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

J. McHowat, P. J. Kell, H. B. O'Neill, M. H. Creer

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)14921-14931
Number of pages11
JournalBiochemistry
Volume40
Issue number49
DOIs
StatePublished - Dec 11 2001

All Science Journal Classification (ASJC) codes

  • Biochemistry

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