Role of platelet-activating factor in aggregation of leukocytes and platelets in cerebral ischemia

Leukocyte and platelet aggregation stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP) was measured in 32 patients with cerebral ischemia and in 15 controls, using a whole blood aggregometer. The increases in impedance and the reductions in leukocyte and platelet counts were significantly greater in stroke patients than in controls. Aggregation was inhibited by oral ticlopidine, but not by oral aspirin. The effects were clearly counteracted by platelet-activating factor (PAF) antagonists, and counteracted in part by a 5-lipoxygenase inhibitor. The results suggest that platelets tend to be activated by PAF and leukotrienes liberated from hyperaggregable leukocytes in patients with ischemic stroke. 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 lipoprotein lipase and blood lipids

We investigated the effect of platelet-activating factor (PAF) and of the PAF specific antagonist CV-6209 on plasma lipid metabolism, and particularly on post-heparin plasma lipolytic activity in male Wistar rats. Lipoprotein lipase (LPL) activity was enhanced by intravenous injection of PAF before intravenous injection of heparin when the PAF dose was low (0.2 μg/kg). PAF activated hepatic triacylglycerol lipase (HTGL) activity dose-dependently. Plasma triacylglycerols (TG) significantly decreased with the activation of LPL and/or HTGL. Plasma total cholesterol (TC) and phospholipid (PL) levels decreased at a low dose of PAF (0.2 μg/kg), but increased when higher doses were used. The PAF antagonist CV-6209 partially reversed the PAF induced effects on HTGL, TC and PL. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Metabolism of lysophosphatidylcholine by swine platelets

Incubation of intact platelets from Sinclair(S-1) miniature swine with³²P-labeled lysophosphatidylcholine (lyso PC) indicated the presence of an active lysophospholipase with a pH optimum of 8.0 for hydrolysis of the substrate. However, lyso PC was incorporated into the membrane phosphatidylcholines by the acyltransferase pathway upon addition of ATP, Mg⁺⁺ and CoA to the platelet suspension. These results suggest that intact platelets are able to resist the cytotoxic effects of lyso PC in plasma, and the phospholipids in platelet membranes are not readily affected by the lipid environment of the plasma. The acyltransfer reaction apparently is saturated with endogenous free fatty acids since arachidonic acid added exogenously did not further enhance the incorporation activity. Neither the acyltransferase nor the lysophospholipase activity was affected by Ca⁺⁺, but divalent metal ions such as Zn⁺⁺ inhibited the lysophospholipase activity. Cholesterol but not cholesteryl esters elicited a biphasic effect on both enzymes, stimulating at low concentration but inhibiting at a cholesterol to lyso PC ratio greater than 1. Serum albumin inhibited the lysophospholipase but gave a small biphasic effect to the acyltransferase.     

The hormonal regulation of platelet-activating factor acetylhydrolase activity in plasma

We have previously reported that certain fetal tissues including the lung and kidney have an increased platelet-activating factor (PAF) content and enzymatic mechanism for its elevated biosynthesis during the latter stages of pregnancy. In contrast, in the maternal plasma compartment of both the rabbit and human, a decreased capacity to inactivate PAF has been demonstrated. The PAF acetylhydrolase in the fetal plasma is also suppressed. The present study was undertaken to determine the mechanism(s) involved in the regulation of PAF acetylhydrolase. The 17α-ethynylestradiol was administered (intraperitoneal [i.p.] 2.5 mg/kg body wt 5 days) to female and male rats. The plasma PAF acetylhydrolase activity decreased 5-fold. A decrease was observed when a concentration of the estrogen as low as 50 μg/kg was employed. The injection of dexamethasone (i.p., 1.3 mg/kg body wt, 5 days) to male and female rats resulted in a 3-fold increase in the plasma PAF acetylhydrolase activity. The activity returned to the values prior to hormone treatment 4 days after cessation of treatment. Testosterone and progesterone were without effect on plasma acetylhydrolase activity. The change in PAF acetylhydrolase activity caused by estrogen and the glucocorticoid was reflected by a change in the activity in the HDL fraction and not due to the presence of an inhibitor or activator in the plasma of the hormone-treated animals. Human serum obtained from a group of women, in which the 17β-estradiol concentration was elevated in preparation for anin vitro fertilization procedure, showed an inverse relationship between the plasma estrogen concentration and the PAF acetylhydrolase activity. It is suggested that estrogen is responsible for the regulation of PAF acetylhydrolase and the decrease in the plasma PAF acetylhydrolase during the latter stages of pregnancy in both the maternal and fetal plasma caused by the hyperestrogenic state that occurs during this period. The observed increase in PAF acetylhydrolase by dexamethasone may account for, in part, the known anti-inflammatory properties of this steroid by decreasing the concentration of this potent autacoid. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Analysis of alpha tocopherol in blood plasma and platelets by gas liquid chromatography

A method is described for the analysis of α-tocopherol by gas liquid chromatography (GLC) with 0.3% Apiezon L as liquid phase. Impurities that interfere with GLC are removed by saponification. Cholesterol and other nonsaponifiables are separated from α-tocopherol by GLC.     

