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We have previously demonstrated that thrombin stimulation of endothelial cells results in increased membrane-associated, Ca(2+)-independent phospholipase A2 (iPLA2) activity, accelerated hydrolysis of membrane plasmalogen phospholipids, and production of several biologically active phospholipid metabolites, including prostacyclin and platelet-activating factor (PAF) that is abolished by pretreatment with the iPLA2-selective inhibitor bromoenol lactone. This study was designed to further investigate the role of alternative PLA2 inhibitors, including methyl arachidonyl fluorophosphonate (MAFP, an inhibitor of cytosolic PLA2 isoforms), on phospholipid turnover and PAF production from thrombin-stimulated human coronary artery endothelial cells (HCAECs). Paradoxically, pretreatment of HCAEC with MAFP (5-25 microM) resulted in a significant increase in PAF production in both unstimulated and thrombin-stimulated cells that was found to be a direct result of inhibition of PAF acetylhydrolase (PAF-AH) activity. Pretreatment with MAFP did not significantly inhibit HCAEC PLA2 activity, possibly due to the localization of PLA2 activity in the membrane fraction rather than the cytosol. Bromoenol lactone did not inhibit PAF-AH activity, even at concentrations as high as 20 microM. We conclude that MAFP augments thrombin-stimulated PAF production by inhibition of PAF catabolism without affecting membrane-associated iPLA2 activity.

In the present study, phospholipase A(2) (PLA(2))-catalyzed hydrolysis of platelet membrane phospholipids was investigated by measuring PLA(2) activity, phospholipid hydrolysis, arachidonic acid release and choline lysophospholipid production in thrombin-stimulated human platelets. Thrombin-stimulated platelets demonstrated selective hydrolysis of arachidonylated plasmenylcholine and plasmenylethanolamine, with little change in diacyl phospholipids. Accelerated plasmalogen hydrolysis was accompanied by increased arachidonic acid and thromboxane B(2) release and increased lysoplasmenylcholine production. Thrombin stimulation caused an increase in PLA(2) activity measured in the cytosolic fraction with plasmenylcholine only; no increase in activity was measured with phosphatidylcholine. No change in membrane-associated PLA(2) activity was observed with either substrate tested. Pretreatment with the Ca(2+)-independent PLA(2)-selective inhibitor, bromoenol lactone, inhibited completely any thrombin-stimulated phospholipid hydrolysis. Thus, thrombin stimulation of human platelets activates a cytosolic PLA(2) that selectively hydrolyzes arachidonylated plasmalogen phospholipids.

We demonstrated previously that thrombin stimulation of endothelial cells activates a membrane-associated, Ca2+-independent phospholipase A2(iPLA2) that selectively hydrolyzes arachidonylated plasmalogen phospholipids. We report that incubation of human coronary artery endothelial cells (HCAEC) with phorbol 12-myristate 13-acetate (PMA) to activate protein kinase C (PKC) resulted in hydrolysis of cellular phospholipids similar to that observed with thrombin stimulation (0.05 IU/ml; 10 min). Thrombin stimulation resulted in a decrease in arachidonylated plasmenylcholine (2.7 ± 0.1 vs. 5.3 ± 0.4 nmol PO4/mg of protein) and plasmenylethanolamine (7.5 ± 1.0 vs. 12.0 ± 0.9 nmol PO4/mg of protein). Incubation with PMA resulted in decreases in arachidonylated plasmenylcholine (3.2 ± 0.3 nmol PO4/mg of protein) and plasmenylethanolamine (6.0 ± 1.0 nmol PO4/mg of protein). Incubation of HCAEC with the selective iPLA2inhibitor bromoenol lactone (5 mM; 10 min) inhibited accelerated plasmalogen phospholipid hydrolysis in response to both PMA and thrombin stimulation. Incubation of HCAEC with PMA (100 nM; 5 min) resulted in increased arachidonic acid release (7.1 ± 0.3 vs. 1.1 ± 0.1%) and increased production of lysoplasmenylcholine (1.4 ± 0.2 vs. 0.6 ± 0.1 nmol PO4/mg of protein), similar to the responses observed with thrombin stimulation. Downregulation of PKC by prolonged exposure to PMA (100 nM; 24 h) completely inhibited thrombin-stimulated increases in arachidonic acid release (7.1 ± 0.6 to 0.5 ± 0.1%) and lysoplasmenylcholine production (2.0 ± 0.1 to 0.2 ± 0.1 nmol PO4/mg of protein). These data suggest that PKC activates iPLA2in HCAEC, leading to accelerated plasmalogen phospholipid hydrolysis and increased phospholipid metabolite production.