In vivo metabolism of platelet-activating-factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by the protozoan Tetrahymena pyriformis.

The metabolism of exogenous platelet-activating-factor was studied in the protozoan Tetrahymena pyriformis in vivo. When the cells are exposed to 1.10(-6) M PAF, the molecule is rapidly metabolized to 1-O-alkyl-2-acyl(long chain)-GPC, a major component of the protozoan membranes. The appearance of lyso-PAF from the first minutes even in low levels provides evidence that deacetylation is an intermediate step. After incubation for 30 min, transformation to aminoethyl phosphonolipids is also observed. The fate of PAF in concentrations 1.5.10(-11) M or 1.10(-8) M PAF, was the same. An amount of PAF depending on the external PAF concentration remained intact in the cell even after 1 h incubation. Our results suggest that the easily cultured protozoan can be a useful model for studying PAF's metabolism.

Studies on the subcellular distribution of 1-O-alkyl-2-acetyl-sn-glycero phosphocholine (PAF) and on the enzymic activities involved in its biosynthesis within the ciliate Tetrahymena pyriformis.

The ciliated protozoan Tetrahymena pyriformis contains platelet-activating factor (PAF) as a physiological minor lipid. Its subcellular localization was found as follows: 13.7% in the pellicles, 24.9% in mitochondria, 56.5% in microsomes and 7.1% in the cytosol. Succinate dehydrogenase was used as marker enzyme. PAF remains cell-associated unless bovine serum albumin is included in the extracellular medium. In this case 15% of total PAF, portion comparable to that found in the pellicles, is released. Investigation of the principal enzymic activities involved in PAF formation showed that PAF-acetyltransferase (2.3.167) is totally absent from the protozoan. This means that the 'remodelling' pathway occurring in pro-inflammatory cells does not contribute in PAF formation in our system. A dithiothreitol (DTT)-insensitive CDPcholine phosphocholinetransferase activity involved in PAF biosynthesis is shown for the first time to be responsible for PAF production in T. pyriformis. It uses exogenous alkyl-acetyl-glycerol as substrate and is saturated over substrate concentration 250 microM. It can also use endogenous lipids as substrate. It is distributed mainly in mitochondria and microsomes, much less is found in the pellicles and it is totally absent from the cytosol. Its insensitivity to DTT, its selectivity to alkyl-acetyl-G and its different distribution compared to the enzymic activity involved in PC formation (EC 2.7.8.2) suggest that a different enzyme, specific for PAF formation (EC2.7.8.16) via the de novo pathway exists in the protozoan.

Platelet-activating factor formation during oxidative modification of low-density lipoprotein when PAF-acetylhydrolase has been inactivated.

A PAF aggregating activity corresponding to 427 +/- 91, 668 +/- 111 and 1319 +/- 217 pg/mg protein was detected when LDL was preincubated at pH 3.5 or with 4 mM PMSF or both for 30 min (treatments that inactivate PAF-AH) and then oxidized with 20 microM Cu2+ at 37 degrees C for 24 h. This molecule was characterized as PAF by its chromatographic behavior on TLC and other established methods and was further characterized as 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16: PAF) by its retention time on reverse phase HPLC and by fast atom bombardment-mass spectroscopy. Native LDL incubated under non oxidizing conditions, even when PAF-AH has been inactivated, or oxidized in the absence of PAF-AH inactivating agents or after pretreatment with 0.5 mM pBPB, does not produce detectable amounts of PAF. The kinetics of PAF formation in relation to PAF-AH activity, show that the apparent rate of PAF formation as well as its total amount depends on both the existence of oxidative conditions and the remaining PAF-AH activity the first hours following the onset of oxidation. Peroxidation of the phosphatidylcholine (PC) content of native LDL produces PAF-like aggregating activity much lower than that produced when intact LDL is oxidized and is not inhibited by BN 52021 as effectively as PAF produced by LDL peroxidation. Our results provide evidence that C16: PAF is formed during LDL peroxidation when PAF-AH has been inactivated and it does not result as a product of peroxidation of the LDL-PC content.

Highly constrained cyclic (S,S) -CXaaC- peptides as inhibitors of fibrinogen binding to platelets.

