Determination of platelet-activating factor by a chemiluminescence method and its application to stimulated guinea pig neutrophils

A simple, rapid and sensitive chemiluminescence method has been developed to measure platelet-activating factor (PAF). Hydrogen peroxide generated from PAF, upon phospholipase D cleavage, by choline oxidase is determined as chemiluminescence by a luminol-microperoxidase system. The detection limit of PAF by this method is 5 pmol/tube. The method is reproducible with a 5.5% coefficient of variation at 10 pmol of PAF (n=5). Lipids were extracted from guinea pig neutrophils after stimulation with cytochalasin B andN-formyl-methionyl-leucylphenylalanine, and PAF was isolated by high-performance liquid chromatography and determined by chemiluminescence measurements. The amount of PAF detected was 96.1±39.7 (mean ± SD, n=7) pmol/10⁸ cells. This highly sensitive method could be useful for the determination of PAF generated under pathophysiological conditions. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Characterization of the coronary vascular responses to platelet-activating factor in the isolated perfused heart

Platelet-activating factor (PAF) is a potent phospholipid mediator with diversein vivo andin vitro coronary vascular effects. In the present study, the coronary vascular responses to bolus injections of PAF were compared in rat hearts perfused under constant flow and under constant pressure. Low levels of PAF (1 pmol) produced vasodilatation only, while higher PAF concentrations (100 pmol) produced initial vasodilatation which was followed by a vasoconstriction under both experimental conditions. To determine species differences in PAF action, the effect of PAF was tested on perfused guinea pig hearts. Unlike in perfused rat hearts, only a dosedependent vasoconstrictor response was observed in perfused guinea pig hearts following a bolus injection of 1 fmol to 10 pmol of PAF. The results from repeated injections of PAf indicated that depletion of vasoactive mediators induced by PAF or receptor desensitization may explain a failure of a second injection of PAF to initiate a vasoconstrictor response. After PAF injection, the coronary vascular response to leukotriene was not altered, indicating that the reduced vasoconstrictor effect of a second injection of PAF cannot be due to a reduced ability of the smooth muscle to constrict. The study demon-strates that similar coronary vascular responses to PAF are observed in perfused rat hearts under either constant flow rate or constant pressure and that some of the variable coronary vascular responses reported may be due to the difference between animal species.     

A sensitive and specific radioimmunoassay for platelet-activating factor

A platelet-activating factor (PAF) analog with a reactive ω-aldehyde group at thesn-1 position was synthesized. The hapten-thyroglobulin conjugate was used to immunize rabbits to produce specific antibodies to PAF. The purified immunoglobulin G (IgG) fraction was found to bind stereo-specifically to tritiated PAF and to crossreact minimally with lysoPAF, plasmalogens, and other phospholipids. The radioimmunoassay detected as little as 20 pg of PAF per assay tube and was used to explore agonist-induced synthesis of PAF in rabbit neutrophils. Calcium ionophore A23187 at 1 μM induced PAF synthesis peaking at 2 min and reaching basal levels after 5 min.N-Formyl-Met-Leu-Phe (FMLP) at 0.1 μM also stimulated rapid synthesis and degradation of PAF with a peak at 5 min. Both A23187 and FMLP stimulated PAF synthesis in a dose-dependent manner. The radioimmunoassay should be applicable to the quantitation of PAF in biological samples. Based on a paper originally presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor detected in bronchoalveolar lavage fluids from an asthmatic patient

It recently has been recognized that platelet-activating factor (PAF) may be a mediator of asthma exacerbation. We had the opportunity to analyze bronchoalveolar lavage fluids from an asthmatic infant, which were characterized by neutrophil infiltration. The patient's lungs were washed on three occasions with saline during asthmatic attacks. PAF was found in each case on the basis of its ability to cause the immediate aggregation of washed rabbit platelets. The PAF detected was equivalent to 1–1.4 pmol of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, three quarters of which were recovered in cell-associated form. By contrast, we did not detect PAF in bronchoalveolar exudates from patients with larungeal stenosis or with respiratory distress syndrome. LysoPAF, the direct precursor as well as initial metabolite of PAF, was also analyzed after being converted to PAF by acetylation. There was a wide variation in the amount of lysoPAF present in individual patients, suggesting that lysoPAF levels cannot be taken as an indicator for the presence of PAF. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Presence of platelet-activating factor in pyuria in humans

