Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195, USA.
J Biol Chem. 2011 Oct 7;286(40):34820-9. doi: 10.1074/jbc.M111.267161. Epub 2011 Aug 15.
Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A(2) with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A(2) synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood.
阿司匹林（乙酰水杨酸）预防可抑制主要心血管不良事件，但它的快速转化限制了血小板环氧化酶活性和血栓的抑制作用。尽管它很重要，但仍不清楚哪种酶（如果有的话）可以水解循环中的阿司匹林乙酰基，而这种酶必须是现有的酶。我们发现，循环中的阿司匹林在红细胞内被广泛水解，色谱分析表明这些细胞含有单一的水解活性。通过超过 1400 倍的纯化和测序，鉴定出血小板激活因子（PAF）乙酰水解酶的 I 型的 PAFAH1B2 和 PAFAH1B3 亚基，这是一种对 PAF 乙酰基具有选择性的磷脂酶 A2，是阿司匹林乙酰水解酶的候选酶。Western blot 显示，I 型酶的催化性 PAFAH1B2 和 PAFAH1B3 亚基与纯化的红细胞阿司匹林水解活性共迁移。重组的 PAFAH1B2，但不是其家族成员血浆 PAF 乙酰水解酶，水解了阿司匹林，并且 PAF 竞争性地抑制了纯化或重组红细胞酶对阿司匹林的水解。用 PAFAH1B2 或 PAFAH1B3 转染的 HEK 细胞水解了阿司匹林，并且竞争性的 I 型 PAF 乙酰水解酶抑制剂 NaF 降低了红细胞对阿司匹林的水解。将阿司匹林暴露于红细胞中会阻止其抑制血栓素 A2 合成和血小板聚集的能力。并非所有个体或人群都能平等地从阿司匹林预防中获益，阿司匹林抵抗现象，以及阿司匹林在红细胞中的水解在个体之间变化了 3 倍，这与 PAFAH1B2 而不是 PAFAH1B3 相关。我们的结论是，细胞内的 I 型 PAF 乙酰水解酶是人类血液中阿司匹林的主要水解酶。
