O'Kane Peter, Xie Liping, Liu Zhen, Queen Lindsay, Jackson Graham, Ji Yong, Ferro Albert
Department of Cardiology, Guy's and St Thomas' NHS Foundation Trust, London, UK.
Cardiovasc Res. 2009 Jul 1;83(1):123-30. doi: 10.1093/cvr/cvp120. Epub 2009 Apr 17.
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.
Platelets isolated from the blood of healthy human subjects were exposed in vitro to vehicle or aspirin at different concentrations (5 micromol/L-4 mmol/L). Changes in intraplatelet Ca(2+) concentration were determined from fura-2 fluorescence. Following immunoprecipitation of NOS-3 from platelet lysates, its activity was determined from l-[(3)H]arginine to l-[(3)H]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-(14)C]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 Ca(2+) 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.
急性给予阿司匹林可增加血小板中一氧化氮（NO）的合成，这一效应其他非甾体抗炎药并不具备。本研究的目的是确定阿司匹林急性增加3型一氧化氮合酶（NOS-3）活性的机制，NOS-3是血小板中表达的主要NOS亚型，具体而言，这是否通过其乙酰化增加而发生。
从健康人类受试者血液中分离的血小板在体外暴露于赋形剂或不同浓度（5 μmol/L - 4 mmol/L）的阿司匹林。通过fura-2荧光测定血小板内Ca(2+)浓度的变化。从血小板裂解物中免疫沉淀NOS-3后，通过l-[(3)H]精氨酸向l-[(3)H]瓜氨酸的转化来测定其活性，并通过蛋白质印迹法对其丝氨酸磷酸化进行定量。通过将放射性从[乙酰-(14)C]阿司匹林掺入免疫沉淀的酶中来评估血小板中NOS-3的乙酰化。用NOS-3转染HeLa细胞后，通过环磷酸鸟苷测量来确定对阿司匹林的NO生物合成反应，并通过液相色谱-串联质谱法确定NOS-3乙酰化位点。在所有测试浓度下，阿司匹林均增加了血小板中NOS-3的活性。这与血小板内Ca(2+)浓度的任何可测量变化均无关。阿司匹林孵育后，血小板中NOS-3的丝氨酸磷酸化降低，丝氨酸-1177磷酸化尤为明显，而NOS-3的乙酰化增加。用NOS-3转染的HeLa细胞在暴露于阿司匹林后NO生物合成增加，这与该酶在丝氨酸-765和丝氨酸-771上的乙酰化有关。
阿司匹林在血小板中急性乙酰化NOS-3，这导致其活性增加以及磷酸化减少。阿司匹林也有可能通过乙酰化血小板内的其他底物间接影响NOS-3活性，但这仍有待确定。
