A New Platelet-Aggregation-Inhibiting Factor Isolated from Bothrops moojeni Snake Venom.

This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor from Bothrops moojeni snake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP). BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni snake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.

Steroidal saponins from Tribulus terrestris.

Sixteen steroidal saponins, including seven previously unreported compounds, were isolated from Tribulus terrestris. The structures of the saponins were established using 1D and 2D NMR spectroscopy, mass spectrometry, and chemical methods. They were identified as: 26-O-ß-d-glucopyranosyl-(25R)-furost-4-en-2α,3ß,22α,26-tetrol-12-one (terrestrinin C), 26-O-ß-d-glucopyranosyl-(25R)-furost-4-en-22α,26-diol-3,12-dione (terrestrinin D), 26-O-ß-d-glucopyranosyl-(25S)-furost-4-en-22α,26-diol-3,6,12-trione (terrestrinin E), 26-O-ß-d-glucopyranosyl-(25R)-5α-furostan-3ß,22α,26-triol-12-one (terrestrinin F), 26-O-ß-d-glucopyranosyl-(25R)-furost-4-en-12ß,22α,26-triol-3-one (terrestrinin G), 26-O-ß-d-glucopyranosyl-(1→6)-ß-d-glucopyranosyl-(25R)-furost-4-en-22α,26-diol-3,12-dione (terrestrinin H), and 24-O-ß-d-glucopyranosyl-(25S)-5α-spirostan-3ß,24ß-diol-12-one-3-O-ß-d-glucopyranosyl-(1→4)-ß-d-galactopyranoside (terrestrinin I). The isolated compounds were evaluated for their platelet aggregation activities. Three of the known saponins exhibited strong effects on the induction of platelet aggregation.

Platelet-activating factor changes in phospholipid extracts from the plasma, peripheral blood mononuclear cells and bone marrow mononuclear cells of patients with acute leukemia--a 31P MRS in vitro study.

The aim of this investigation was to evaluate the changes in PAF concentrations in the plasma, PBMC and BMMC of patients with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The plasma was from 23 healthy volunteers (HV) and 44 patients with AL (16 ALL, 28 AML). The PBMC were from 15 HV and 55 patients with AL (18 ALL, 37 AML), and the BMMC from 40 patients with AL (11 ALL, 29 AML). Methanol-chloroform phospholipid extraction from 60 x 10(6) cells (PBMC or BMMC) was performed according to a modified version of Folch's method. (31)P MRS data was obtained on an AMX 300 Bruker spectrometer (7.05 T). The PAF concentration in the plasma of the patients with ALL or AML was lower than that for the healthy volunteers. The PAF concentration in the plasma of the patients with ALL did not differ significantly from that of the patients with AML. In the case of both the PBMC and BMMC, the PAF concentration was significantly diminished in patients with ALL relative to the concentration for those with AML and for the healthy volunteers. No differences were observed in the PAF concentrations for the AML patients and the healthy volunteers.

Platelet-activating factor.

Platelet-activating factor (PAF) is a potent mediator that occurs at very low concentrations in cells and tissues. Accurate quantitation of PAF has always been difficult because of the physicochemical properties of PAF and its structural similarity to several much more abundant phospholipids. Numerous assays for PAF have been developed, all of which have their strengths and limitations. Herein, this chapter describes a high-pressure liquid chromatography (HPLC)-tandem mass spectrometry assay for PAF. Major strengths of the method are its sensitivity (detection limit = 1 pg) and selectivity. Another advantage is that, by using liquid instead of gas chromatography, sample derivatization is avoided. The limitations of the method are its use of expensive instrumentation and the requirement of performing two HPLC runs per sample. Detailed technical advice on application of the method to various types of samples is given.

Movement of monoglyceride derived from hydrolysis of fluorescence-labeled lyso platelet-activating factor by lysophospholipase C through plasma membranes of porcine kidney epithelial cell line LLC-PK1.

