Inhibition of Platelet-Activating Factor (PAF) Acetylhydrolase by Methyl Arachidonyl Fluorophosphonate Potentiates PAF Synthesis in Thrombin-Stimulated Human Coronary Artery Endothelial Cells

We have previously demonstrated that thrombin stimulation of endothelial cells results in increased membrane-associated, Ca²⁺-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 iPLA ₂-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 μM) 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 μM. We conclude that MAFP augments thrombin-stimulated PAF production by inhibition of PAF catabolism without affecting membrane-associated iPLA₂ activity.