The relationship between the occurrence of platelet-activating factor (PAF) and neutrophils in urine from patients with urinary tract infection was examined. PAF was detected in human pyuria, when leukocyte levels reached at least 300 cells/μL (n=45), but not in normal urine (n=12). The amount of PAF found in pyuria, measured by platelet aggregation assay, was 0.01 to 13.3 pmol/mL. A close correlation was seen between the amount of PAF present and the number of urinary leukocytes (p<0.01, r=0.70). The leukocytes in pyuria consisted almost entirely of neutrophils (96±4%, mean ±S.D.). Our findings suggest that the occurrence of PAF is associated with the accumulation of neutrophils in urine. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor stimulates expression of IL-1 beta mRNA in THP-1 cells

A human monocytic cell line (THP-1) was used to study the effects of PAF (platelet-activating factor) on the expression of IL-1 beta mRNA. THP-1 cells were incubated with 10 pM PAF in the presence or absence of 0.1 μg/mL endotoxin for 4 hr, after which cytoplasmic RNA was extracted and subjected to Northern hybridizations. PAF, alone and in combination with endotoxin, caused an increase in mRNA levels for IL-1 beta. The magnitude of the effects of PAF on IL-1 beta mRNA levels matched closely the effects seen at the level of protein synthesis, suggesting that the effects of PAF on IL-1 beta release may result largely from its effects on IL-1 beta mRNA levels.     

Effect of platelet-activating factor on cortisol and corticosterone secretion by perfused dog adrenal

Administration of platelet-activating factor (PAF) to perfused adrenal increased cortisol and corticosterone secretion. With hexadecyl PAF (C₁₆PAF; 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), the increase was significant at 1 nM and maximal at 10 nM. The responses to 10 nM octadecyl PAF (C₁₈PAF; 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine) were one fourth of those to 10 nM C₁₆PAF. The addition of C₁₆PAF to dispersed adrenal cells significantly increased cortisol and corticosterone production at 0.1 nM and 10 nM, respectively. C₁₆PAF was about 1000 times more potent than histamine on a molar basis in respect to cortisol response in both perfused adrenal and dispersed adrenal cells. The results suggest that PAF induces cortisol release from dog adrenal. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989. The present data were also reported at the VIIth International Congress on Hormonal Steroids, Madrid, Spain, September, 1986 (J. Steroid Biochem. 25, 76S, 1986, Abstract).     

Effect of the hetrazepinoic platelet-activating factor antagonist bepafant (WEB 2170) in models of active and passive anaphylaxis in mice and guinea pigs

The selective hetrazepinoic platelet-activating factor (PAF) antagonist WEB 2170 (Bepafant) was used to study the pathophysiological role of PAF in several models of anaphylaxis in mice and guinea pigs. In actively sensitized mice, the PAF antagonist WEB 2170 (1.0–10 mg/kgp.o.) protected mice from anaphylactic death in a dosedependent manner when the anaphylactic response was potentiated by the beta-receptor antagonist propranolol. When active anaphylaxis in guinea pigs was induced intravenously by 100 mg/kg ovalbumin (OA) in the presence of small doses of the antihistamine mepyramine, additional treatment with oral or intravenous WEB 2170 protected the guinea pigs from anaphylactic death. Also, the remaining anaphylactic bronchoconstriction and blood pressure changes (including anaphylactic hypotension) were attenuated. When guinea pigs were passively sensitized with a heterologous antibodyvia the tracheal route and then challenged by ovalbumin (100 mg/kgi.v.) 24 hr after sensitization in the presence of 0.003 mg/kgi.v. mepyramine, additional treatment with tracheal WEB 2170 at 0.1–1 mg/kg protected the guinea pigs dosedependently not only from anaphylactic death but also from a further decrease of respiratory flow and changes of blood pressure. Increased levels of PAF-like activity (20–50 ng PAF/whole lung) were detected in lungs removed from antigen-challenged animals. The results suggest a causative role for PAF in active and passive anaphylaxis. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Effect of platelet-activating factor on tumor necrosis factor-induced superoxide generation from human neutrophils. Possible involvement of G proteins

