Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation

The present study was performed to characterize thrombin-stimulated phospholipase A₂ (PLA₂) activity and the resultant release of lysophospholipids from endothelial cells. The majority of PLA₂ activity in endothelial cells was membrane associated, Ca²⁺ independent, and arachidonate selective. Incubation with thrombin increased membrane- associated PLA₂ activity using both plasmenylcholine and alkylacyl glycerophosphocholine substrates in the absence of Ca²⁺, with no increase in activity observed with phosphatidylcholine substrate. The increased PLA₂ activity was accompanied by arachidonic acid and lysoplasmenylcholine (LPlasC) release from endothelial cells into the surrounding medium. Thrombin-induced changes were duplicated by stimulation with the thrombin- receptor-directed peptide SFLLRNPNDKYEPF. Pretreatment with the Ca2+- independent PLA₂ inhibitor bromoenol lactone blocked thrombin-stimulated increases in PLA₂ activity, arachidonic acid, and LPlasC release. Stimulation of protein kinase C (PKC) increased basal PLA₂ activity and LPlasC production. Thrombin-stimulated PLA₂ activity and LPlasC production were enhanced with PKC activation and completely prevented with PKC downregulation. Thus thrombin treatment of endothelial cells activates a PKC- activated, membrane-associated, Ca²⁺-independent PLA₂ that selectively hydrolyzes arachidonylated, ether-linked phospholipid substrates, resulting in LPlasC and arachidonic acid release.