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.     

Influence of platelet-activating factor,its cell analogs,and antagonist on the production of superoxide radicals by blood leukocytes of healthy and hypercholesterolemic individuals

The influence of the phospholipid platelet-activating factor (PAF), its cell analogs, and lipid PAF antagonist on the production of superoxide radicals by leukocytes isolated from the blood of healthy and hypercholesterolemia IIA individuals was studied. It was found that endogenous superoxide production level in the leukocytes of hypercholesterolemic individuals more than 4-5 times higher than in the leukocytes of healthy individuals. Exogenous PAF stimulates the superoxide production in the leukocytes of healthy individuals but significantly inhibits the superoxide production in the leukocytes of hypercholesterolemic individuals. The compounds 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-PAF) and 1-alk-1"-enyl-2-acetyl-sn-glycero-3-phosphocholine (1-alkenyl-PAF) only slightly inhibited the endogenous superoxide production in the leukocytes of hypercholesterolemia individuals. However, pretreatment of leukocytes by 1-alkenyl-PAF or PAF-antagonist (1-O-alk-1"-enyl-2-(2"-acetoxybenzoyl)-sn-glycero-3-phosphocholine) results in a 50% inhibition of the PAF-induced superoxide production by leukocytes of healthy individuals. This PAF-antagonist alone or in combination with PAF induces a substantial (65-70%) inhibition of superoxide production in the leukocytes of hypercholesterolemic individuals. It is concluded that superoxide production by leukocytes of healthy individuals and especially by leukocytes of hypercholesterolemic individuals is process that depends on PAF or PAF-like lipids.     

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.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

Synthesis of a fluorescently-labeled platelet activating factor

The synthesis of fluorescently labelled PAF-acether, 1-alkyl-2-acetyl-sn-glycero-3-phospho-[N-(9-anthrylmethyl)-N, N-dimethylethanolamine] with the label in the choline moiety is described, plasmalogen lysophosphatidylcholine of bovine heart being used as starting material.     

A novel CoA-independent transacetylase produces the ethanolamine plasmalogen and acyl analogs of platelet-activating factor (PAF) with PAF as the acetate donor in HL-60 cells.

In this study, we demonstrate the presence of a unique membrane-associated transacetylase that transfers the acetate group from platelet-activating factor (PAF) to lysoplasmalogen (in the presence of EDTA and sodium acetate) with the formation of 1-alk-1-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alk-1-enylacetyl-GPE). The identity of alk-1-enylacetyl-GPE was confirmed by acid hydrolysis, phospholipases A2 or C treatment and derivatization by fluorodinitrobenzene. The transacetylase has no requirement for Ca2+, Mg2+, or CoA and a broad pH optimum (7.0-8.0) with Km values of 12.0 microM for PAF and 106.4 microM for lysoplasmalogens. The enzyme activity from the isolated membrane fraction is not changed when whole cells are supplemented with 20:4, induced to differentiate into granulocytes, or treated with ionophore A23187. Radyllyso-sn-glycero-3-phosphocholine (GPC), radyllyso-GPE, acyllyso-sn-glycero-3-phosphoserine (GPS), acyllyso-sn-glycero-3-phosphoinositol (GPI), alkyllyso-sn-glycero-3-phosphate (GP), acyllyso-GP, or cis-9-octadecen-1-ol can also serve as acetate acceptors, whereas alkylglycerol, acylglycerol, or cholesterol are inactive. Differences in substrate acceptor specificity, sensitivity toward phenylmethylsulfonyl fluoride, and response to temperature suggest that the CoA-independent transacetylase and the CoA-independent transacylase that transfers long-chain acyl moieties are two separate enzymes. With intact differentiated HL-60 cells, [3H]acetate from [3H]PAF can be incorporated into alk-1-enylacetyl-GPE in the presence of ionophore A23187, but not in its absence. Moreover, phospholipase A2 inhibitors (p-bromophenacyl bromide and mepacrine) block the transacetylation process in whole cell system. These results indicate the production of alk-1-enyllyso-GPE is a rate-limiting factor for the subsequent transacetylation step during cell activation. We conclude that the transacetylase may participate in the biosynthesis of ethanolamine plasmalogen and acyl analogs of PAF, in vivo, fine-tuning of PAF biological responses, and cross-talk between de novo and remodeling pathways of PAF biosynthesis.     

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.     

