Platelets isolated from the blood of healthy human subjects were exposed in vitro to vehicle or aspirin at different concentrations (5 µmol/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-[³H]arginine to l-[³H]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-¹⁴C]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.

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.