Cardiovascular Research Advance Access [Accepted Manuscript] published online on April 17, 2009
Cardiovascular Research, doi:10.1093/cvr/cvp120
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Aspirin acetylates nitric oxide synthase type 3 in platelets thereby increasing its activity
1 Department of Cardiology, Guy's and St Thomas’ NHS Foundation Trust, London, UK
2 Key Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing 210029, China
3 Department of Clinical Pharmacology, Cardiovascular Division, King's College London, London, UK
* Corresponding authors: Yong Ji, Key Laboratory of Human Functional Genomics, Atherosclerosis Research Centre, Nanjing Medical University, Nanjing 210029, China, Tel: +86 25 8686 2886; Fax: +86 25 8650 8960; E-mail: yongji{at}njmu.edu.cn; and Albert Ferro, King's College London, Cardiovascular Division, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK, Tel: +44 20 7848 4283; Fax: +44 20 7848 3743; E-mail: albert.ferro{at}kcl.ac.uk
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: Platelets isolated from 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 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.
Results: At all concentrations tested, aspirin increased 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, whilst 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.
Conclusions: 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.
KEYWORDS aspirin; nitric oxide; nitric oxide synthase; acetylation; phosphorylation
Time for primary review: 17 Days