To investigate the mechanisms of the release of lyso platelet-activating factor (PAF), an alkyl ether-linked lysophosphatidylcholine, from the kidney epithelial cell line LLC-PK1, the cell monolayer was incubated with a fluorescence-labeled lysoPAF analog, Bodipy-lysoPAF, on either the basolateral or apical side. The fluorescent lipids in the culture media mixed with or without bovine serum albumin at a final concentration of 2% were analyzed by thin layer chromatography. In both cases, two major bands, assignable to Bodipy-lysoPAF and Bodipy-monoglyceride (MG), were detected in the culture medium to which Bodipy-lysoPAF had been added, whereas the culture medium at the opposite side exhibited only the major band of Bodipy-MG. Our results suggest that lysoPAF was degraded by high ecto-lysophospholipase C activity. The possible physiological significance of this metabolic pathway is discussed.

Isolation and identification of hydroxyl-platelet-activating factor from natural sources.

Platelet-activating factor (PAF), a potent inflammatory mediator that has previously been detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid (GCF) and in saliva, is implicated in periodontal disease. The biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl-PAF analogue. In a preliminary study this bioactive molecule was detected for the first time in human blood derived from volunteers with chronic periodontitis as well as from periodontally healthy volunteers. Compounds isolated from natural sources as well as synthetic ones have been reported as biologically active lipids with physiological importance based on the fact that they induce platelet aggregation with EC50 values ranging from 100 to 0.01 microM through interaction with G-protein-coupled receptors like the PAF receptor, leading to altered signal transduction. In this study, the existence of hydroxyl-PAF analogue in human blood was further studied as well as its distribution in plasma and in blood components. The existence of hydroxyl-PAF analogue was also investigated in samples from rabbit blood hen's egg yolk. The hydroxyl-PAF analogue was purified by high-performance liquid chromatography, detected by biological assays and identified by electrospray MS analysis. Quantitative determination of PAF and hydroxyl-PAF analogue (expressed as PAF-like activity) showed a statistically significant increase in the ratio of plasma hydroxyl-PAF analogue levels to plasma PAF levels in volunteers with periodontitis. Moreover, hydroxyl-PAF analogue was also detected in rabbit blood and hen's egg yolk samples. These data support that this bioactive lipid may play a role in oral inflammation and suggest PAF as a member of a lipid molecule family with different structures and from different sources which share the same or similar biological activities, apparently with different physiological roles in human and animals.

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom.

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA2, crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 microM/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA2, thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Non-enzymatic platelet-activating factor formation by acetylated proteins.

Substantial amounts of platelet-activating factor (PAF 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine), the potent phospholipid mediator of allergic and inflammatory reactions, are formed upon incubation of acetylated low-density lipoprotein, acetylated bovine serum albumin (BSA) and acetylated apolipoprotein A-I with 1-0-hexadecyl-sn-glycero-3-phosphocholine (lyso-PAF). Acetylated BSA produced 0.3 nmol PAF/mg of protein after a 6 h incubation period with 40 microM lyso-PAF. The transfer of acetate bound to acetylated proteins to lyso-PAF was non-enzymatic. Chemical PAF formation by acetylated proteins, involved in lipid metabolism and transport, could lead to complication of inflammatory and allergic events.

Supplementation with Saccharomyces boulardii ameliorates hypoxia/reoxygenation-induced necrotizing enterocolitis in young mice.