Bradykinin-stimulated release of [3H]arachidonic acid from phospholipids of HSDM1C1 cells: Comparison of diacyl phospholipids and plasmalogens as sources of prostaglandin precursors

Ethanolamine plasmalogens (1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [³H]arachidonate-labeled phospholipids of HSDM₁C₁ cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE₂. HSDM₁C₁ cells labeled with [³H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids (16%). Bradykinin treatment stimulated a rapid hydrolysis of [³H]arachidonate from the cellular lipids and conversion of the released acid to PGE₂, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM₁C₁ cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE₂ and to a lesser degree inhibited the release of [³H]-arachidonate from the cellular lipids into the medium.     

Metabolic conversion of platelet-activating factor into ethanolamine plasmalogen in an amnion-derived cell line.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) labeled with 3H in the alkyl side chain was taken up rapidly by amnion-derived WISH cells in culture. The radioactivity was found in a number of cellular metabolites, principally 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-acyl-GPC) which was labeled at a rapid rate. No intracellular accumulation of lyso-PAF was detected. At longer time periods, a substantial proportion of the radioactivity was found in association with the phosphatidylethanolamine fraction extracted from the cells. This fraction contained a high proportion of the corresponding 1',2'-alkenyl derivative (plasmalogen), as judged by the formation of long-chain fatty aldehyde after exposure to acid. The magnitude of the conversion of PAF into ethanolamine plasmalogen is suggestive of a correlation between plasmalogen content and exposure to PAF in some tissues. The exact sequence of reactions leading from alkyl-acyl-GPC to the ethanolamine derivatives is yet to be established.     

Bradykinin-stimulated release of [H]arachidonic acid from phospholipids of HSDM1C1 cells: Comparison of diacyl phospholipids and plasmalogens as sources of prostaglandin precursors

Ethanolamine plasmalogens (1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [³H]arachidonate-labeled phospholipids of HSDM₁C₁ cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE₂. HSDM₁C₁ cells labeled with [³H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids (16%). Bradykinin treatment stimulated a rapid hydrolysis of [³H]arachidonate from the cellular lipids and conversion of the released acid to PGE₂, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM₁C₁ cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE₂ and to a lesser degree inhibited the release of [³H]-arachidonate from the cellular lipids into the medium.     

The hydrolysis of the alkenyl group from enthanolamine-plasmalogen by oligodendroglial cell-enriched fractions

The rate of hydrolysis of the 1-0-alkenyl group of sn-1-alk-1′-enyl-2-acyl-glycerylphosphorylethanolamine (alkenyl, acyl-GPE; ethanolamine plasmalogen) by plasmalogenase is higher in oligodendroglial cell-enriched fractions from bovine brain compared with fractions enriched in neuronal perikarya and astroglia. The distribution of plasmalogenase activity in membrane fractions isolated from bovine oligodendroglia has been compared with that of ‘marker’ enzymes. The highest specific activity was in a fraction enriched in plasma membranes, whilst most activity was recovered in an endoplasmic reticulum membrane fraction. In bovine oligodendroglial cell homogenates, the enzyme had a neutral pH optimum, had no requirement for divalent cations and its activity towards 1-alkenyl-GPE (lysoplasmalogen) was half that with alkenyl, acyl-GPE. C₁₆ alkenyl groups were hydrolysed more rapidly than C₁₈ alkenyl groups. With ³H-labelled alkenyl, acyl-GPE as substrate, radioactivity in released aldehydes appeared in fatty acids esterified in phospholipid while the oxidation of fatty aldehydes was blocked by the addition of NADH. An NAD-dependent aldehyde dehydrogenase was found to be present in oligodendroglia which exhibited highest activity towards C₁₄C₁₈ aldehydes (K_m, 2 μM).     

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.     

1-O-hexadec-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine and its biological activity

1-O-Alk-1'-enyl analog of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, alkylacetyl-GPC) was prepared semi-synthetically from choline plasmalogens of beef heart muscle. The main compound was identified mass spectrometrically as 1-hexadec-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine (16:O alk-1'-enylacetyl-GPC, 16:O vinyl form of PAF) and its platelet aggregation activity was about one-fifth of that of the corresponding 16:O alkylacetyl-GPC. The irreversible platelet aggregation activity induced by 5X10(-10) M 16:O alk-1'-enylacetyl-GPC was completely inhibited by 5X10(-7) M CV-3988 and 1X10(-7) M L-652, 731, specific PAF antagonists, and more than 99% of the activity was also lost by acid treatment. The hydrogenated product, alkylacetyl analog, showed quite same activity as that of authentic 16:O alkylacetyl-GPC. The platelets desensitized with 16:O alkylacetyl-GPC and with 16:O alk-1'-enylacetyl-GPC were not aggregated with 5X10(-10) M 16:O alk-1'-enylacetyl-GPC, suggesting that alk-1'-enylacetyl-GPC occupied the same receptor site of alkylacetyl-GPC.     

