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The activity of the acetyltransferase capable of transferring the acetyl moiety of acetyl-CoA onto 2-lyso PAF-acether (1-alkyl-sn-glycero-3-phosphocholine) to form PAF-acether was compared in ionophore A23187-stimulated and in non-stimulated rat peritoneal cells. Stimulation resulted in a doubling of the acetyltransferase activity within 30 s. This effect was abolished in the presence of EDTA (1 mM) or EGTA (1 mM) and restored by addition of Ca2+ (10 mM). The specificity of acetyltransferase measured in ionophore-stimulated as well as in untreated cells is the same. In both situations we observed the same Km values for acetyl-CoA, whereas the Vmax values were different. The wide similarities of the two enzyme preparations lead us to conclude that stimulation by the ionophore involves an increase in the number of enzyme molecules rather than a change in the kinetic parameters of the acetyltransferase.

The metabolism of bioreactive lipid mediators was studied in two types of activated macrophages (M phi). We compared the capacity of resident and activated M phi to release, upon a zymosan challenge, cyclooxygenase and lipoxygenase products as well as PAF-acether (platelet-activating factor) and its 2-lyso precursor. Activated M phi were obtained from mice injected intraperitoneally either with nonviable C74 streptococci (St-M phi) or with trehalose dimycolate, a defined immunostimulant isolated from Mycobacterium tuberculosis (TDM-M phi). Both activated populations exhibited common features: conversion of endogenous [14C]arachidonic acid into prostaglandin E2 and thromboxane A2 rather than into prostaglandin I2 and low biosynthesis of PAF-acether, probably due to an impairment of the acetylation step. However, contrary to St-M phi, TDM-M phi did not display a marked overall reduction of arachidonate metabolism. In addition, as compared to resident M phi, TDM-M phi presented a ratio of thromboxane B2/6-ketoprostaglandin F1 alpha 30-fold higher, a better conversion of leukotriene C to leukotriene D and a higher capacity to release the PAF-acether they synthesize. These macrophages thus seem to be valuable tools for studying the formation of mediators and for determining specific markers of an activated state.

Platelet-activating factor (PAF-acether; 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is released from murine peritoneal adherent cells by inflammatory and non-inflammatory stimuli. We have found, in extracts from these cells, an enzyme activity that synthesizes. PAF-acether from synthetic lyso-PAF-acether by transferring the acetyl moiety of acetyl-coenzyme A onto the lyso-PAF-acether molecule. The enzyme is stabilized by 1 mM dithiothreitol, is calcium-dependent, has an apparent Km of 172 microM for acetyl-CoA and is active in a 6-8 pH range. When the acetyl-CoA substrate is replaced by propionyl-CoA, an ether lipid is produced which turns out to be as potent an aggregating agent as PAF-acether. In all cases, the products of the reaction were characterized by their behaviour in platelet-aggregation tests and their high-pressure liquid chromatography (HPLC) elution profiles. The precise definition of this acetyl-transferase is of primary importance for the development of new pharmacological agents capable of moduling a potent platelet aggregating factor.

The regulating mechanisms of PAF-acether (platelet-activating factor) biosynthesis in cultured human vascular endothelial cells stimulated with thrombin were investigated. The formation of PAF-acether was maximal at 5 min after stimulation and gradually decreased for up to 30 min. Thrombin induced a rapid 3-4-fold increase in the activity which was maximal by 1 min after stimulation and returned progressively to basal level within 10 min. The thrombin-induced enhancement in acetyltransferase activity was due to an increase of the Vmax of the acetylation reaction without a significant effect on the apparent Km of the enzyme for acetyl-CoA. Human endothelial cells also exhibited a basal PAF-acether acetylhydrolase activity which was not altered upon thrombin stimulation. The pretreatment with 2 mM phenylmethylsulfonyl fluoride (PMSF), a serine proteinase inhibitor reported to block the acetylhydrolase, induced about 2-times more PAF-acether production in response to 2.5 U/ml thrombin stimulation. However, this enhancement of PAF-acether formation seems to be not only due to the inhibition of the acetylhydrolase, but also to the influences on the activities of the acetyltransferase and other enzymes such as phospholipase A2. These results suggest a key role for acetyltransferase and acetylhydrolase in the regulation of PAF-acether formation and catabolism in thrombin-stimulated human endothelial cells.

We have compared for rabbit platelet aggregating and desensitizing activity two different preparations of platelet-activating factor (PAF-acether) (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) and of its enantiomer (3-O-alkyl-2-O-acetyl-sn-glycero-1-phosphocholine). After phospholipase A2 treatment, the unnatural enantiomers appeared about 3000-fold less active than PAF-acether, a result which definitively establishes the stereospecificity of the mode of action of this mediator. A new structural analog of PAF-acether, 1-O-hexadecyl-3-O-acetyl-sn-glycero-2-phosphocholine, was isolated and characterized. It was a weak platelet agonist, stressing further the importance for PAF-acether activity of the acetyl group at position 2 of the glycerol.

The role of paf-acether as an intracellular mediator was recently challenged by studies showing that it remained cell-associated in several cell types. We showed that the level of paf-acether found in extracellular medium was strongly reduced when extracellular pH decreased and when cell concentration increased. Therefore the number of cells and extracellular pH should be taken into consideration before speculating on the release vs. retention of paf-acether.

A new method of synthesis of octadecyl platelet-activating factor (PAF; 1-O-octadecyl 2-O-acetyl sn-glycero-3-phosphorylcholine) is described. Its advantage is to proceed by way of the 'lyso-PAF' which may be substituted by various groups, while avoiding the inconveniences of the total synthesis already described. Moreover, the intermediates in synthesis are easier to purify, with better yields. The platelet-aggregating activity of synthetic PAF, is enantiomer (3-O-octadecyl) and racemic mixtures were 2.7 . 10(-10), 1.7 . 10(-7) and 2.2 . 10(-10) M, respectively. These results indicate the stereospecificity of platelet activation induced by PAF.