The Arg-Gly-Asp RGD motif of adhesive proteins is recognized by the activated platelet integrin alpha(IIb)beta3. Binding of fibrinogen (Fg) to activated alpha(IIb)beta3 causes platelet aggregation and thrombus formation. Highly constraint cyclic (S,S) -CXaaC- containing peptides incorporating the (S,S) -CDC- and (S,S) -CRC- motifs were tested for their ability to inhibit platelet aggregation and Fg binding. Our results suggest that the above cyclic scaffolds stabilize a favorable structure for the antiaggregatory activity (IC50-values ranged from 1.7 to 570 microm). The peptides inhibited Fg binding with IC50-values up to 30-fold lower than those determined for the inhibition of the adenosine diphosphate (ADP)-induced platelet aggregation. Importantly, peptides (S,S) PSRCDCR-NH2 (peptide 11) and (S,S) PRCDCK-NH2 (peptide 10) did not inhibit PAC-1 binding to the activated platelets at a concentration in which they completely inhibited Fg binding. Moreover, (S,S) PSRCDCR-NH(2) (peptide 11), one of the more active peptides, inhibited ADP-induced P-selectin exposure. By contrast, peptide (S,S) Ac-RWDCRC-NH2, incorporating the inverse (S,S) -DCRC- sequence (peptide 16), failed to inhibit P-selectin exposure whereas at the same concentration, it effectively inhibited PAC-1 and Fg binding. It is concluded that peptides containing the (S,S) -CDC- as well the (S,S) -CRC- sequences, exhibit a broad range of activities toward platelets, and could be helpful tools for elucidating the structural interaction of Fg with the integrin receptor alpha(IIb)beta3, in its activated form. Furthermore, the (S,S) -RCDC- sequence can be used as a scaffold for developing potent non-RGD-like Fg-binding inhibitors.

Platelet aggregatory response to platelet activating factor (PAF), ex vivo, and PAF-acetylhydrolase activity in patients with unstable angina: effect of c7E3 Fab (abciximab) therapy.

OBJECTIVE: Platelet activation and aggregation is a dominant feature in the pathophysiology of unstable angina. The final step of platelet aggregation is mediated through the platelet integrin glycoprotein IIb/IIIa (GP IIb/IIIa), while abciximab (ReoPro) is one of the most potent inhibitors of this receptor. Platelet-activating factor (PAF) is a potent platelet agonist which is degraded and inactivated by PAF-acetylhydrolase (PAF-AH). The plasma form of PAF-AH is associated with lipoproteins. We studied the platelet response to the aggregatory effect of PAF, ex vivo, in relation to the plasma PAF-AH activity in 32 patients with unstable angina, as well as the effect of abciximab therapy on the above parameters. METHODS: Thirty two patients with unstable angina and 25 sex- and age-matched healthy controls participated in the study. On the day of admission (day 1) 17 patients received a bolus of abciximab (0.25 mg/kg) followed by a 12-h infusion (10 micrograms/min). Platelet aggregation to both PAF and ADP, in platelet rich plasma, was successively studied in both patients receiving abciximab or remaining untreated. The plasma and HDL-associated PAF-AH activity was also determined at the same times. RESULTS: In the untreated patients, the PAF EC50 values were significantly lower on the day of admission, whereas the maximal percentage of aggregation was significantly higher compared to controls (p < 0.01 for both comparisons). Similar behaviour of the platelets was observed in the aggregatory effect of ADP. This aggregatory response was not significantly altered 4 days, 7 days or 1 month afterwards. In the 17 patients who received abciximab, platelet aggregation to both PAF and ADP was inhibited by 90 +/- 5 and 96 +/- 3%, respectively, 1 h after bolus. At 2 and 3 days after treatment, platelet aggregation to both agonists was significantly recovered being similar to controls. However, it was fully restored 6 days after bolus, still being significantly higher compared to controls (p < 0.01 for PAF and p < 0.003 for ADP). The total plasma PAF-AH activity in both patient groups was not different from that of controls, whereas the HDL-associated PAF-AH activity was significantly lower. The total plasma or HDL-associated enzyme activity was not altered at any time interval studied, and it was not influenced by abciximab. CONCLUSIONS: The increased aggregatory response of platelets to PAF and the low plasma levels of HDL-cholesterol and HDL-associated PAF-AH activity in patients with unstable angina may contribute to the severe atherosclerosis and to acute thrombosis found in these patients. Abciximab therapy may protect platelets from PAF action in vivo the first days after drug administration, but it fails to permanently restore the enhanced aggregatory response observed.