CV-3988 - a specific antagonist of platelet activating factor (PAF)

CV-3988, rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate was shown to be a specific inhibitor of platelet activating factor (PAF). This compound in concentrations of 3 x 10(-6) to 3 x 10(-5)M inhibited aggregation of rabbit platelets induced by PAF (3 x 10(-8)M), while it had no effect on the aggregation induced by arachidonic acid, ADP, collagen or A-23187. CV-3988 alone even at a concentration of 10(-3)M had no effect on platelet aggregation. The inhibitory action of CV-3988 on the PAF-induced aggregation was independent of the formation of micelles. The PAF (0.1 to 1.0 micrograms/kg, i.v.)-induced hypotension in anesthetized rats was also inhibited dose-dependently by the i.v. administration of CV-3988 (1 and 10 mg/kg), while the hypotensive actions induced by the i.v. administration of acetylcholine (1 micrograms/kg), arachidonic acid (1 mg/kg), bradykinin (10 micrograms/kg), isoproterenol (1 microgram/kg) and histamine (100 micrograms/kg) were not altered by CV-3988 (10 mg/kg, i.v.). All these findings indicate that CV-3988 specifically inhibits the action of PAF in vitro and in vivo. This is the first report of a PAF antagonist which can specifically inhibit the PAF-induced hypotension as well as the PAF-induced platelet aggregation.     

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.     

Susceptibility of plasmenyl glycerophosphoethanolamine lipids containing arachidonate to oxidative degradation

Plasmenyl phospholipids (1-alk-1′-enyl-2-acyl-3-glycerophospholipids, plasmalogens) are a structurally unique class of lipids that contain an α-unsaturated ether substituent at the sn-1 position of the glycerol backbone. Several studies have supported the hypothesis that plasmalogens may be antioxidant molecules that protect cells from oxidative stress. Because the molecular mechanisms responsible for the antioxidant properties of plasmenyl phospholipids are not fully understood, the oxidation of plasmalogens in natural mixtures of phospholipids was studied using electrospray tandem mass spectrometry. Glycerophosphoethanolamine (GPE) lipids from bovine brain were found to contain six major molecular species (16:0p/18:1-, 18:1p/18:1-, 18:0p/20:4-, 16:0p/20:4, 18:0a/20:4-, and 18:0a/22:6-GPE). Oxidation of GPE yielded lyso phospholipid products derived from plasmalogen species containing only monounsaturated sn-2 substituents and diacyl-GPE with oxidized polyunsaturated fatty acyl substituents at sn-2. The only plasmalogen species remaining intact following oxidation contained monounsaturated fatty acyl groups esterified at sn-2. The mechanism responsible for the rapid and specific destruction of plasmalogen GPE may likely involve unique reactivity imparted by a polyunsaturated fatty acyl group esterified at sn-2. This structural feature may play a central role determining the antioxidant properties ascribed to this class of phospholipids.     

Pharmacological actions of Y-24180, a new specific antagonist of platelet activating factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-t hieno [3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC50 0.84 nM) was more potent than WEB 2086 (IC50 4.21 nM) and etizolam (IC50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC50 value of 3.50, 9.35 and 29.5 nM, respectively. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180 (1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 microM. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

Pharmacological actions of Y-24180, a new specific antagonist of Platelet Activating Factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of ³H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC₅₀ 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC₅₀ values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC₅₀ 0.84 nM) was more potent than WEB 2086 (IC₅₀ 4.21 nM) and etizolam (IC₅₀ 998 nM). Y-24180, WEB 2086 and etizolam displaced ³H-PAF binding from the washed-platelets of rabbits with an IC₅₀ value of 3.50, 9.35 and 29.5 nM, respective- ly. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180(1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced ³H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 μ M. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

Effect of prostacyclin on platelet-activating factor induced rabbit platelet aggregation

In this paper, the effect of prostacyclin (PGI₂) on the aggregation induced by Platelet-activating factor (PAF), a phospholipid mediator of anaphylaxis, was studied. Synthetic PGI₂ and PGI₂-like activity generated from rabbit aorta were demonstrated to be effective inhibitors of PAF-induced rabbit platelet aggregation and release of ³H-serotonin (³H-5HT).     

Nicotinic-type unitary currents in Helix neurons

1. The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10(-4) M), while inducing no aggregation, caused the release of 1248 +/- 1147 pg/ul (mean +/- SD) of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 from stimulated platelet. The addition of 1 x 10(-4) M ADP to platelets caused suboptimal aggregation and the release of only 25 +/- 10 pg TXB2/microliters. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.