Existence of endogenous inhibitors of platelet-activating factor (PAF) with PAF in rat uterus

Two kinds of phospholipids in normal rat uterus were found to inhibit the aggregation of washed rabbit platelets induced by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and were named Inhibitor I and Inhibitor II and identified by mass spectrometry. Inhibitor I was a mixture of 1-acyl (16:0, 18:0, 18:1, 18:2, and 20:4)-2-lyso-sn-glycero-3-phosphocholine (acyllyso-GPC) and 1-alkyl (16:0, 18:0, and 18:1)-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC). 16:0 acyllyso-GPC was the most inhibitory, followed by 18:1, 18:2, 20:4, and 18:0 acyllyso-GPCs and 16:0 alkyllyso-GPC. Their IC50 values were in the range of 1-4 X 10(-5) M against the platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC, indicating that they were about 100 times weaker inhibitors than CV-3988. Inhibitor II was a mixture of N-acyl sphing-4-enyl phosphocholine (18:1/18:0, 18:1/20:0, 18:1/24:0, and 18:1/24:2). The most inhibitory of these components were 18:1/20:0 and 18:1/24:0, followed by 18:1/24:2 and 18:1/18:0, and their IC50 values were in the range of 4-5 X 10(-5) M against platelet aggregation induced by the alkylacetyl-GPC. Quantitatively, about 10(5) times higher concentrations of these inhibitors should be necessary to inhibit platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC. In fact, the contents of Inhibitors I and II, respectively, were approximately 10(5) times (4.7 X 10(-2) and 7.1 X 10(-2) mol/mol lipid-phosphorus of the original uterine phospholipids) than that of 16:0 alkylacetyl-GPC (1.4 X 10(-6) mol/mol lipid-phosphorus). The role of alkylacetyl-GPC in normal rat uterus is uncertain, but it coexists in situ with two kinds of endogenous inhibitors, choline containing lysoglycerophospholipids and sphingophospholipids.     

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.     

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.     

Differential responsiveness of human neutrophils to the autocrine actions of 1-O-alkyl-homologs and 1-acyl analogs of platelet-activating factor.

The phlogistic actions of six molecular species of platelet-activating factor (PAF) (1-O-alkyl-PAF homologs, 16:0-, 18:0- and 18:1-alkyl-PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and their respective 1-acyl-PAF analog counterparts, 16:0-, 18:0- and 18:1-acyl-PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (AGPC)) were assessed relative to five human neutrophilic polymorphonuclear leukocyte (PMN) functional responses: 1) lysosomal enzyme secretion; 2) specific desensitization to 16:0-AGEPC-induced lysosomal enzyme secretion; 3) O2- production; 4) chemotaxis; and 5) priming for enhanced O2- production. With respect to inducing lysozyme secretion, 18:0-AGEPC was 30- and 75-fold less potent than 16:0-AGEPC and 18:1-AGEPC, respectively, and was 25- and 40-fold less potent for inducing beta-glucuronidase secretion. 18:0-AGEPC was also 10-fold less active than 18:1- and 16:0-AGEPC for inducing O2- production. Thus, the rank order of potency of the alkyl-PAF homologs for inducing both lysosomal enzyme secretion and O2- production was 18:1- greater than or equal to 16:0- much greater than 18:0-AGEPC. In contrast, these three alkyl-PAF homologs had the same potency for desensitizing PMN to subsequent 16:0-AGEPC-induced lysosomal enzyme secretion and for priming PMN for augmented O2- production in response to FMLP or human recombinant C5a. Paradoxically, however, the rank order of potency of the alkyl-PAF homologs for effecting PMN chemotaxis was 18:0- greater than 18:1- much greater than 16:0-AGEPC. At concentrations as high as 1.0 microM, the acyl-PAF analogs did not initiate PMN lysosomal enzyme secretion, O2- production, or chemotaxis. However, the acyl-PAF analogs induced partial PMN desensitization to 16:0-AGEPC. A novel finding of potential (patho)-physiologic significance was the ability of acyl-PAF at nM concentrations to prime PMN for significantly enhanced O2- production after stimulation with FMLP or human recombinant C5a. The priming action of acyl-PAF was due to an increase in the rate as opposed to a prolongation of O2- production. The differing rank orders of potency of the alkyl-PAF homologs and acyl-PAF analogs for stimulating several physiologic responses of the same target cell, the human PMN, support the premise that there may be more than one PAF receptor subtype on the PMN and/or that differences in the biophysical properties of the various molecular species of PAF modulate their interaction with PAF receptor(s) linked to stimulus-response coupling.     