The effect of platelet-activating factor (PAF) and of two specific PAF antagonists on tumor necrosis factor (TNF) induced superoxide production by human polymorphonuclear neutrophils (PMN) was examined. PAF alone (0.1 pM to 0.1 nM) failed to evoke superoxide production; however, when PAF was added for 10 min to cells upon prior incubation with 10 ng/mL TNF for 50 min, superoxide production was significantly enhanced as compared to that induced by TNF alone. Maximum amplification (+30%) was obtained with 10 pM PAF; however, the effect was completely abolished by two structurally unrelated PAF antagonists, BN 52021 and BN 52111. The antagonists also decreased by 25% the superoxide production elicited solely by TNF, implicating the involvement of endogenous PAF in this process. Pretreatment of the PMN with either pertussis or cholera toxin attenuated the PAF amplified superoxide production in TNF stimulated cells, suggesting that G proteins sensitive to these toxins may be involved in the mechanisms controlling amplification. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May, 1989.     

Vitamin E. Deficiency increases the synthesis of platelet-activating factor (PAF) in rat polymorphonuclear leucocytes

Vitamin E deficiency was found to stimulate FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine)-induced biosynthesis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in polymorphonuclear leucocytes (PMN) from rat peritoneum. In three separate experiments each, the amounts of PAF synthesized during 6min and 12 min incubation of PMN cells from control, vitamin E-supplemented, and vitamin E-deficient rats were 129–240, 131–227 and 248–354 pmol/10⁶ cells, respectively. The activity of the acetyl-transferase, which transfers the acetyl moiety of [³H]acetyl-CoA to 2-lysoPAF (1-O-alkyl-sn-glycero-3-phosphocholine) to form [³H]PAF, was higher in PMN homogenates from vitamin E-deficient rats (2.28±0.07 nmol/min/mg protein) than in those from E-supplemented rats (1.06±0.10 nmol/min/mg protein). However, there was no difference between the two groups in the activity of acetylhydrolase (4.26±0.71 and 4.26±0.06 nmol/min/mg protein, respectively), measured as degradation of [³H]PAF to [³H]lysoPAF.In vitro addition of α-tocopherol did not inhibit the increased activity of acetyl-transferase in vitamin E-deficient rats, in-dicating that the enzyme in vitamin E-supplemented rats was not directly inhibited by α-tocopherol. The acetyltransferases of the two groups showed similar Km values for acetyl-CoA, but different Vmax values (225 μM and 6.4 nmol/min/mg protein in vitamin E-deficient rats, and 216 μM and 3.6 nmol/min/mg protein in vitamin E-supplemented rats), suggesting that the enzyme was not activated but increased in amount in vitamin E deficiency.     

Transient activation of 1-O-alkyl-sn-glycero-3-phosphocholine: Acetyl-CoA acetyltransferase during the incubation of macrophages

The activity of the platelet-activating factor (PAF)-synthesizing enzyme, 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF):acetyl-CoA acetyltransferase (EC 2.3.1.67) in alveolar macrophage lysate was found to be elevated after warming the cells to 37°C. Such an increase in enzyme activity was detectable only when intact cells were warmed. The stimulation was transient, reaching a peak at 2 min, and then gradually decreased to the control level. We could not find increased PAF formation in warmed cells which had increased acetyltransferase activity, even though substantial amounts of lysoPAF were shown to be present within cells. In contrast, considerable amounts of PAF were formed after treatments of the cells with exogenous lysoPAF. These results suggest that the activation of acetyltransferase is not sufficient to induce PAF formation and that the increased availability of substrates, especially lysoPAF, in the cells is indispensable for triggering PAF biosynthesis in this type of cells.     

