Mechanisms of interaction of a plasmalogenic analog of platelet-activating factor with human platelets.

The interaction of a plasmalogenic analog of platelet-activating factor (1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphocholine; 1-alkenyl-PAF) with human platelets was studied. 1-Alkenyl-PAF induced an increase in intracellular Ca2+ concentration and inhibition of adenylate cyclase at significantly higher concentrations than PAF. 1-Alkenyl-PAF inhibits PAF-induced platelet aggregation but has no effect on ADP- or thrombin-induced aggregation of human platelets. In contrast to PAF, 1-alkenyl-PAF increases [3H]PGE1 binding with human platelets. The properties of 1-alkenyl-PAF as an agonist or antagonist of PAF receptors apparently depend on its concentration in the cell medium. Under physiological conditions 1-alkenyl-PAF might be a natural PAF antagonist acting in the human cardiovascular system.     

The effect of aldehydogenic and acyl structural analogs of platelet activating factor on the formation of superoxide radicals by human blood leukocytes]

The effects of plasmalogenic and acyl structural analogs of the platelet activation factor (PAF) on the superoxide radical production by human blood leukocytes were studied. It was found that 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine and 1-O-(1'-alkenyl)-2-acetyl-sn-glycero-3-phosphocholine stimulates the production of leukocyte superoxide radicals, their activity being comparable with that of PAF. Stimulation of superoxide radical production strongly depends on the structure of the polar heads of PAF analogs. It is supposed that choline-containing plasmalogenic and acyl PAF analogs may act as specific lipid mediators of the neutrophil function.     

Histamine release from human leukocytes after treatment with 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) and its structural analogs

The influence of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, a platelet activating factor (PAF), and its structural analogs--1-acyl-2-acetyl-sn-glycero-3-phosphocholine and 1-(1'-alkenyl)-glycero-3-phosphocholine--on the histamine release from human leukocytes of healthy and allergic individuals was investigated. It was found that within the concentration range of 10(-10) to 10(-7) M PAF and its analogs induce a moderate histamine release from the leukocytes. However, at higher concentrations (greater than 10(-7) M) PAF induces an enhanced release of histamine from the leukocytes of allergic patients as compared to healthy individuals. PAF and its analogs significantly potentiate the allergens-induced release of histamine from the leukocytes of allergic patients. It was assumed that PAF induces the expression or demasking of additional numbers of IgE receptors on the surface of basophils, which leads tot he stimulation of histamine release from the leukocytes in the presence of allergens.     

Inhibition of Human Platelet Aggregation by Amides and Ester of Salicylic Acid with Platelet-Activating Factor Analogs

The influence of acetyl salicylic acid (ASA) derivatives with platelet-activating factor (PAF) lipid analogs on PAF-induced human platelet aggregation has been studied. It was found that the ASA amide with an ethanolamine plasmalogen PAF analog (1-0-alk-1"-enyl-2-acetyl-sn-glycero-3-phospho-(N-2"-acetoxybenzoyl)ethanolamine) and the ASA ester with a choline plasmalogen PAF analog (1-0-alk-1"-enyl-2-(2"-acetoxybenzoyl)-sn-glycero-3-phosphocholine) at concentrations of 10^–7-10^–6 M effectively inhibit PAF-induced aggregation of human platelets. In contrast to these compounds, the ASA amide with an alkyl PAF analog (1-0-alkyl-2-acetyl-sn-glycero-3-phospho-(N-2"-acetoxybenzoyl)ethanolamine) did not inhibit PAF-induced platelet aggregation. As possible mechanisms of action of the studied compounds, the blockade of PAF-receptor and cyclooxygenase inhibition are proposed.     

Influence of acyl and plasmalogenic analogs of platelet activating factor on chemotaxis of human leukocytes in vitro and their inflammatory and antiinflammatory activity in vivo

The effects of platelet activating factor (PAF) and its cell analogs 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine (1-alkenyl-PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-PAF) on chemotaxis of human leukocytes in vitro and their inflammatory and antiinflammatory activities in vivo were studied. Both analogs stimulated chemotaxis of human leukocytes in agarose gel. PAF and 1-alkenyl-PAF induced rat paw edema in the range of doses 0.1-10 and 10-100 g per paw, respectively. Paw edema induced by 1-acyl-PAF (10-100 g per paw) was more pronounced than that induced by PAF or 1-alkenyl-PAF. The latter also exhibited significant antiinflammatory effect by inhibiting PAF- or car-rageenan-induced rat paw edema, and this effect exceeded that of dexamethasone. In these models of inflammation 1-acyl-PAF did not exhibit any antiinflammatory activity. The data suggest that PAF is not the only cell phospholipid mediating inflammation—its cell analogs, 1-acyl-PAF and 1-alkenyl-PAF, may also be involved into the inflammatory response. Possible interrelationships between cellular synthesis of 1-acyl-PAF, its formation in oxidized LDL, biological effects of lysolecithin, and penetration of LDL into the arterial wall are discussed.     