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.     

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.     

Metabolism of platelet-activating factor in human platelets. Transacylase-mediated synthesis of 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine

The present study demonstrates that inactivation of exogenous 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC; platelet-activating factor) by human platelets is mediated by the sequential action of two enzymes, 1) a Ca2+-independent acetylhydrolase recovered in the cytosolic fraction of platelets that deacylates alkylacetyl-GPC forming alkyllyso-GPC and 2) a CoA-independent, N-ethylmaleimide-sensitive transacylase associated with platelet membranes that incorporates a long-chain fatty acid into alkyllyso-GPC to produce alkylacyl-GPC. Separation of platelet phospholipids and subsequent resolution into individual molecular species by high-performance liquid chromatography revealed that the newly formed alkylacyl-GPC was exclusively alkylarachidonoyl-GPC and that the arachidonoyl group for acylation of alkyllyso-GPC was provided by phosphatidylcholine. We conclude that the previously described platelet arachidonoyl transacylase (Kramer, R.M., and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811) may play an important role in the metabolism of platelet-activating factor.     

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.     

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.     

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.     

Activation of guinea pig peritoneal macrophages by platelet activating factor (PAF) and its agonists

The platelet activating factor (PAF: 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) and its analogs were examined to determine their effects on guinea pig peritoneal macrophages. PAF activated macrophages, but its effect on macrophages was much weaker than that observed on platelets: the concentration required for 50% maximum activation was 8.5 X 10(-6) M for macrophages and 2.9 X 10(-10) M for platelets. Three PAF agonists, 1-O-octadecyl-2-O-(N,N-dimethylcarbamoyl)-glycero-3-phosphocholine (Compound I), 1-O-octadecyl-2-acetamido-2-deoxy-glycero-3-phosphocholine (Compound II), and 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (Compound III), showed higher activity in stimulating macrophage function than PAF. The abilities of these non-metabolizable PAF agonists to activate macrophage paralleled their relative potency to induce platelet activation. The sn-3 enantiomers of PAF and Compound III exhibited activity, while the sn-1 did not. By comparing the activities of derivatives of Compound III, it was shown that the long-chain alkyl-ether group in the glycerol-1 position, a relatively small size of the substituent on the hydroxy group at the sn-2 position, and the choline moiety in the glycerol-3 position must play critical roles in the process of macrophage activation. A specific PAF antagonist, CV3988, which inhibits PAF-induced platelet activation and hypotension, inhibited the activation of macrophages caused by PAF and its agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding sites for platelet activating factor in human lung tissues

Specific and saturable binding of [3H]-labeled 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine (PAF) to membrane preparations of human lung tissues is demonstrated. The equilibrium dissociation constant (KD) was determined by Scatchard analysis to be 4.9 (+/- 1.7) X 10(-10)M and the maximal number of binding sites was estimated to be 140 (+/- 37) fmole/mg protein. The binding site is PAF specific and its selectivity toward PAF analogs is very similar to that in rabbit platelets. Two PAF receptor antagonists, kadsurenone and ginkgolide B, previously characterized in platelet systems, also displace the binding of [3H]-PAF to human lung homogenates. These data indicate that human lung tissues contain PAF specific receptors, and binding of PAF to these receptor sites may be the first step to initiate PAF-induced lung pathophysiology.     