1-Acyl-2-acetyl-sn-glycero-3-phosphocholine from stimulated human polymorphonuclear leukocytes

1-Acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl GPC) was found in the fraction of platelet-activating factor obtained from stimulated human polymorphonuclear leukocytes (PMN). The amount of 1-acyl-2-acetyl GPC obtained from 1×10⁷ PMN stimulated with ionophore A23187 at 37 C for 15 min ranged from 8 to 56 pmol (32±10 pmol, mean±standard error; n=4). The main species was 16∶0 palmitoyl (17±5 pmol), followed by 18∶0 stearoyl (8±3 pmol) and 18∶1 oleoyl (7±3 pmol). Although the physiological significance is unknown, 1-acyl-2-acetyl GPC was always detected when 1-alkyl-2-acetyl GPC was detected.     

Dietary α-linolenate suppresses endotoxin-induced platelet-activating factor production in rat kidney

In comparison with dietary high-linoleate safflower oil, high α-linolenate perilla oil decreased alkylacyl-and alkenylacyl-glycerophosphocholine (GPC) content in rat kidney by roughly 30 and 25%, respectively. The fatty acid composition was also modified by high α-linolenate oil; arachidonic acid (AA) level in alkylacyl-GPC, a platelet-activating factor (PAF) precursor, decreased by 30% along with concomitant increases in the n-3 fatty acid levels. PAF contents under resting conditions were similarly low in the two dietary groups. Fifteen minutes after endotoxin administration, PAF and lyso-PAF contents increased significantly, and the PAF content in the high α-linolenate group was 60% lower than in the high linoleate group; the lyso-PAF contents also tended to be lower. Lyso-PAF acetyltransferase and CoA-independent transacylase activities in kidney microsomes increased significantly after endotoxin administration, while PAF acetylhydrolase activity in the cytosol was relatively unchanged. The lyso-PAF acetyltransferase and PAF acetylhydrolase activities did not differ between the two dietary groups, but the CoA-independent, transacylase activity was roughly 30% lower in the high α-linolenate group. In agreement with in vitro study, our present study demonstrates that dietary high α-linolenate suppresses PAF production in rat kidney during systemic endotoxemia, and which is mainly due to the decrease in alkylacyl-GPC content, altered fatty acid compositions of the precursor lipids and lower CoA-independent transacylase activity.     

Platelet activating factor-induced pulmonary accumulation of111indium-oxine labelled neutrophils in anesthetized guinea pigs

The thoracic accumulation of neutrophils labelled with¹¹¹Indium-oxine in response to infusion of platelet activating factor (PAF, 18 ng/kg/min × 5 min, i.v.) was studied using an automated isotope monitoring system (AIMSplus) in anesthetized guinea-pigs. Loss of cell associated radioactivityin vitro was less than 1% over 4 hr. Labelled neutrophils maintained their functional capacity (oxidative response to the cell stimulants N-formyl-L-methionine-L-leucine-L-phenylalanine and phorbol myristate acetate) and >95% viability (ethidium bromide/acridine orange stain)in vitro. Total thoracic radioactivity increased significantly from baseline in response to PAF with a slight tachyphylaxia in the neutrophil-accumulation after a repeat PAF infusion. The highest ratios of radiolabel (tissue/blood) were found in the spleen >>liver>lung. Based in part on a paper presented at the Third Internatioinal Conference on Platelet-Activating-Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor modulates phospholipid acylation in human neutrophils