A PAF-acetylhydrolase activity in Tetrahymena pyriformis cells.

Our study provides evidence for the existence of an acylhydrolase activity in Tetrahymena pyriformis cells, capable of hydrolyzing the sn-2 ester bond of the PAF molecule. This activity is mainly distributed in the microsomal fraction (76.5% of total) and has properties similar to the mammalian PAF-acetylhydrolase since it is Ca(2+)-independent, acid-labile, is inhibited by DFP and PMSF but it is not affected by egg yolk phosphatidylcholine. This microsomal acylhydrolase has apparent Km and Vmax values of 1.56 microM and 373 pmols.mg.min respectively. This is the first report of the existence of a PAF-acetylhydrolase activity in a non-mammalian cell.

Placenta-specific growth factor production by splenic cells during pregnancy.

Within the last 5 years strong evidence has correlated the successful outcome of pregnancy with various cytokines, which interfere with sperm mobility, fertilization, implantation, trophoblast outgrowth, as well as maternal immunoregulation. The newly arising antigens on the extra-embryonic membranes initiate many mechanisms protective to the fetus and not harmful to the mother, one of which is novel protein synthesis. These events are apparent in many different sites of the maternal organism including the decidual cap, uterine walls, draining lymph nodes, spleen etc. Working on a murine model, in the present study we concentrated on the growth factor production by spleen cells isolated from syngeneically pregnant mice on the 11th day of gestation. Focusing our interest on the proteins that have a stimulatory effect on placental cells, we fractionated 24 h spleen cell supernatants through a G-25 Sephadex followed by a Heparin-Sepharose affinity column and isolated pregnancy specific growth factors capable of inducing placental cell proliferation. In this study we focused on three growth factors, interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF) and colony stimulating factor-1 (CSF-1), which have been previously shown to play an important role in placental growth. CSF-1 and IL-3 were detected in single Heparin-Sepharose fractions, whereas GM-CSF was found dispersed in essentially three fractions. Although we were able to detect these three growth factors in specific affinity column fractions, other proteins, which we have not yet characterized, showed significant biologic activity. Such biologic activity could not be detected from non-pregnant spleen cell supernatants similarly fractionated.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation, chemical characterization, and subcellular distribution of 1-O-alkyl-2-acetyl-sn-glycerol in Tetrahymena pyriformis cells.

1-O-Hexadecyl-2-acetyl-sn-glycerol, the immediate precursor of platelet- activating factor (PAF) in its de novo formation, was detected in the protozoon Tetrahymena pyriformis. It was purified from the total lipid extract by TLC, after successive developments in two different solvent systems. Characterization was assessed by (a) gas-liquid chromatography with electron capture detection, and (b) gas chromatography combined with mass spectrometry in selected ion monitoring mode, after derivatization with heptafluorobutyric acid anhydride and tert-butyldimethylchlorosilane/imidazole, respectively. Its quantity was found to be 0.1 nmol/10(7) cells from the GC-MS, using authentic alkylacetylglycerol as external standard. Cell fractionation revealed that alkylacetylglycerol is located exclusively in the microsomal fraction of the protozoon. Previously, we have reported the occurrence of PAF in the microsomal fraction, as well as a dithiothreitol-insensitive CDP-choline: cholinephosphotransferase activity that utilizes exogenous alkylacetylglycerol as substrate in the mitochondrial and microsomal fractions. The above findings indicate that PAF can be formed in the cell by the de novo pathway.

Effect of ethanol on the sterols of the fission yeast Schizosaccharomyces pombe.