Pooling of blood in postischemic shock is modulated by platelet-activating factor (PAF)

In experiments on dogs,i.v. administration of platelet-activating factor (PAF) (500 ng/kg) was shown to induce hypotension which, apart from decreased myocardial contractility, was characterized by blood pooling in veins (82.6±6.8 mL/kg). This was accompanied by restriction of venous return to the heart and reduction of cardiac output (CO). During postischemic shock the cardio- and hemodynamic disturbances were similar to those induced byi.v. administration of PAF. In the postischemic shock model, preliminary blockage of PAF receptors with the PAF receptor antagonist BN 52021 (6 mg/kg,i.v.) significantly decreased the amount of blood pooled in shock from 38.7±5 to 18.3±2 mL/kg (p<0.01). Simultaneously, the reduction of CO and blood pressure, induced by reperfusion of the continuously ischemized tissues of a rear limb, was less significant in pretreatedvs. the nontreated group. The data suggest that PAF may be involved in postischemic blood pooling and that PAF antagonists could be used to correct postischemic cardio- and hemodynamic disturbances. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Studies on the role of platelet-activating factor in blood pressure regulation

Circulating levels of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C₁₆PAF) in human subjects were measured by gas chromatography/mass spectrometry using negative ion chemical ionization. The mean (±S.D.) circulating C₁₆PAF levels in patients with essential hypertension (18.1±5.3 pg/mL, n=16) were not significantly different from those in normotensive subjects (17.2±7.2 pg/mL, n=14). During a salt balance study, high salt intake (20 g/day) significantly increased the circulating level of C₁₆PAF, and changes in circulating C₁₆PAF significantly and positively correlated with changes in mean arterial blood pressure (r=0.47, p<0.05). Changes in C₁₆PAF also correlated with changes in creatinine clearance (r=0.55, p<0.05), but did not correlate with changes in plasma sodium concentration, plasma chloride concentration and plasma volume. An intravenous injection of 50 μg of human atrial natriuretic peptide (hANP) decreased circulating C₁₆PAF levels from 20.0±2.7 to 13.9±2.4 pg/mL of blood (n=10, p<0.01) in healthy subjects. The data appear to indicate that C₁₆PAF levels are changed by salt intake-induced mild increase in blood pressure, and that hANP may be an endogenous factor which lowers circulating C₁₆PAF. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Production of platelet-activating factor by human normodense and hypodense eosinophils

Normodense eosinophils and neutrophils from normal donors produced considerable amounts of plateletactivating factor (PAF) when stimulated with ionophore A23187. PAF produced by eosinophils appeared to be degraded more rapidly than PAF formed by neutrophils, suggesting a higher activity of PAF-degrading enzyme in eosinophils. Substantial proportions of PAF newly formed by both eosinophils and neutrophils were shown to be cell-associated. By comparison, hypodense eosinophils obtained from a patient with idiopathic hypereosinophilic syndrome produced an extremely large amount of PAF and released much of it into the incubation medium. The accelerated formation of PAF in hypodense eosinophils may be related to various cardiovascular complications associated with hypereosinophilic syndrome. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Radioimmunoassay for platelet-activating factor

A radioimmunoassay (RIA) for measurement of platelet-activating factor (PAF) was developed. At a final antiserum dilution of 1∶640, the lowest detection limit of PAF was 0.1 pmol (50 pg). The standard curve obtained was suitable for measurement of PAF in amounts ranging from 0.1 pmol to 30 pmol. The antiserum showed high specificity. Cross-reaction for lysoPAF, lysophosphatidylcholine and long-chain phosphati-dylcholines was very low (less than 0.025%). 1-Palmitoyl-2-acetyl-sn-glycero-3-phosphocholine cross-reacted slightly (6.25%). PAF exogenously added to macrophage suspensions was quantitatively determined by RIA after solvent extraction and high-performance liquid chromatographic separation. RIA was also used to estimate PAF formation after stimulation of rabbit alveolar macrophages in suspension with calcium ionophore A23187.     

