. Following transfection of HeLa cells with NOS-3, NO biosynthesis in response to aspirin was determined from cyclic GMP measurement, and sites of NOS-3 acetylation were ascertained by liquid chromatography-tandem mass spectrometry. At all concentrations tested, aspirin increased the activity of NOS-3 from platelets. This was not associated with any measurable change in intraplatelet Ca2+ concentration. Serine phosphorylation of NOS-3 in platelets was decreased, and this was especially marked for serine-1177 phosphorylation, whereas acetylation of NOS-3 was increased, by aspirin incubation. HeLa cells transfected with NOS-3 exhibited an increase in NO biosynthesis following aspirin exposure, and this was associated with acetylation of the enzyme on both serine-765 and serine-771. Conclusion Aspirin acetylates NOS-3 acutely in platelets, and this causes an increase in its activity as well as a decrease in its phosphorylation. It is also possible that aspirin indirectly affects NOS-3 activity by acetylating other substrates within the platelet, but this remains to be determined.