Development of a novel scintillation proximity radioimmunoassay for platelet-activating factor measurement: comparison with bioassay and GC/MS techniques

A novel, facile and sensitive scintillation proximity radioimmunoassay (SPRIA) for quantitation of PAF has been developed. No separation of antibody bound [3H]PAF from free [3H]PAF is required as the assay employs protein A - coated fluomicrospheres (beads containing scintillant). The assay system was suitable for the quantitation of 0.03 to 2 pmol of 1-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine. The cross-reactivity was high with 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine but was very low with PAF analogs such as 1-alkyl- and 1-acyl-2-lyso-sn-glycero-3-phosphocholine, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine. The specificity of SPRIA was higher than that of bioassay (platelet degranulation assay). PAF receptor antagonists (L-652,731, WEB2086, and FR900452) at up to 10 nmol per tube had no affect on the SPRIA. These observations indicate that the specificity of the PAF antibody is quite different from that of the platelet receptor. The values obtained using SPRIA for the measurement of PAF produced in polymorphonuclear leukocytes with stimuli are comparable to those obtained by SIM/GC/MS analysis.     

Platelet activating factor is a potent stimulant of the production of active oxygen species by human monocyte-derived macrophages

Platelet activating factor (PAF; C16), 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine) stimulated the production of active oxygen species by human monocyte-derived macrophages in culture. An optimal response was observed at a concentration of 13 microM PAF with half-maximal stimulation at 5 microM. The generation of superoxide ion (O2-) and hydrogen peroxide (H2O2) in response to PAF was inhibited specifically by a PAF-antagonist (1-O-Hexadecyl-2-acetyl-sn-glycero-3-phospho (N,N,N,-trimethyl) hexanolamine; such generation varied with the degree of maturation of cultured monocytes into macrophages. Production of active oxygen species increased progressively to reach a maximal level between days 4 to 6 of culture and remained maximal to day 12, after which it decreased progressively. Phorbol 12-myristate-13-acetate (PMA) and opsonized zymosan also stimulated generation of O2- and H2O2. PAF was however distinguished by its potent capacity to stimulate O2- and H2O2 production even at late stages of macrophage maturation (18 days), at which time both PMA and zymosan lacked significant effect. These findings suggest that PAF is a factor of potential relevance to the inflammatory role of the macrophage in atherogenesis.     

Subcellular localization of enzyme activities involved in the metabolism of platelet-activating factor in rainbow trout leukocytes

The subcellular distribution of an alkyllyso-GPC: acetyl-CoA acetyltransferase (EC 2.3.1.67) and transacylase, two important enzyme activities involved in the remodeling pathway for the biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) have been examined in leukocytes isolated from the pronephros of the rainbow trout, Oncorhynchus mykiss. Contrary to mammalian systems, in which the acetyltransferase is localized to intracellular membranes, the subcellular distribution of an acetyltransferase activity in rainbow trout leukocytes was localized to the plasma membrane. Analysis of the acetyltransferase products by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed synthesis of two subclasses of PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. The transacylase activity in this study was detected in membrane fractions in two domains of the intermediate density region which also contained the NADH dehydrogenase activity, a marker enzyme for the endoplasmic reticulum. Acylation of lysoPAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) exhibited approximately 95% specificity for omega-3 fatty acids. Acylation patterns were not significantly different in either domain of the endoplasmic reticulum. A model is proposed herein for the metabolism of PAF in rainbow trout leukocytes.     