Tumor necrosis factor release by human monocytes stimulated with platelet-activating factor

Platelet-activating factor (PAF) is a low molecular weight phospholipid which enhances human monocyte cytotoxicity for tumor cells. In the current studies, the capacity of PAF to stimulate release of tumor necrosis factor α (TNFα) by human monocytes was assessed. PAF induced maximal TNFα synthesis 2–3 hr after monocyte stimulation as assessed by dot blotting of cell-associated TNFα using polyclonal anti-TNF antibody. Maximal net release of TNFα occurred 5–16 hr after monocyte stimulation, as assessed by a specific ELISA. Dose-response studies revealed that a maximal two- to three-fold increase in release of TNFα occurs at 10–100 pM PAF. LysoPAF and the optical isomer of PAF did not stimulate release of TNFα, suggesting that stimulation is mediated by specific PAF receptors. Scatchard analysis of [³H]PAF binding to monocyte membranes revealed 651±495 binding sites/monocyte with a Kd of 4.7±4.2×10⁻¹⁰M. PAF is a structurally unique activator of monocytes whose interactions with TNFα and other cytokines may be critical to host defense against tumors. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Cardiovascular effects of platelet-activating factor

Sudden release of platelet-activating factor (PAF) into the circulation can cause hypotension, tachycardia, and circulatory collapse. To further examine this response, we performed detailed studies of cardiovascular function after PAF administration to young domestic pigs and newborn piglets. Our results indicate that circulatory dysfunction after PAF reflects severe constriction of pulmonary resistance vessels and consequent acute right ventricular failure. Although PAF-induced coronary artery constriction and contractile depression may be complicating problems, left ventricular underperfusion and dysfunction after PAF are mainly the result of systemic arterial hypotension and diminished left ventricular filling. The adverse hemodynamic effects of PAF are accompanied by substantial release of thromboxane A₂ (TxA₂). These effects are mimicked by the TxA₂ agonist U-46619 and partially blocked by specific and nonspecific inhibitors of TxA₂ synthesis (OKY-046 and indomethacin). Even more potent blockade of PAF action is exerted by the TxA₂ receptor blocker, SQ 29,548. Taken together, these findings indicate that severe pulmonary vascular constriction and hemodynamic collapse soon after intravenous PAF are at least partially mediated by PAF-induced TxA₂ release. Tachyphylaxis to PAF influence has been observed in studies of leukocyte and platelet function. We hypothesized that tachyphylaxis to PAF might also occur in our studies of constrictor responses in pulmonary vessels. Recently, we have examined the capacity of PAF to produce sustained pulmonary vasoconstriction in openchested, anesthetized newborn piglets. Infusions sufficient to produce 100% increase in mean pulmonary artery pressure after 3 min showed no loss of efficacy when sustained for 30 min. The same was true for infusions of U-46619. Thus, the pulmonary vasoconstrictor influence of PAF or U-46619 is not readily diminished by tachyphylaxis. These findings favor the viewpoint that PAF or TxA₂ release during inflammatory processes could have prolonged adverse actions on pulmonary and systemic circulations. 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 opinions or assertions contained here are those of the authors. They do not reflect the views of the Department of Defense or the Uniformed Services University of the Health Sciences. The experiments reported here were conducted according to the principles set forth in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, U.S. Department of Health and Human Services (NIH Publ. 85-23, 1985).     

A simple and precise method for the routine determination of platelet-activating factor in blood and urine

A simple and precise method is described for the measurement of platelet-activating factor (PAF) in blood and urine. The method involves the isolation of PAF from blood samples by two successive steps. In the first step, blood proteins are precipitated with ethanol and the “free” PAF, i.e. the PAF which is extractable with ethanol, is recovered. In the second step, “bound” PAF, i.e., PAF not extractable with ethanol, is extracted from the protein precipitate with chloroform/methanol/water. The extraction of PAF from urine samples requires only the ethanol extraction step. “Free” and “bound” PAF are then each fractionated by silicic acid column chromatography, and the methanol/water eluent containing PAF is then further fractionated by high-performance liquid chromatography using an isocratic solvent system of acetonitrile/methanol/water. PAF is then quantitated by measuring its ability to induce platelet aggregation in an aggregometer. Application of the method to blood and urine samples from twenty-three healthy volunteers revealed PAF levels in blood of 140–480 pg/mL (630–254.4 pg “free” PAF/mL and 64–225.6 pg “bound” PAF/mL), and of 1.2–4.0 pg PAF/mL in urine. The method overcomes various technical problems and was shown to be very precise. It should prove useful for monitoring PAF levels in various disease conditions.     