The present study showed that platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, PAF), but not lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) rapidly (within 15 sec) stimulated the incorporation of both [1-¹⁴C]arachidonate and [1-¹⁴C]docosahexaenoate into phosphatidylinositol (PI) and phosphatidylcholine (PC) in human neutrophils. Concomitantly, it inhibited the formation of labeled phosphatidic acid from both fatty acids. The magnitude of stimulation (percentage of control) was greater in PI than in PC for the incorporation of arachidonate and vice versa for the incorporation of docosahexaenoate. It reached a maximum at 10⁻⁷ M and started to decline at 10⁻⁶ M. Extracellular Ca²⁺ was not essential for the action of PAF on phospholipid acylation. The distribution of labeled arachidonate in the molecular species of PC was not altered by PAF after 1 min incubation, suggesting that the increased formation of arachidonyl-PC during the early stage of neutrophil-PAF interaction was not originated from the added PAF. No measurable changes in the mass of each phospholipid were detected in neutrophils challenged by PAF from 15 sec to 2 min. The data suggest that the increased incorporated of extracellular fatty acids into PI and PC elicited by PAF may be secondary to increased deacylation of these phospholipids, and the magnitude of stimulation reflects the specificity of acyltransferase catalyzing the acylation of lysoPI and lysoPC by fatty acyl-CoA.     

Platelet-activating factor type activity in plasma from patients with septicemia and other diseases

The purpose of the present study was to determine whether increased levels of platelet-activating factor (PAF) type activity can be detected in plasma from patients with septicemia and other diseases. A level of PAF below 0.5 ng/mL of plasma was considered normal. We found that plasma from a patient with adverse anaphylactoidic reaction to intravenous analgetics contained 2.1 ng PAF/mL. In seven patients with septicemia, including urosepsis, endocarditis and peritonitis, and with positive blood culture, increased plasma PAF levels (1–20 ng PAF/mL) were observed. Other patients with clinical indications of septicemia had negative blood cultures and/or increased levels of C-reactive protein (CRP). Yet, in the plasma from these patients, no increased PAF levels were detected under the assay conditions used. Two patients with allergic asthma, requiring treatment with steroids, had no measurable plasma PAF. In the plasma from a patient with idiopathic thrombocytopenic purpura (ITP) only an “endogenous” inhibitor of PAF induced platelet aggregation was initially observed. In spite of this, the patient responded to treatment with the PAF antagonist WEB 2086 with a dramatic increase in platelet count (Lohmannet al., Lancet ii, 1147, 1988). Thereafter, also increased PAF levels (3.3 ng PAF/mL) were detected in plasma, although some “endogenous” inhibitor of PAF was still present. In conclusion, increased PAF levels in plasma from patients support a role of PAF in certain human disease states, such as in anaphylactoid reaction, sepsis and septic shock. The type, relevance and specificity of endogenous inhibitors of PAF deserve further study. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor regulates phospholipid metabolism in human neutrophils

This study extended the earlier finding that platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) promotes arachidonic acid incorporation into neutrophil phosphatidylinositol (PI) and phosphatidylcholine (PC). In the present study the effect of PAF on fatty acid uptake by human neutrophils and the incorporation of extracellular linoleic acid and palmitic acid into phospholipids were investigated. Incubation of 10⁻⁷ M PAF with neutrophils and radiolabeled arachidonic acid or linoleic acid or palmitic acid for 1–10 min resulted in an increased rate of loss of label from the incubation medium. PAF stimulated the incorporation of linoleic acid and palmitic acid most significantly into PI and PC. The magnitude of stimulation was greater in PI than in PC for the incorporation of linoleic acid, and vice versa for the incorporation of palmitic acid. The positional distribution of linoleic acid and palmitic acid in PI and PC and the mass of these phospholipids were not altered in PAF-stimulated neutrophils. An increased incorporation of all three fatty acids into both diacyl and alkylacyl species of PC was demonstrated after a two minute incubation of cells with PAF. While more radioactivity was recovered in the diacyl species, the magnitude of increase of radioactivity in the alkylacyl species was more pronounced than that in the diacyl species of PC. These results suggest that both increased fatty acid uptake and increased available lysophospholipids may be contributory to the increased phospholipid acylation induced by PAF.     