Intestinal bacterial proliferation is an important aspect of gastrointestinal injury in neonatal necrotizing enterocolitis (NEC). In the present investigation, we examined the protective action of oral supplementation with Saccharomyces boulardii (S. boulardii), non-pathogen probiotic yeast, against hypoxia-reoxygenation (H/R)-induced NEC in young mice. Young mice were divided into three groups; Group 1 mice (untreated) were subjected only to hypoxia-reoxygenation; Group 2 mice were subjected to hypoxia-reoxygenation and were then given lyophilized S. boulardii (10 mg suspended in 0.5 ml saline) twice a day by orogastric intubation for 10 days. Group 3 mice served as controls. Hypoxia was induced by placing young mice in a 100 % CO (2) chamber for 5 min. After hypoxia, the young mice were reoxygenated for 10 min with 100 % oxygen. We examined the intestinal lesions by light microscopy and measured intestinal generation of PAF and TNF-alpha in the H/R-induced model of NEC. In the probiotic group, NEC-induced intestinal tissue damage was greatly attenuated, with necrosis partially limited to the mucosa. Both intestinal tissue PAF and TNF-alpha concentrations were significantly higher in the untreated group than in controls (p < 0.001). S. boulardii-supplemented young mice showed a significant decrease in intestinal generation of PAF compared with untreated young mice (p < 0.05). On the other hand, no significant difference was observed in the intestinal concentration of TNF-alpha between untreated and probiotic groups ( p > 0.05). The present study suggests that hypoxia/reoxygenation plays an important role in the pathogenesis of NEC and supports hypothesis that especially PAF and TNF-alpha are involved in the pathophysiological mechanism of H/R-induced NEC. This study also demonstrates that dietary supplementation with S. boulardii ameliorates the histologic evidence of H/R-induced intestinal injury. Based on these findings, the beneficial effects of probiotic S. boulardii in this model of NEC are mediated via mechanisms inhibiting intestinal proinflammatory mediator release.

Additional bioactive Lyso-PAF congeners from the sponge Spirastrella abata.

A known (1) and four new (2--5) lyso-PAF (platelet activating factor) derivatives were isolated from the sponge Spirastrella abata. Two of them are unprecedented in having a methoxy group at C-2'. The structures have been determined by combined spectroscopic methods. Their inhibitory effect on the biosynthesis of cholesterol and cytotoxicity against human solid tumor cell lines are reported.

Lyso-PAF analogues and lysophosphatidylcholines from the marine sponge Spirastrella abata as inhibitors of cholesterol biosynthesis.

A series of phospholipids, including previously undescribed compounds 4-7, were isolated by a bioactivity-guided fractionation from the marine sponge Spirastrella abata as inhibitors of cholesterol biosynthesis in human liver cells. These compounds were identified as lyso-PAF analogues (1-5) and lysophosphatidylcholines (6, 7) based on NMR and MS analyses. Compounds 1-7 specifically blocked the conversion of lanosterol into cholesterol in the Chang liver cell.

Interleukin-12 is synthesized by mesangial cells and stimulates platelet-activating factor synthesis, cytoskeletal reorganization, and cell shape change.

Preliminary studies indicate the involvement of interleukin (IL)-12 in experimental renal pathology. In the present study, we evaluated whether cultured glomerular mesangial cells are able to produce IL-12 and whether IL-12 may regulate some of their functions, including the cytoskeletal reorganization, the change in cell shape, and the production of platelet-activating factor (PAF). The results obtained indicate that pro-inflammatory stimuli, such as tumor necrosis factor-alpha and bacterial polysaccharides, induce the expression of IL-12 mRNA and the synthesis of the protein by cultured mesangial cells. Moreover, cultured mesangial cells were shown to bind IL-12 and to express the human low-affinity IL-12 beta1-chain receptor. When challenged with IL-12, mesangial cells produced PAF in a dose- and time-dependent manner and superoxide anions. No production of tumor necrosis factor-alpha and IL-8 was observed. Moreover, we demonstrate that IL-12 induced a delayed and sustained shape change of mesangial cells that reached its maximum between 90 and 120 minutes of incubation. The changes in cell shape occurred concomitantly with cytoskeletal rearrangements and may be consistent with cell contraction. As IL-12-dependent shape change of mesangial cells was concomitant with the synthesis of PAF, which is known to promote mesangial cell contraction, we investigated the role of PAF using two chemically different PAF receptor antagonists. Both antagonists inhibited almost completely the cell shape change induced by IL-12, whereas they were ineffective on angiotensin-II-induced cell shape change. In conclusion, our results suggest that mesangial cells can either produce IL-12 or be stimulated by this cytokine to synthesize PAF and to undergo shape changes compatible with cell contraction.

Platelet-activating factor (PAF) and PAF acetylhydrolase activity in rat uterus and placenta during the late stages of pregnancy.