Bioactive amide of prostaglandin E1 and ethanolamine plasmalogen analog of platelet-activating factor inhibits several pathways of human platelet aggregation

The influence of an amide of prostaglandin E1 and ethanolamine plasmalogen platelet-activating factor analog 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-11alpha, 15alpha-dioxy-9-keto-13-prostenoyl)ethanolamine (PGE1-PPAF) on platelet-activating factor (PAF)-, ADP-, and thrombin-induced human platelet aggregation has been studied. It was found that PGE1-PPAF inhibits the PAF-, ADP-, and thrombin-induced platelet aggregation in platelet-rich plasma. 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine inhibited PAF-induced aggregation up to 50% but had no influence on platelet aggregation induced by ADP or thrombin. The ethanolamine plasmalogen analog of PAF 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolami ne, having a palmitoyl residue instead of PGE1, did not inhibit platelet aggregation induced by PAF, ADP, or thrombin. We propose that inhibition of human platelet aggregation by PGE1-PPAF is mediated by its action on platelet PAF-receptors and the adenylate cyclase system.     

Acyl and plasmalogen analogs of platelet activating factor--new lipid cellular bioregulators]

Recent data concerning two structural platelet-activating factor (PAF) analogs-1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine (acyl-PAF) and 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine (vinyl-PAF) identified in some cells and tissues are reviewed. Isolation, identification, biosynthesis, and metabolism of acyl-PAF and vinyl-PAF are considered. The activity of acyl-PAF and vinyl-PAF towards platelets, leukocytes, isolated myocardium, and ileum as well as its in vivo activity are discussed. The influence of acyl-PAF and vinyl-PAF on PAF platelet interaction, Ca2+ mobilization, and platelet adenylate cyclase activity is considered. It is concluded that similar to PAF, acyl-PAF and vinyl-PAF should be regarded as a family of PAF lipid bioregulators.     

Platelet-activating factor in normal rat uterus

Platelet-activating factor (PAF) was found in normal rat uterus and identified as 1-0-hexadecyl/octadecenyl-2-acetyl-sn-glycero-3-phosphocholine. PAF was purified by several successive chromatographic procedures. It showed platelet aggregating activity, which was inhibited by CV 3988, and had no effect on platelets desensitized with 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine. The tert-butyldimethyl-silylderivative of 1-0-alkyl-2-acetyl-sn-glycerol, which was obtained by hydrolysis of uterine PAF with phospholipase C, was analyzed by gas chromatography-mass spectrometry. One rat uterus contained approximately 21.3 ng of 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine. This is the first report of the occurrence of a significant amount of PAF in a normal animal tissue.     

Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family

Four naturally occurring platelet-activating factor (PAF) analogs, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine, 1-hexade-canoyl-2-acetyl-sn-glycero-3-phosphocholine, 1-octadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, stimulated human neutrophils (PMN) to mobilize Ca²⁺, degranulate, and produce Superoxide anion. They were, respectively, 5-, 300-, 500-, and 4000-fold weaker than PAF in each assay; inhibited PMN-binding of [³H]PAF at concentrations paralleling their biological potencies; and showed sensitivity to the inhibitory effects of PAF antagonists. PAF and the analogs, moreover, desensitized PMN responses to each other but not to leukotriene B₄ and actually increased (or primed) PMN responses to N-formyl-MET-LEU-PHE. Finally, 5-hydroxyicosatetraenoate-enhanced PMN responses to PAF and the analogs without enhancing the actions of other stimuli. It stereospecifically raised each analog's potency by as much as 100-fold and converted a fifth natural analog, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine from inactive to a weak stimulator of PMN. PAF and its analogs thus represent a structurally diverse family of cell-derived phospholipids which can activate, prime, and desensitize neutrophils by using a common, apparently PAF receptor-dependent mechanism.     

Metabolism of platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-alkyl-2-acetyl-sn-glycerol by human endothelial cells

The metabolism of platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol was studied in cultures of human umbilical vein endothelial cells. Human endothelial cells deacetylated 1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine to the corresponding lyso compound (1-[1,2-3H]alkyl-2-lyso-sn-glycerol-3-phosphocholine) and a portion was converted to 1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine. Lyso platelet activating factor (lyso-PAF) (1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine) was detected in the media very early during the incubation and the amount remained higher than the level of the lyso product observed in the cells. Cellular levels of 1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine were significantly higher than the acylated product (1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine) at all times during the 60-min incubation period, which suggests that the ratio of acetylhydrolase to acyltransferase activities is greater in endothelial cells than in most other cells. When endothelial cells were incubated with 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol, a known precursor of PAF, 1-[1,2-3H]alkyl-sn-glycerol was the major metabolite formed (greater than 95% of the 3H-labeled metabolites during 20- and 40-min incubations). At least a portion of the acetate was removed from 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol by a hydrolytic factor released from the endothelial cells into the medium during the incubations. Only negligible amounts of the total cellular radioactivity (0.2%) was incorporated into platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine); therefore, it is unlikely that the previously observed hypotensive activity of 1-alkyl-2-acetyl-sn-glycerols can be explained on the basis of the conversion to platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells. Results of this investigation indicate that endothelial cells play an important role in PAF catabolism. Undoubtedly, the endothelium is important in the regulation of PAF levels in the vascular system.     