Observations on the critical micellar concentration of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and a series of its homologs and analogs

In a study designed to explore the physical chemical characteristics of platelet activating factor (PAF), or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, the critical micellar concentration of this compound, as well as the propionyl, butyryl and hexanoyl homologs was determined. In addition, an analogous series of compounds, in which the 1-O-alkyl was replaced by a 1-O-hexadecanoyl or a 1-O-octadecanoyl group, were examined for their critical micellar concentration. A variety of analytical techniques including NMR, gas liquid chromatography, infra-red spectrometry, thin layer chromatography, phosphorus, choline, glyceryl ether and fatty acid analyses were used to confirm the high purity of the individual derivatives. A dye binding assay and a surface tension technique were compared as to their suitability for determination of the critical micellar concentration of these compounds. Whereas the dye binding method proved highly variable, the surface tension procedure proved to be a facile, reproducible technique and was the assay of choice. The critical micellar concentration of the 1-O-alkyl and the 1-O-acyl derivatives showed comparable values for each short chain substituent at carbon 2, with values, in microM, ranging from 1.3 +/- 0.03 for 1-O-hexadecanoyl-2-acetyl-sn-glycero-3-phosphocholine and 1.1 +/- 0.10 for 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine to 0.22 +/- 0.01 for 1-O-hexadecanoyl-2-hexanoyl-sn-glycero-3-phosphocholine and 0.18 +/- 0.03 for 1-O-hexadecyl-2-hexanoyl-sn-glycero-3-phosphocholine. The data show that at the molar concentration usually employed in biological studies with these compounds, i.e., 1 X 10(-7) to 10(-11) M, one can assume that they will be present as monomolecular species. Thus, it seems unlikely that the widely diverse biological activities of these compounds can be explained by this physical parameter.     

Vertebrate class distribution of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase in serum

Serum from numerous mammals and lower vertebrates contains an enzyme activity that is specific for the hydrolysis of the acetate moiety of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF, platelet activating factor). Acetylhydrolase (EC 3.1.1.47, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase) was found in all mammalian sera with activity ranging from 11 (fetal calf) to 178 (rabbit) pmol acetate liberated/microliter serum/min. The enzyme is not present in avian serum but is a constituent of reptiles and bony fishes.     

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.     

A new de novo pathway for the formation of 1-alkyl-2-acetyl-sn-glycerols, precursors of platelet activating factor. Biochemical characterization of 1-alkyl-2-lyso-sn-glycero-3-P:acetyl-CoA acetyltransferase in rat spleen

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC, platelet activating factor (PAF] can be biosynthesized either by acetylation of alkyllyso-GPC through a remodeling pathway or by the transfer of phosphocholine to alkylacetyl-sn-glycerol (alkylacetyl-G) via a putative de novo pathway involving a dithiothreitol-insensitive cholinephosphotransferase. However, the relevance of the de novo pathway in the biosynthesis of PAF depends on the existence of enzymes that can directly synthesize alkylacetyl-G from 1-alkyl-2-lyso-sn-glycero-3-P (alkyllyso-GP) or some other source. In this study, we demonstrated that microsomal preparations of rat spleen can synthesize alkylacetyl-GP by an alkyllyso-GP:acetyl-CoA acetyltransferase and that this intermediate is subsequently dephosphorylated by an alkylacetyl-GP phosphohydrolase to generate alkylacetyl-G. The properties of alkyllyso-GP:acetyl-CoA acetyltransferase were characterized under conditions where the contaminating activity of alkylacetyl-GP phosphohydrolase was minimal; this was accomplished by inhibiting the phosphohydrolase with the addition of sodium vanadate and sodium fluoride to the assay mixtures and incubating at relatively low temperatures (23 degrees C). Alkyllyso-GP:acetyl-CoA acetyltransferase had a pH optimum of 8.4 at 23 degrees C and was located in the microsomal fraction. The apparent Km for acetyl-CoA under these conditions was 226 microM and the optimal concentration of alkyllyso-GP ranged between 16 and 25 microM. Based on pH optima, substrate inhibition studies, and sensitivities to preincubation temperatures of the microsomes, it appears that alkyllyso-GP:acetyl-CoA acetyltransferase differs from the acetyltransferase responsible for the transfer of acetate from acetyl-CoA to alkyllyso-GPC to form PAF. A variety of tissues had high activities of alkyllyso-GP:acetyl-CoA acetyltransferase, which indicates that this pathway is operational in many cell types. Our results document the existence of a complete de novo biosynthetic pathway for the assembly of PAF, and this route could be responsible for maintaining physiological levels of platelet activating factor for normal cell function.     