We evaluated the roles of platelet-activating factor (PAF) and PAF-acetylhydrolase (PAF-AH) activity in late pregnancy. Uterine and placental concentrations of PAF were determined by the washed rabbit platelet aggregation bioassay. Uterine, placental, and plasma PAF-AH activities were also assayed. PAF concentration in the uterus increased 4-fold between Days 15 and 21 of pregnancy. PAF was also determined in the placenta on Days 15 and 21. In contrast to findings in the uterus, the concentration in the placenta was decreased by 75%. Platelet aggregation caused by uterine and placental PAF was inhibited by the PAF receptor antagonists CV-3988 and TCV-309. Plasma and uterine PAF-AH activities decreased significantly between Days 15 and 21. In contrast, the placental PAF-AH activity significantly increased during this same time period. On the basis of these findings, it is suggested that the PAF concentration in the uterus and placenta may be regulated by intracellular PAF-AH and/or plasma PAF-AH activities. Increased PAF activity in the pregnant rat uterus may be related to the initiation of labor due to its known effect on myometrial contraction. Decreased PAF concentration in the placenta may contribute to the fetoplacental circulation due to its known hypotensive activity and the increase in vascular permeability.

Purification and partial characterization of a novel human platelet aggregation factor in the extracellular products of Streptococcus mitis, strain Nm-65.

A human blood platelet aggregation factor was purified from the extracellular products (ECP) of Streptococcus mitis, strain Nm-65 by sequential chromatography on DEAE-Sepharose CL-6B, hydroxyapatite and Superdex 75 columns. The purified factor (S. mitis-derived human platelet aggregation factor, Sm-hPAF) gave a single band with a molecular weight of 66 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Sm-hPAF showed a peak absorption at 278 nm and an isoelectric point of around 8.5. Chemical analyses revealed that Sm-hPAF contained no sugars and that its first 15 amino-terminal amino acid residues were H-DEQGNRPVETENIAR. Platelet aggregation activity of Sm-hPAF was abolished by heating at 45 degrees C for 10 min. Platelet aggregation by Sm-hPAF was accompanied by a release of prostaglandin E2 (PGE2) in a dose-dependent manner. The platelet aggregation was not inhibited by either prostaglandin E1 (PGE1) or Gly-Arg-Gly-Asp-Ser (GRGDS), that inhibit the platelet aggregation induced by collagen. Twenty (77%) platelet rich-plasma (PRP) specimens derived from 26 healthy volunteers were aggregated by Sm-hPAF, but the remaining 6 (23%) were not reactive. A preliminary study suggested the presence of an inhibitory factor against Sm-hPAF in the plasma from a non-reactive donor.

Endotoxin induces PAF production in the rat ileum: quantitation of tissue PAF by an improved method.

PAF (platelet-activating factor) is an endogenous mediator of endotoxin (LPS) shock and intestinal injury. In the present study we used an improved method to quantitate intestinal PAF after LPS injection. Both column and thin layer chromatography (TLC) were used to purify PAF. We found that using C18 column eluted sequentially with 10% acetic acid, ethyl acetate and 70% ethanol, yielded consistent results. TLC yielded falsely high PAF values, possibly from an unknown tissue lipid which co-migrated with PAF, or from toxic ingredients in the silica gel. Moreover, addition of optimal amounts of Tween-20 or ethanol in the bioassay samples enhanced PAF solubility and markedly improved PAF recovery. Lastly, dilution and heparinization of platelet-rich plasma greatly improved the sensitivity of the bioassay. The overall PAF recovery under these optimal conditions was 70-80%. We found that LPS (2-10 mg/kg, iv, 90 min) stimulated PAF production in the rat ileum, but not in the jejunum and colon. The difference in PAF production did not correlate to the numbers of sequestered neutrophils (reflected by myeloperoxidase levels) after LPS injection. This selective PAF production may account for the special vulnerability of the ileum to develop injury during endotoxemia.

[The measurement of PAF (platelet activating factor) in human saliva: standardization of the method]. / Misurazione del PAF (fattore attivante le piastrine) nella saliva umana: standardizzazione del metodo.