Ergosterol, lanosterol and two further unidentified sterols were detected and quantified in Schizosaccharomyces pombe cell extracts. In cells grown under anaerobic conditions, the levels of these sterols were dramatically reduced with a concomitant increase of their squalene precursor as compared with cells growing under aerobic conditions. Presence of ethanol resulted in a decrease in the sterol content under aerobic conditions. On the contrary, under anaerobic conditions presence of ethanol resulted in a three-fold increase of total sterols. Lanosterol was the main constituent of this elevation. It is suggested that lanosterol in parallel with unsaturated fatty acids is responsible for maintaining membrane integrity of S. pombe cells growing in the presence of ethanol.

LDL subfractions in patients with myocardial infarction: effect of smoking and beta-blocker treatment.

The aim of the present case-control study was to estimate, by density gradient ultracentrifugation, LDL heterogeneity in myocardial infarction, and to evaluate the effect of smoking and beta-blocker treatment on LDL subfraction profile. Our results show that patients who survive myocardial infarction have an abundance of small, dense LDL in their plasma, compared with controls. Patients who were on beta-blockers and those who smoked showed a more atherogenic LDL subfraction profile than the rest. In patients on beta-blocker treatment, the proportion of LDL3 was positively correlated with triglyceride concentration and body mass index. Dense LDL predominates in patients irrespective of smoking or beta-blocker treatment. The relative risk, calculated by logistic regression as the odds ratio of high LDL3, was 7.5 (95% confidence interval 2.5-22.1) and was not significantly influenced when smoking, beta-blocker treatment, triglycerides or the other parameters of the study were included in the statistical model.

Involvement of phospholipids in apolipoprotein B modification during low density lipoprotein oxidation.

An increased amount of phospholipids remained attached on delipidated apolipoprotein B originated from oxidized low density lipoprotein (LDL). 31P nuclear magnetic resonance analysis of such apolipoprotein showed an organic phosphorus peak at -0.55 ppm, which suggests the formation of adducts (most probably Schiff bases) of oxidized phospholipids with apolipoprotein B. The above reaction occurs in parallel with the hydrolysis of oxidized phospholipids, catalyzed by the LDL-attached platelet-activating factor acetylhydrolase, and may contribute to the proatherogenic effect of oxidatively modified low density lipoprotein.

Inhibition by cardiolipins of platelet-activating factor-induced rabbit platelet activation.

Evidence is presented that cardiolipin, a naturally occurring phospholipid, inhibits the aggregatory effect of platelet-activating factor (paf) on rabbit platelets in vitro. Bovine heart cardiolipin was shown to inhibit the aggregation of washed rabbit platelets induced by 1 x 10(-10) M and 2 x 10(-10) M paf with IC50 values (doses for half-maximal inhibition) of 8.4 +/- 0.8 x 10(-7) M and 2.6 +/- 0.6 x 10(-6) M, respectively. Phosphonocardiolipin was also able to inhibit platelet aggregation induced by 1 x 10(-10) M paf with an IC50 value of 3 +/- 1 x 10(-7) M. Both compounds, in concentrations up to 1 x 10(-5) M, were unable to aggregate washed rabbit platelets and failed to inhibit the aggregation induced by 0.9 and 1.8 microM adenosine diphosphate or 0.2-1.0 microM arachidonic acid. By contrast, the acetylated derivative of cardiolipin exerted an aggregatory effect on aspirin-treated rabbit platelets in the presence of creatine phosphate/creatine phosphokinase. This aggregation was inhibited by the specific paf antagonists BN 52021 and WEB 2086. Also, platelets treated with acetyl-cardiolipin were insensitive to the aggregatory effect of paf. Phosphatidic acid, phosphatidylglycerol, bis(dipalmitoylglycero)phosphate and their phosphono analogues were totally inactive. Similar data were obtained when platelet-rich plasma was used instead of washed rabbit platelets. Our results support the hypothesis that the effect of cardiolipin is mediated through specific paf receptors that act on the rabbit platelet membrane.

Semisynthetic preparation of 1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor).

