Camacho M, Vila L
Laboratory of Inflammation Mediators, Institute of Research of Santa Creu i Sant Pau Hospital, Barcelona, Spain.
Thromb Res. 2000 Jul 15;99(2):155-64. doi: 10.1016/s0049-3848(00)00241-3.
Despite an almost total suppression of platelet cyclooxygenase (COX) by aspirin, as monitored ex vivo, incomplete suppression of thromboxane (Tx)A(2) metabolite excretion has been detected in some patients with unstable angina treated with low doses of aspirin. A plausible explanation for this finding is the transcellular formation of TxA(2) by platelets from prostaglandin H(2) released by endothelial cells. We recently reported that probably only COX and PGI-synthase (PGIS) are involved in the biosynthesis of prostanoids in endothelial cells. The present work was thus focused to ascertain the dependence of the transcellular biosynthesis of TxA(2), by endothelial cells and aspirin-treated platelets, on the relative activity of these enzymes. Synthesis of eicosanoids from exogenous and endogenous arachidonic acid (AA) by mixed incubations of human umbilical vein endothelial cells (HUVEC) in culture and aspirin-treated platelets were determined by HPLC and enzyme immune assay. The ratio of COX to PGIS activities was modified in HUVEC by treatment with interleukin-1beta (IL-1beta). Transcellular formation of TxA(2) was only relevant when HUVEC overexpressed COX-2 (monitored by RT-PCR and Western blotting), and in these conditions TxA(2) formation started 2 minutes after substrate addition. Progression curves showed that half-times (t(1/2)) of the COX and PGIS activity were 2.73 and 0.47 minutes, respectively, in resting HUVEC, whereas these values for IL-1beta-treated cells were 1.33 and 0.07 minutes, respectively, indicating that expression of COX-2 increased the rate of PGIS "suicide" inactivation. Collectively, these results indicated that not only enhanced COX activity but also substantial PGIS inactivation was required for significant transcellular biosynthesis of TxA(2).
尽管通过体外监测发现阿司匹林几乎完全抑制了血小板环氧化酶（COX），但在一些接受低剂量阿司匹林治疗的不稳定型心绞痛患者中，仍检测到血栓素（Tx）A2代谢产物排泄未被完全抑制。对此发现的一个合理的解释是，血小板从内皮细胞释放的前列腺素H2跨细胞形成TxA2。我们最近报道，在内皮细胞中，可能只有COX和前列环素合酶（PGIS）参与类前列腺素的生物合成。因此，本研究的重点是确定内皮细胞和经阿司匹林处理的血小板跨细胞生物合成TxA2对这些酶相对活性的依赖性。通过高效液相色谱法（HPLC）和酶免疫测定法，测定了培养的人脐静脉内皮细胞（HUVEC）与经阿司匹林处理的血小板混合孵育时，从外源性和内源性花生四烯酸（AA）合成类花生酸的情况。通过用白细胞介素-1β（IL-1β）处理，改变了HUVEC中COX与PGIS活性的比例。只有当HUVEC过表达COX-2时（通过逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法监测），TxA2的跨细胞形成才具有相关性，并且在这些条件下，添加底物后2分钟开始形成TxA2。进展曲线显示，静息HUVEC中COX和PGIS活性的半衰期（t1/2）分别为2.73分钟和0.47分钟，而经IL-1β处理的细胞的这些值分别为1.33分钟和0.07分钟，这表明COX-2的表达增加了PGIS“自杀”失活的速率。总的来说，这些结果表明，TxA2的显著跨细胞生物合成不仅需要增强COX活性，还需要大量的PGIS失活。