Platelet-activating factor (PAF) stimulates the lysoPAF acetyltransferase in leukocyte-rich plasma: Use in PAF antagonist studies

Addition of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to leukocyte-rich plasma from several species resulted in the rapid and pronounced activation of the PAF biosynthetic enzyme acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67). Activation of acetyltransferase by PAF occurred in leukocyte-rich plasma from human, chimpanzee, rhesus monkey, and dog. The neutrophil was indicated to be the major cellular source of the activabable acetyltransferase in leukocyte-rich plasma. The induction of acetyltransferase was substantial with 10 nM PAF, and maximal at 10–30 seconds. Measurable acetyltransferase activation was significantly greater when the PAF-activated cells were separated from the plasma by centrifugation before the acetyltransferase assay. This may be due in part to the removal of the PAF-specific acetylhydrolase present in plasma which can cleave the acetyl group from PAF. Measuring PAF activation of acetyltransferase in leukocyte-rich plasma can be useful to determine the potency of PAF antagonists with neutrophils in plasma compared to isolated neutrophils in aqueous buffer, and as anex vivo assay to determine the efficacy and plasma concentration equivalents of antagonists administered to whole animals. The PAF antagonist L-659,989 was shown to be 3–5 times more potent in inhibiting PAF induction of acetyltransferase in isolated human neutrophils than in human leukocyte-rich plasma, with IC₅₀ values of 10 nM and 40 nM, respectively. In theex vivo assay, oral administration of the PAF antagonist L-667, 131 to dogs resulted in very substantial inhibition of PAF induction of acetyltransferase in the leukocyte-rich plasma. Utilizing theex vivo assay, oral administration of 1 mg/kg L-659,989 to rats was found to result in plasma concentration equivalents of approximately 200–300 nM L-659,989. Our findings offer a new approach for charagerizing thein vitro andin vivo efficacy of PAF receptor antagonists and demonstrate that PAF may be able to activate neutrophils in the bloodin vivo, further enhancing PAF synthesis. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Intracellular Ca2+ concentration and H2O2 production in mouse peritoneal macrophages are stimulated by platelet activating factor

The intracellular calcium concentration ([Ca²⁺]i) in mouse peritoneal macrophages cultured on a coverglass was measured in the superfusion system using fura-2 as a fluorescent calcium probe and platelet activating factor (PAF) as a stimulant. In the presence of extracellular Ca²⁺, 10⁻⁷M PAF sharply increased [Ca²⁺]i from a basal level of 90 nM to 340 nM. Thereafter, the [Ca²⁺]i level gradually decreased in two phases, in an initial rapid phase and a subsequent slow phase of decrease. The calcium response was dependent on the PAF concentration. In the absence of extracellular Ca²⁺, a single sharp peak was observed, suggesting two different modes of Ca²⁺ movement, one from intracellular stores and the other from the extracellular medium. A simple, sensitive fluorometric assay system was developed for measuring H₂O₂ in the superfusate of macrophages after stimulation by use of immobilized peroxidase and 3-(p-hydroxyphenyl)propionic acid as a fluorogenic substrate. With this system, as little as 2 pmol of H₂O₂ could be measured. PAF (1 μM) increased H₂O₂ production in peritoneal macrophages in the presence of extracellular Ca²⁺, but H₂O₂ production was not observed in the absence of extracellular Ca²⁺. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Role of platelet-activating factor (PAF) in superoxide production by human polymorphonuclear leukocytes

The effect of platelet-activating factor (PAF) in superoxide production by human polymorphonuclear leukocytes (PMN) was studied. Cypridina luciferin analog (CLA) dependent chemiluminescence was used to detect superoxide anion radicals. PAF induced superoxide generation in human PMN in a dose-dependent manner. Preincubation with a small amount of PAF (5 x 10⁻⁹ M) enhanced PMN superoxide release induced by various stimuli, such as phorbol myristate acetate (PMA), opsonized zymosan (OZ), calcium ionophore (A23187) andN-formyl-methionyl-leucyl-phenylalanine (FMLP). The PAF antagonist, CV-6209, inhibited superoxide production induced by PAF, but not that induced by other stimuli. These findings would indicate that PAF may play an important role at inflammatory reaction sites and that CV-6209 may inhibit excessive inflammatory reaction.