Turnbull Catriona M, Cena Clara, Fruttero Roberta, Gasco Alberto, Rossi Adriano G, Megson Ian L
Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh.
Br J Pharmacol. 2006 Jun;148(4):517-26. doi: 10.1038/sj.bjp.0706743. Epub 2006 May 15.
Incorporation of a nitric oxide (NO)-releasing moiety in aspirin can overcome its gastric side effects. We investigated the NO-release patterns and antiplatelet effects of novel furoxan derivatives of aspirin (B8 and B7) in comparison to existing antiplatelet agents. Cyclooxygenase (COX) activity was investigated in purified enzyme using an electron paramagnetic resonance-based technique. Concentration-response curves for antiplatelet agents +/- the soluble guanylate cyclase inhibitor, ODQ (50 microM) were generated in platelet-rich plasma (PRP) and washed platelets (WP) activated with collagen using turbidometric aggregometry. NO was detected using an isolated NO electrode. The furoxan derivatives of aspirin (B8, B7) and their NO-free furazan equivalents (B16, B15; all 100 microM) significantly inhibited COX activity (P < 0.01; n = 6) in vitro and caused aspirin-independent, cGMP-dependent inhibition of collagen-induced platelet aggregation in WP. B8 was more potent than B7 (PRP IC(50) = 0.62 +/- 0.1 microM for B8; 400 +/- 89 microM for B7; P < 0.0001. WP IC(50)s = 0.6 +/- 0.1 and 62 +/- 10 microM, respectively). The NO-free furazan counterparts were less potent antiplatelet agents (WP IC(50)s = 54 +/- 3 microM and 62 +/- 10 microM, respectively; P < 0.0001, B8 vs B16). Of the hybrids investigated, only B8 retained antiplatelet activity in PRP.NO release from furoxan-aspirin hybrids was undetectable in buffer alone, but was accelerated in the presence of either plasma or plasma components, albumin (4%), glutathione (GSH; 3 microM) and ascorbate (50 microM), the effects of which were additive for B7 but not B8. NO generation from furoxans was greatly enhanced by platelet extract, an effect that could largely be explained by the synergistic effect of intracellular concentrations of GSH (3 mM) and ascorbate (1 mM). We conclude that the decomposition of furoxan-aspirin hybrids to generate biologically active NO is catalysed by endogenous agents which may instil a potential for primarily intracellular delivery of NO. The blunting of the aspirin effects of furoxan hybrids is likely to be due to loss of the acetyl moiety in plasma; the observed antiplatelet effects are thereby primarily mediated via NO release. Compounds of this class might represent a novel means of inhibiting platelet aggregation by a combination of NO generation and COX inhibition.
在阿司匹林中引入释放一氧化氮（NO）的部分可以克服其胃部副作用。我们研究了阿司匹林新型呋咱衍生物（B8和B7）与现有抗血小板药物相比的NO释放模式和抗血小板作用。使用基于电子顺磁共振的技术在纯化酶中研究环氧化酶（COX）活性。使用比浊法在富含血小板血浆（PRP）和用胶原激活的洗涤血小板（WP）中生成抗血小板药物±可溶性鸟苷酸环化酶抑制剂ODQ（50μM）的浓度-反应曲线。使用分离的NO电极检测NO。阿司匹林的呋咱衍生物（B8、B7）及其不含NO的呋咱类似物（B16、B15；均为100μM）在体外显著抑制COX活性（P < 0.01；n = 6），并在WP中引起与阿司匹林无关的、cGMP依赖性的胶原诱导血小板聚集抑制。B8比B7更有效（B8的PRP IC50 = 0.62±0.1μM；B7为400±89μM；P < 0.0001。WP的IC50分别为0.6±0.1和62±10μM）。不含NO的呋咱类似物是效力较弱的抗血小板药物（WP的IC50分别为54±3μM和62±10μM；P < 0.0001，B8与B16相比）。在所研究的杂合物中，只有B8在PRP中保留抗血小板活性。呋咱-阿司匹林杂合物在单独缓冲液中未检测到NO释放，但在血浆或血浆成分、白蛋白（4%）、谷胱甘肽（GSH；3μM）和抗坏血酸盐（50μM）存在时释放加速，其中对B7的作用是相加的，但对B8不是。血小板提取物极大地增强了呋咱类化合物产生NO的能力，这种作用在很大程度上可以用细胞内浓度的GSH（3mM）和抗坏血酸盐（1mM）的协同作用来解释。我们得出结论，呋咱-阿司匹林杂合物分解产生生物活性NO是由内源性物质催化的，这可能为NO的主要细胞内递送带来潜力。呋咱杂合物的阿司匹林效应减弱可能是由于血浆中乙酰部分的丢失；观察到的抗血小板效应因此主要通过NO释放介导。这类化合物可能代表了一种通过产生NO和抑制COX相结合来抑制血小板聚集的新方法。