Role for platelet-activating factor in asthma

Recent studies of the effects of platelet-activating factor (PAF) on huan and animal airways would support a putative role for this lipid mediator in asthma. PAF can induce many aspects of the clinical and pathological features seen in asthmatic airways such as airway oedema, eosinophil accumulation in the airway wall, and bronchial hyperresponsiveness. PAF has potent activity as a chemotactic agent and as an activator of eosinophils, which are prominent cells in asthmatic airways, through the activation of specific surface receptors. The interaction between PAF and eosinophils may be crucial in the pathogenesis of bronchial hyperresponsiveness in asthma. A role for PAF in asthma can now be studied using the recently developed antagonists of the PAF receptor. 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 effect of inhibitors of platelet aggregation on the metabolism of platelet-activating factor (PAF) in washed rabbit platelets

The rabbit platelet metabolizes platelet-activating factor (PAF) intracellulary. PAF is deacetylated to produce lysoPAF which, in turn, can be acylated to produce 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl GPC). Some PAF receptor antagonists have been shown to inhibit this metabolic conversion. In the present study we examined whether the PAF receptor antagonists SRI 63-441 and WEB 2086 would inhibit the metabolism of PAF by intact rabbit platelets. In addition, we examined whether iloprost, a stable analogue of prostaglandin I₂ (PGI₂), and a potent inhibitor of platelet activation induced by a range of agonists, would also inhibit PAF metabolism. We found that SRI 63-441 and WEB 2086 caused an almost complete inhibition of the conversion of PAF to alkylacyl GPC. Iloprost caused up to a 50% inhibition of PAF metabolism compared to antagonist-free controls. Iloprost (and PGI₂) is thought to inhibit platelet response by elevation of cAMP, while receptor antagonists act by blocking PAF binding to its receptor. Since iloprost caused partial inhibition of PAF metabolism, the results of this study suggest that inhibition of PAF metabolism does not occur solely due to competitive inhibition of PAF binding to its receptor. 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 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).     

Significance of platelet-activating factor in mesenteric ischemia-reperfusion

Reperfusion of the ischemic mesenterium is frequently followed by acute circulatory collapse. This review focuses on the possible role of platelet-activating factor (PAF) in ischemia-induced damage. It provides evidence that (i) PAF concentrations are elevated in the mesenteric circulation following temporary ischemia; (ii) administration of exogenous PAF into the superior mesenteric vein mimics many events observed during reperfusion; and (iii) pretreatment of the experimental animals with specific PAF receptor antagonists prevent the circulatory collapse. These findings suggest that PAF may play an important role in the development of circulatory collapse caused by mesenteric ischemia-reperfusion. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

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.     

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.     

Involvement of platelet-activating factor in zymosan-induced rat pleurisy

The role of platelet-activating factor (PAF) in inflammatory reactions was studied in zymosan-induced rat pleurisy. Pleurisy was induced by injection of a 2% zymosan suspension into the pleural cavity of rats. The time course of pleural exudate accumulation, the exudation rate, and exudate leukocyte numbers were followed then for 96 hr. Peak pleural exudate accumulation was about 3 mL at 24 hr, whereas the exudation rate increased biphasically with peaks at 0.5 hr and 5 hr. The migration of leukocytes into the pleural cavity increased with time up to 48 hr. The polymorphonuclear leukocytes were the dominant white cells in the exudate between 5 and 16 hr, but mononuclear leukocytes started to outnumber them around 24 hr. Pretreatment with cyproheptadine (5 mg/kg), an inhibitor of both histamine and seotonin, significantly suppressed pleural fluid accumulation and the exudation rate at 0.5 hr. The PAF antagonist CV-6209 (1 mg/kg) significantly suppressed pleural fluid accumulation and the exudation rate at both 0.5 and 5 hr. At either time point, the parameters were not suppressed by indomethacin. We detected PAF activity in the high-performance liquid chromatography (HPLC) fraction (with a retention time corresponding to that of authentic PAF) of the exudates at 0.5 hr, 5 hr, and 16 hr using an aggregation bioassay with washed rabbit platelets. The results suggest that in zymosan-induced rat pleurisy, histamine and/or serotonin are the main mediators of exudation at 0.5 hr and that PAF may be partly responsible for exudation at 0.5 hr and later at 5 hr to 16 hr. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Bioactions of 5-hydroxyicosatetraenoate and its interaction with platelet-activating factor

In a variety of stimulated cells, platelet-activating factor (PAF) and numerous arachidonate derivatives are coproducts that form as a consequence of receptor-mediated phospholipid mobilization. These lipid co-products produce a plethora of biological effects in a wide variety of cell systems. Furthermore, they often have a fascinating, although less widely appreciated, interaction. 5-HETE, at submicromolar concentrations, exerts relatively few direct bioactions. It does, however, potently (16–160 nM) raise cytosolic free calcium [Ca²⁺]_i and augment PAF-induced responses in human polymorphonuclear neutrophils (PMN) by as much as 100- to 1000-fold. 5-HETE acts on PMN by a structurally specific, stereospecific and pertussis toxin-inhibitable mechanism. In addition, PMN exposed to 5-HETE exhibit homologous but not heterologous desensitization. These findings suggest that 5-HETE, like PAF, may bind to its own specific plasmalemmal receptors to exert its unique set of bioactions. However, further investigation is required to demonstrate any putative 5-HETE receptors. Other potential mechanisms of 5-HETE-induced bioactions together with the possible effects of 5-HETE on PAF transduction mechanisms are also discussed. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.