Platelet activating factors (AGEPC) from epidermal secretions of the Arabian Gulf catfish, Arius bilineatus, which stimulate wound healing

High levels of platelet activating factor (PAF) activity were demonstrated by platelet aggregation and serotonin release assays to be present in fright induced epidermal secretions of the Arabian Gulf catfish, Arius bilineatus (Valenciennes, 1840). The PAF activity was purified by thin-layer chromatography. Mass spectral analysis combined with chemical and enzymatic modification of the purified PAF and inhibitor studies indicated that PAF activity was due to the presence of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC) molecules. The total AGEPC concentration in the epidermal secretions based on PAF assays was 8 x 10(8) M, well above the threshold level for platelet activation which is near 5 x 10(-11) M. Thus, stimulated epidermal secretory cells of Arius bilineatus supply platelet activating molecules at physiologically high concentrations to sites of injury.     

Platelet-activating factor promotes arachidonate incorporation into phosphatidylinositol and phosphatidylcholine in neutrophils

Platelet-activating factor (1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) promotes the incorporation of [1-14C]arachidonic acid most significantly into phosphatidylinositol (PI) and phosphatidylcholine (PC) during the early stages of guinea pig neutrophil-PAF interaction. The stimulation reached a maximum at 10(-7) M and started to decline at 10(-6) M. No changes in the mass of each phospholipid were detected in neutrophils challenged by PAF for 1 to 5 minutes. The stimulation by PAF on the formation of [14C]arachidonoyl-PC but not [14C]arachidonoyl-PI was dependent on the presence of external Ca2+. These results suggest that the increased acylation of PI and PC elicited by PAF is secondary to an increased deacylation of these phospholipids and the mechanisms by which PAF stimulates the deacylation of PI and PC may be different.     

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.     

Metabolism of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human promyelocytic leukemic HL60 cells. Stimulated expression of phospholipase A2 and acetyltransferase requires differentiation

Human promyelocytic leukemia (HL60) cells can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide. The addition of N-formylMet-Leu-Phe or the Ca2+ ionophore A23187 to these differentiated cells generated 15-30 pmol of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC)/10(6) cells as quantified by platelet aggregation assays. Under identical conditions, uninduced cells produced little alkylacetyl-GPC. Upon the addition of ionophore A23187, differentiated cells, and not uninduced ones, released [14C]arachidonate from prelabeled phospholipids including ether-linked phosphatidylcholines, formed both 3H-labeled 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC) and [3H]alkylacetyl-GPC from endogenous 3H-labeled 1-O-alkyl-2-(long chain) acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), and incorporated exogenously added [3H]acetate or [3H]alkyllyso-GPC into alkylacetyl-GPC. These results are suggestive that both phospholipase A2 and acetyltransferase activities are involved in alkylacetyl-GPC biosynthesis by HL60 cells and that these activities appear during differentiation. However, when measured in cell extracts, the activities of phospholipase A2 and acetyltransferase of uninduced cells were virtually indistinguishable from those of differentiated cells. Uninduced cells exhibited enhanced incorporation of [3H]alkyllyso-GPC or [3H]alkylacetyl-GPC into alkylacyl-GPC and of [14C]arachidonate and [14C]oleate into various phospholipids including phosphatidylcholine. However, such enhanced expression of acylation reactions could not account for the lack of accumulation of arachidonate or of alkylacetyl-GPC by uninduced cells. Furthermore, analyses of phospholipid classes by phosphorus determination showed no significant alterations in phospholipid composition of HL60 cells during differentiation. Together these data are suggestive that mechanisms regulating the activation of phospholipase A2 and acetyltransferase activities are defective in uninduced cells and that an increased concentration of cytosolic free Ca2+ alone is not a sufficient requirement for these mechanisms.