Endothelial cell PAF synthesis following thrombin stimulation utilizes Ca(2+)-independent phospholipase A(2).
Endothelial cell PAF synthesis following thrombin stimulation utilizes Ca(2+)-independent phospholipase A(2).
复制标题
凝血酶刺激后内皮细胞 PAF 合成利用 Ca(2 ) 独立的磷脂酶 A(2)。
DOI：
10.1021/bi0156153
复制
发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Creer,MH
中科院分区：
文献类型：
--
作者：
McHowat,J;Kell,PJ;O'Neill,HB;Creer,MH
血小板激活因子（PAF）是一种有效的脂质自体蛋白，在凝血酶刺激后快速合成并呈现在内皮细胞表面。 PAF 的产生可能通过从头合成或通过磷脂酶 A2(PLA2) 和乙酰辅酶 A:lyso-PAF 乙酰转移酶的联合直接作用或通过重塑途径发生。本研究旨在明确经凝血酶处理的人脐动脉内皮细胞 (HUAEC) 中 PLA2 和缩醛磷脂水解在 PAF 合成中的作用。 HUAEC 中的基础 PLA2 活性主要被发现是 Ca2+ 独立的 (iPLA2)、膜相关的，并且对花生酰化血浆胆碱底物具有选择性。 HUAEC 的凝血酶刺激导致利用纤浆胆碱底物的膜相关 iPLA2 活性优先增加 3 倍，而使用烷基酰基甘油磷脂时活性增加最小。在凝血酶刺激的 HUAEC 中，未观察到囊性 iPLA2 活性的变化。凝血酶刺激的 iPLA2 激活和相关的缩醛磷脂水解伴随着花生四烯酸水平的增加（从 1.1 ± 0.1 到 2.8 ± 0.1%）和前列环素释放（从 38 ± 12 到 512 ± 24%）以及溶浆胆碱产生水平的增加（从 0.6 ± 0.1 到2.1 ± 0.3 nmol/mg 蛋白质）、溶血磷脂酰胆碱（0.3 ± 0.1 至 0.6 ± 0.1 nmol/mg 蛋白质）和 PAF（790 ± 108 至 3380 ± 306 dpm）。用溴烯醇内酯抑制 iPLA2 会导致 iPLA2 活性、缩醛磷脂水解、胆碱溶血磷脂的产生和 PAF 合成受到抑制。这些数据表明，PAF 的产生需要在凝血酶刺激的 HUAEC 中 iPLA2 激活，并且可能通过不依赖 CoA 的转酰基酶重塑途径发生，而不是 PLA2 催化膜烷基酰基甘油磷酸胆碱水解的直接结果。
Platelet activating factor (PAF) is a potent lipid autocoid that is rapidly synthesized and presented on the surface of endothelial cells following thrombin stimulation. PAF production may occur via de novo synthesis or by the combined direct action of phospholipase A2(PLA2) and acetyl-CoA:lyso-PAF acetyltransferase or via the remodeling pathway. This study was undertaken to define the role of PLA2and plasmalogen phospholipid hydrolysis in PAF synthesis in thrombin-treated human umbilical artery endothelial cells (HUAEC). Basal PLA2activity in HUAEC was primarily found to be Ca2+-independent (iPLA2), membrane-associated, and selective for arachidonylated plasmenylcholine substrate. Thrombin stimulation of HUAEC resulted in a preferential 3-fold increase in membrane-associated iPLA2activity utilizing plasmenylcholine substrates with a minimal increase in activity with alkylacyl glycerophospholipids. No change in cystolic iPLA2activity in thrombin-stimulated HUAEC was observed. The thrombin-stimulated activation of iPLA2and associated hydrolysis of plasmalogen phospholipids was accompanied by increased levels of arachidonic acid (from 1.1 ± 0.1 to 2.8 ± 0.1%) and prostacyclin release (from 38 ± 12 to 512 ± 24%) as well as an increased level of production of lysoplasmenylcholine (from 0.6 ± 0.1 to 2.1 ± 0.3 nmol/mg of protein), lysophosphatidylcholine (from 0.3 ± 0.1 to 0.6 ± 0.1 nmol/mg of protein), and PAF (from 790 ± 108 to 3380 ± 306 dpm). Inhibition of iPLA2with bromoenol lactone resulted in inhibition of iPLA2activity, plasmalogen phospholipid hydrolysis, production of choline lysophospholipids, and PAF synthesis. These data indicate that PAF production requires iPLA2activation in thrombin-stimulated HUAEC and may occur through the CoA-independent transacylase remodeling pathway rather than as a direct result of the PLA2-catalyzed hydrolysis of membrane alkylacyl glycerophosphocholine.