Production of platelet-activating factor by washed rabbit platelets

Production of platelet-activating factor by washed rabbit platelets under stimulation with the ionophore A23187 was investigated utilizing two groups of platelet preparations. The first platelet preparation contained 0.03 +/- 0.02% contaminating white cells, while the second preparation contained 0.48 +/- 0.27% white cells. The latter preparation produced platelet-activating factor, mainly 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 8.3 +/- 6.3 pmol (mean +/- standard deviation) with a range of 2.6 to 21.4 pmol (n = 9), followed by small quantities of 1-octadecenyl- and 1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine. In contrast, there was no production of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by the former platelet preparation having 0.03% leukocytes. These quantitative analyses were carried out by the selected ion monitoring technique and it was concluded that it is necessary to consider the presence of contaminating white cells in studies on the production of platelet-activating factor by platelets.     

Molecular and mechanistic characterization of platelet-activating factor-like bioactivity produced upon LDL oxidation

Oxidation of LDL is thought to be involved in both initiating and sustaining atherogenesis through the formation of proinflammatory lipids and the covalent modification of LDL particles. Platelet-activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator involved in inflammation. Upon oxidation of LDL, oxidized phospholipids with PAF-like structure are generated, and some of them may act via the PAF receptor. We evaluated the contribution of 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) and of other PAF analogs on the PAF-like bioactivity formed upon Cu2+-initiated oxidation of LDL. Reverse-phase HPLC purification and electrospray ionization-MS analyses showed that upon oxidation of LDL with inactivated PAF-acetylhydrolase (PAF-AH), C16:0 PAF accounted for >30% of PAF-like biological activity and its sn-2 butenoyl analog accounted for >50%. However, upon LDL oxidation in the presence of exogenous 1-0-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) without PAF-AH inactivation, C16:0 PAF formation accounted for >90% of the biological activity recovered. We suggest that the C16:0 PAF, despite being a minor constituent of the LDL peroxidation products, may contribute substantially to the bioactivity formed in oxidized LDL. The higher bioactivity of C16:0 PAF, and the higher selectivity of the LDL-attached lyso-PAF transacetylase toward very short acyl chains [acetate (C2) vs. butanate (C4)], may explain the contribution described above.     

High performance liquid chromatography of platelet-activating factors

Silica and C18 reverse phase high performance liquid chromatography (HPLC) were used to fractionate synthetic molecular species of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and semi-synthetic platelet-activating factor (PAF) synthesized from beef heart plasmalogens. A single coincident peak from silica HPLC was observed for either a mixture of synthetic AGEPC's with alkyl chain lengths from C12 to C18 or for beef heart-derived PAF. This peak was well separated from other classes of phospholipid standards including 2-lysophosphatidylcholine and 3H-labeled lyso-PAF. Subsequently, the synthetic AGEPC mixture or beef heart PAF was separated into individual species on a C18 reverse phase column. Beef heart-derived PAF was fractionated into at least four molecular species of PAF activity which had similar retention times as the radioactivity of 3H-labeled beef heart PAF. Approximately 56% of the radioactivity of 3H-labeled PAF was found in the fraction with a similar retention time as 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 10% as 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine, 11% as 1-O-pentadecyl-2-acetyl-sn-glycero-3-phosphocholine, and 13% in an unidentified fraction which eluted after C-16-AGEPC. The unidentified fraction did not correspond to any of the homologous series of synthetic AGEPCs with saturated alkyl chain lengths from C12 to C18. Recoveries of radioactive phospholipids from silica or reverse phase columns were greater than 95%.     

