Endothelial cell PAF synthesis following thrombin stimulation utilizes Ca2+-independent phospholipase A(2)

Platelet activating factor (PAF) is a potent lipid autocoid that is rapidly synthesized and presented on the surface of endothelial cells following th rombin stimulation. PAF production may occur via de novo synthesis or by th e combined direct action of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso -PAF acetyltransferase or via the remodeling pathway. This study was undert aken to define the role of PLA(2) and plasmalogen phospholipid hydrolysis i n PAF synthesis in thrombin-treated human umbilical artery endothelial cell s (HUAEC). Basal PLA(2) activity in HUAEC was primarily found to be Ca2+-in dependent (iPLA(2)), membrane-associated, and selective for arachidonylated plasmenylcholine substrate. Thrombin stimulation of HUAEC resulted in a pr eferential 3-fold increase in membrane-associated iPLA(2) activity utilizin g plasmenylcholine substrates with a minimal increase in activity with alky lacyl glycerophospholipids. No change in cystolic iPLA(2) activity in throm bin-stimulated HUAEC was observed. The thrombin-stimulated activation of iP LA(2) and associated hydrolysis of plasmalogen phospholipids was accompanie d 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 in creased 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). I nhibition of iPLA(2) with bromoenol lactone resulted in inhibition of iPLA( 2) activity, plasmalogen phospholipid hydrolysis, production of choline lys ophospholipids, and PAF synthesis. These data indicate that PAF production requires iPLA(2) activation in thrombin-stimulated HUAEC and may occur thro ugh the CoA-independent transacylase remodeling pathway rather than as a di rect result of the PLA(2)-catalyzed hydrolysis of membrane alkylacyl glycer ophosphocholine.