Endothelial cell PAF synthesis following thrombin stimulation utilizes Ca²⁺-independent phospholipase A₂

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 PLA₂ and plasmalogen phospholipid hydrolysis in PAF synthesis in thrombin-treated human umbilical artery endothelial cells (HUAEC). Basal PLA₂ activity 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 iPLA₂ activity utilizing plasmenylcholine substrates with a minimal increase in activity with alkylacyl glycerophospholipids. No change in cystolic iPLA₂ activity in thrombin-stimulated HUAEC was observed. The thrombinstimulated activation of iPLA₂ and 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 iPLA₂ with bromoenol lactone resulted in inhibition of iPLA₂ activity, plasmalogen phospholipid hydrolysis, production of choline lysophospholipids, and PAF synthesis. These data indicate that PAF production requires iPLA₂ activation 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.