The metabolism of platelet-activating factor in human T-lymphocytes

The metabolism of 1-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-[3H]alkyl-2-acetyl-GPC; platelet-activating factor; PAF) was investigated in purified human peripheral blood T-lymphocytes and a human leukemia cell line of T-cell origin (MOLT-4). The major metabolic products of T-lymphocyte PAF metabolism are 1-alkyl-2-acyl-GPC, 1-alkyl-2-lyso-GPC and neutral lipid. The pattern of PAF metabolism in peripheral blood T-lymphocytes and MOLT-4 lymphoblasts was similar, although MOLT-4 lymphoblasts transformed PAF to 1-alkyl-2-acyl-GPC faster than peripheral blood T-lymphocytes (67% vs. 21% of added label after 64 min at 37 degrees C, respectively). Pre-exposure of MOLT-4 lymphoblasts to 1 mM of the serine hydrolase inhibitor phenylmethylsulfonyl fluoride resulted in an inhibition of PAF metabolism. Our results indicate that intact T-lymphocytes actively metabolize this biologically active phospholipid by the deacetylation-transacylation pathway.     

Platelet-activating factor metabolism in human amnion and the responses of this tissue to extracellular platelet-activating factor

It was found previously that platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is undetectable in human amniotic fluid obtained before labor but is present in the majority of samples of amniotic fluid obtained after labor. In the present investigation, the amount of PAF in amnion tissue and the ability of this tissue to produce PAF and respond to PAF were investigated. Amounts of PAF in amnion obtained either during the second trimester of gestation or at term (before labor) were similar. After labor, however, the amount of PAF in amnion increased to 2.5-times that in amnion before labor without any discernible changes in the amounts of two related glycerophospholipids viz., 1-0-alkyl-sn-glycero-3-phosphocholine and 1-0-alkyl-2-acyl-sn-glycero-3-phosphocholine. The Ca2+-ionophore A23187, in the presence of Ca2+, caused an increase in the amount of PAF in amnion tissue disks but PAF did not appear to be released into the incubation medium. The stimulation of PAF formation by A23187 and Ca2+ was not affected by the addition of indomethacin. Addition of PAF to disks of amnion tissue resulted in an increase in the concentration of prostaglandin E2 in the incubation medium. An increase in prostaglandin E2 formation of similar magnitude was induced by A23187. Based on these results it is concluded that PAF can be synthesized in amnion tissue and net production is stimulated by Ca2+. In addition, amnion is receptive to extracellular PAF and exhibits, as one response, an increased production of prostaglandin E2.     

An anti-inflammatory protein secreted from the rat seminal vesicle epithelium inhibits the synthesis of platelet-activating factor and the release of arachidonic acid and prostacyclin

Platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a mediator of inflammation and endotoxic shock produced by a variety of stimulated cells. Since the main biosynthetic pathway of PAF involves acetylation of 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) generated from 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine by phospholipase A2, we suggest a general physiological role played by steroid-induced anti-(phospholipase A2) proteins in the modulation of PAF synthesis. The results of the present study support this hypothesis since an androgen-induced anti-inflammatory protein, SV-IV, secreted from rat seminal vesicles, inhibits PAF synthesis in stimulated polymorphonuclear neutrophils, macrophages and endothelial cells. SV-IV impairs PAF synthesis by inhibiting the activation of phospholipase A2, that also results in the inhibition of arachidonic acid and prostacyclin release, and of acetyl-CoA:lyso-PAF acetyltransferase.     

Increased biosynthesis of platelet-activating factor in activated human eosinophils

1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase catalyzes the conversion of biologically inactive lysophospholipid to bioactive platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) by an acetylation reaction. The activity of this enzyme in eosinophils isolated from patients with eosinophilia is stimulated (up to 4-fold) in a dose-, time-, and Ca2+/Mg2+-dependent manner after exposure to the eosinophil chemotactic factor of anaphylaxis (ECF-A), C5a, formyl-methionylleucylphenylalanine (fMLP), or ionophore A23187. The three naturally occurring chemotactic factors (ECF-A, C5a, and fMLP) cause a rapid and transient increase of enzyme activity, with a maximum at 1 or 3 min, whereas ionophore A23187 maintains an elevated level for up to 15 min. The activity of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase, an enzyme that catalyzes the breakdown of PAF to lyso-PAF, is not affected by C5a, fMLP, or ionophore A23187. The presence of PAF in eosinophils was established by demonstrating the lipid nature of the compound, the RF value being identical with that of synthetic 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine on thin layer chromatograms, and by its ability to induce serotonin release from rabbit platelets. Furthermore, ECF-A, C5a, fMLP, and ionophore A23187 all induce the secretion of PAF from eosinophils. These findings suggest that the generation and release of PAF could be a consequence of eosinophil chemotactic activation and may thus function in inflammatory and allergic reactions in which eosinophils participate.