1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor, PAF), (1.9 mumol) was prepared from the total lipid extract of the protozoan Tetrahymena pyriformis 9 x 10(7) cells. The procedure involved mild alkaline hydrolysis of the total lipids, followed by acetylation and purification of the product by preparative TLC and HPLC. The yield was 60% with respect to the content of 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine in the total lipids, determined after preparative TLC. The alkyl side chain of the semisynthetic PAF was composed of hexadecyl residue. Our product was identified as PAF according to its biological activity, the chromatographic behaviour on TLC and HPLC, the physicochemical properties and the behaviour under treatment with PLA2 and Lipase from Rhizopus arrhizus. The above procedure is proposed as a facile, inexpensive and convenient method.

Interferon-induced class II expression at the spongiotrophoblastic zone of the murine placenta is linked to fetal rejection and developmental abnormalities.

Type II interferon is known to induce a plethora of gene expression involved in the humoral and cellular immunity. One of the multiple sites of action of gamma-IFN is the fetoplacental unit, where its role has not yet been clearly defined. We have previously shown in vitro that gamma-IFN may induce expression of class II MHC antigens on the spongiotrophoblast layer of the murine placenta, which under physiological conditions is negative for these antigens. Indeed, the absence of class II antigens from the placenta could be part of a mechanism evoked by fetal tissues to escape a host vs. graft reaction. In the present study we show that intraperitoneal in vivo administration of low doses of recombinant gamma-IFN to pregnant females specifically induces class II antigens on the spongiotrophoblast zone, increases fetal abortion, causes retardation of eye development in the fetuses and decreases fetal weight. This treatment also affects the maternal pathology as we witness a prominent hypersplenism in the mother accompanied by low levels of haematocrit, elevated IgG production and decreased granulocytic and thrombocytic counts. These results are specifically linked to the pregnant state of the mother, since virgin females do not develop any of the above abnormalities. Our results not only point to a new dimension in gamma-IFN's role during pregnancy, but may be of clinical importance for prophylaxis since administration of gamma-IFN to a pregnant female may lead to abortion, fetal abnormalities or cause haematologic disorders to the mother.

A simple enzyme immunoassay for detecting brucellosis antibodies.

A simple enzyme immunoassay was developed and evaluated for serological diagnosis of brucellosis in 25 patients with various forms of brucellosis and 292 control patients with other conditions and disorders. All brucellosis patients gave a positive test with the initial sample. In 3 acute, febrile brucellosis patients with follow-up sera taken during therapy a sharp drop in specific antibody was noted. There was a less pronounced antibody reduction in 1 chronic and 2 relapse patients and an antibody increase in 1 chronic and 1 relapse case. All control samples gave negative results. In addition, the assay was evaluated as a screening test with 315 sera from 'healthy' individuals living in a brucellosis focus and representing 15% of that population. 11.5% (34/293) of subjects with no reported history of brucellosis and 45% (10/22) of cases treated in the past gave a positive test result. The agreement in those samples between the assay and the serum agglutination test was 95.5%.

Effect of ethanol on the phospholipid and fatty acid content of Schizosaccharomyces pombe membranes.

Ethanol at concentrations up to 5% (v/v) had no effect on the growth of Schizosaccharomyces pombe, whereas concentrations over 7.5% were inhibitory. The major membrane phospholipids in S. pombe cells growing aerobically in the absence of added ethanol were phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine. Oleic acid (18:1) was the main fatty acid. When ethanol (7.5%) was added to aerobically growing cultures, the phosphatidylinositol content increased, whereas the 18:1 content decreased. Similar changes were observed in the membrane phospholipids of cells grown anaerobically without ethanol. However, the presence of ethanol in anaerobically growing cultures had an opposite effect on fatty acids, as the 18:1 content increased. The results support the idea that ethanol tolerance in S. pombe may be connected with a high content of 18:1 fatty acids, and with the ability to maintain a high rate of phospholipid biosynthesis.

Electrocardiographic alterations induced by AGEPC in Wistar rats in relation to its hypotensive and hematologic effects.