The evaluation of the salivary PAF has possible by using the radio immuno assay method (RIA). We wanted to study the presence of such substance in the saliva under physiological conditions and particularly in relation to possible existence of a circadian rhythm or periodical oscillations. The work has been developed in two phases. In the first one we evaluated the daily salivary PAF amount while in the second phase of the study we verified the existence of a possible circadian rhythm. The results encouraged us to extend the study to the typical, different physiological aspects of such phospholipid having as objective the control of the salivary PAF amount in pathological conditions.

Platelet activating factor and lyso-phosphatidylcholines from strawberry.

Lipids from strawberry fruits, leaves, achenes and pollen were separated into classes by TLC, purified by HPLC and tested for biological activity. A lipid fraction from fruits with the same chromatographic behaviour as authentic platelet activating factor (PAF) showed identical biological activity, namely, dose-dependent aggregation of washed rabbit platelets, inhibition of aggregation by CV 3988, platelet desensitization to PAF and vice versa, and loss of activity by alkaline hydrolysis and recovery of activity by reacetylation. The presence of PAF was confirmed by FAB mass spectrometry. Lyso-phosphatidylcholines, including lyso-PAF, were also found in all the plant parts tested.

Evidence for the production of platelet-activating factor by murine embryos and its putative role in the maternal physiology.

Thrombocytopenia has been shown to be an initial maternal response to conception. We investigated a potent bioactive lipid, platelet-activating factor (PAF), as one of the candidates derived from embryos to induce the decrease of platelet counts in maternal blood. First, we examined the potential of murine embryos to liberate PAF. In brief, twenty to thirty murine two-cell embryos of C57BL/6 mice were cultured in Whitten's medium for 24 h. The supernatant of the medium was collected and the total was extracted. After developing the lipid on thin-layer chromatography, the area corresponding to authentic PAF was scraped off and the lipid was extracted. The embryo-derived PAF was quantified by platelet aggregation using rabbit whole blood, and the presence of PAF was further confirmed by the addition of a PAF-receptor antagonist, SRI63-441. The results showed that a significant amount of PAF (ca. 10 ng/ml) was recovered from the lipid extract, whereas little platelet-aggregating activity was observed in the presence of SRI63-441. Second, the physiological function of murine platelets was examined using authentic PAF. When PAF (up to 83 micrograms/ml) was added to the murine platelets suspended in the whole blood, it neither induced platelet aggregation nor reduced the number of platelets. Considering these results, it is considered that PAF released from the embryos does not directly act on maternal platelets, but transduces its signal to the maternal host via some other bioactive substance.

Quantitation of platelet-activating factor in biological samples using liquid chromatography/mass spectrometry with column-switching technique.

A procedure is described for the fully automated quantitative determination of platelet-activating factor (PAF) using liquid chromatography/fast atom bombardment mass spectrometry (LC/FAB-MS) with two types of column-switching techniques. Various parameters in LC/FAB-MS were optimized, and the use of a microbore column allowed highly selective detection of PAF. A column-switching system was incorporated to minimize band broadening and to increase the permissible injection volume. Analysis of authentic PAF indicated that the limit of quantitation was at the low picogram level (about 50 pg/ml). Another system using both a strong cation-exchange column and an ODS column in tandem was developed for the analysis of PAF in blood samples. These methods were validated with standard samples and applied to the determination of PAF in human polymorphonuclear neutrophils stimulated by addition of a calcium ionophore and in human blood.

Platelet-activating factor production by human fetal microglia. Effect of lipopolysaccharides and tumor necrosis factor-alpha.

Since platelet-activating factor (PAF) exerts neurotoxic effects on brain cells, we explored the possibility of PAF production by human fetal microglial cells in vitro. PAF content in pure cultures was assayed and characterized in basic conditions, and after stimulation with growth factors and cytokines. Results showed that microglia cells synthesized PAF when challenged with tumor necrosis factor-alpha and lipopolysaccharides, whereas other molecules, such as gamma-interferon or basic fibroblast growth factor, were ineffective. The induced PAF production was concentration- and time-dependent. These results are in line with the hypothesis that microglia can start a cascade of events leading to tissue damage, thus playing a central role in the pathogenesis of several central nervous system diseases.