AGEPC administration into Wistar rats caused no remarkable thrombocytopenia, slight decrease of the percent count of PMNs in whole blood accompanied by anequal leukocytopenia and a transient increase in hematocrit, due to fluid extraversion. Apart from the dramatic fall in blood pressure caused by AGEPC, relatively sinus bradycardia was recorded at doses over 6 micrograms/kg b.w. S-T segment elevation, mainly evident in II, III and AVF leads, was also recorded within the first minutes after AGEPC administration, at doses over 1 microgram/kg b.w. At lethal doses, various degrees of A-V block resulting in complete A-V block with idioventricular rhythm, or injury pattern resulting in ventricular fibrillation or ventricular flutter, were recorded. At sublethal doses no arrhythic manifestations were recorded, while S-T segment elevation upward inversion became gradually normal.

Study of the glycogenolytic action of platelet activating factor in Tetrahymena pyriformis.

1. A novel action of AGEPC on non-inflammatory cells was revealed, namely the ability to stimulate glycogenolysis in Tetrahymena pyriformis cells. 2. The glycogenolytic effect of AGEPC seems to be dependent on Ca2+ transport and regulation, thus the effects are completely inhibited by Verapamil and partially by EGTA. 3. The influence of Propranolol, Labetalol, Atenolol and Theophylline in the glycogenolytic effect of AGEPC are also studied. 4. Our findings suggest that the AGEPC promoted glycogenolysis in Tetrahymena through a mechanism distinct from that of catecholamines.

Platelet aggregability to platelet activating factor at rest and after exercise in patients with coronary artery disease.

The platelet response to the aggregatory effect of platelet-activating factor (PAF) in relation to blood PAF levels, serum PAF-acetylhydrolase (PAF-AH) activity and to their lipidaemic profile, was studied in 44 patients with coronary artery disease undergoing exercise tests. The PAF EC50 values in 21 patients with positive exercise test results were found to be significantly decreased at rest compared with 21 normal subjects (12.6 +/- 3.9 nM and 24.9 +/- 11.7 nM respectively) (P<0.0001). Moreover, the maximal percentage of aggregation to 50 nM PAF was found to be significantly increased (20.0 +/- 4.3% vs 13.5 +/- 3.6%, respectively) (P<0.0001). By contrast, the PAF EC50 values and the maximal percentage of aggregation in 23 patients with negative exercise test results were not statistically significantly different from the control group (25.2 +/- 11.4 nM and 14.1 +/- 4.7%, respectively). At the end of exercise, the PAF EC50 values and the maximal percentage of aggregation did not change in any group, and there were no significant differences in the whole-blood PAF levels either at rest or at the end of exercise. In patients with positive exercise test results, the PAF-AH activity at rest was significantly higher compared with the control group (37.2 +/- 8.0 nmol.ml(-1).min(-1) vs 32.4 nmol.ml(-1).min(-1), (P<0.03), whereas the enzyme activity did not differ in patients with negative exercise test results compared to controls (33.6 +/- 6.1 nmol.ml(-1).min(-1)). There was no change in PAF-AH activity during exercise in any group. The enzyme activity was positively correlated to the serum total and low density lipoprotein (LDL) cholesterol levels in the control group and in patients with negative exercise test results, whereas no correlation was found between PAF-AH activity and total or LDL cholesterol levels in patients with positive exercise test results.

Platelet response to the aggregatory effect of platelet activating factor (PAF) ex vivo in patients with acute myocardial infarction.

Platelets from patients with acute myocardial infarction exhibit an increased sensitivity to the aggregatory effect of PAF, in vitro, the first 48 h after the onset of the symptoms. This sensitivity, expressed as PAF EC50 values, seems to be transient after the 2 day period. Also, a remarkable decreased sensitivity to the inhibitory effect of PGI2 against the aggregation induced by PAF appears to the platelets of those patients the first hours after the onset of the symptoms, and persists for at least 14 days. Treatment of patients by drugs with a known inhibitory effect on platelet aggregation in vivo and in vitro (aspirin, nifedipine, indomethacin), does not influence the increase in platelet sensitivity to PAF, but inhibits the secondary aggregation induced by the released aggregating factors from the PAF activated platelets. The increase in platelet sensitivity to PAF is not unique to the AMI since it is also observed in patients with acute bacterial pneumonia. However, we cannot support the theory that it is a general phenomenon of acute tissue injury since it is general phenomenon of acute tissue injury since it is not observed in patients with acute muscular injury.