Platelet-activating Factor Content Research Articles

Sensitive densitometry for the determination of platelet-activating factor and other phospholipids in human tears.

A sensitive TLC method is reported for the determination of the platelet-activating factor (PAF) and other phospholipids in human tears. Tear samples absorbed on filter-paper were subjected to diatomite column extraction with chloroform-methanol. The eluent containing phospholipids was spotted on a silica gel plate. After removing lipids other than phospholipids by pre-development with hexane-diethyl ether (4 + 1 v/v), individual phospholipid separation was carried out by development with chloroform-methanol-water (65 + 35 + 7 v/v). Phospholipase C and then alkaline phosphatase solutions were sprayed on the TLC plate at 45 degrees C to liberate phosphate from each phospholipid. By spray application of a mixture of ammonium molybdate and Malachite Green, the liberated phosphates appeared as blue-green spots of molybdophosphate-Malachite Green aggregate on a yellow-brown background. The absorbance of each spot on the plate was measured at 620 nm with a densitometer. The peak area was found to be linearly related to PAF content in the range 2-100 pmol per spot, the RSD being 2% (n = 7). Approximately 86% of standard PAF added to tears was recovered. By this method, lysophosphatidylcholine (lysoPC), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human tears could also be detected with high sensitivity. Each phospholipid in healthy human tears showed a nearly constant content; PAF, lysoPC, PC and PE were present at levels of 26.2, 42.3, 10.0 and 19.7%, respectively.

156 Radiotherapy for head and neck malignancies is associated with increased salivary platelet-activating factor content

156 Radiotherapy for head and neck malignancies is associated with increased salivary platelet-activating factor content

Oxidative stress in gastric mucosal injury: role of platelet-activating factor-activated granulocytes.

Temporal and spatial changes due to oxidative stress in the rat gastric mucosa were visualized and quantified during the process of mucosal hemorrhagic change. The fluorescence associated with dichlorofluorescein (DCF), a hydroperoxide-sensitive fluorochrome, increased 30 min after repeated electrical stimuli to the gastric artery. The increase in the fluorescence was enhanced in the area between two adjacent collecting venules. The content of platelet-activating factor (PAF), the activity of myeloperoxidase (MPO) in the gastric mucosa, the area of mucosal lesions, and the luminol-dependent chemiluminescence activity in zymosan-treated blood samples, obtained from the gastric vein, were measured and found to increase significantly 30 min after the stimuli. The intravenous injection of CV-6209, a PAF antagonist, 5 min prior to the stimuli significantly inhibited the DCF activation, the increases in PAF level and MPO activity, the mucosal hemorrhagic change, and the elevation in chemiluminescence activity. In addition, continuous infusion of superoxide dismutase also inhibited all these changes, except for chemiluminescence activity. These results suggest that oxygen radicals derived from PAF-activated granulocytes induce oxidative stress, and that oxidative changes are actually implicated in the pathogenesis of gastric mucosal injury.

Enhancement by cyclo-oxygenase inhibitors of platelet-activating factor production in thapsigargin-stimulated macrophages.

1. Thapsigargin stimulated the accumulation of cell-associated platelet-activating factor (PAF) and extracellular prostaglandin E2 (PGE2) in rat peritoneal macrophages. PAF in the conditioned medium was less than the detectable amount. To obtain further insight into the mechanism of PAF accumulation, the role of PGE2 in PAF accumulation was investigated. 2. When macrophages were incubated in medium containing thapsigargin (30 ng ml(-1), 46.1 nM) and cyclo-oxygenase inhibitors such as indomethacin, naproxen or ibuprofen, the PAF content of the cells at 10 min was increased in a concentration-dependent manner in accordance with inhibition of PGE2 production. The stimulation by thapsigargin, cyclo-oxygenase inhibitors did not increase PAF accumulation. 3. In thapsigargin-stimulated macrophages, when PGE2(10(-7) M) was added to the medium, the cyclo-oxygenase inhibitor-induced stimulation of PAF accumulation at 10 min was markedly inhibited. 4. The accumulation of PAF induced by thapsigargin alone at 10 min was inhibited by exogenous PGE2 (10(-8) and 10(-7) M), or arachidonic acid (10(-6) and 10(-5) M) in accordance with the increase in PGE2 production. 5. The accumulation of PAF induced by thapsigargin alone or by thapsigargin and indomethacin (10(-6) M) was inhibited by dibutyryl cyclic AMP. 6. These results indicate that the concurrently produced PGE2 in thapsigargin-stimulated macrophages down-regulates PAF accumulation by increasing intracellular cyclic AMP levels, and that cyclo-oxygenase inhibitors increase PAF accumulation by inhibiting PGE2 production.

Inhibitions of platelet-activating factor production and granulocyte oxidative activation by Rikkunshi-To in the process of gastric mucosal injury

Abstract The effects of Rikkunshi-To on gastric mucosal injury were investigated with attention to the mechanisms of oxidative stress. Repeated electrical stimulation (RES) on the gastric artery of male Wistar rats decreased mucosal blood flow, which was measured by laser Doppler flowmetry, and subsequently produced hemorrhagic lesions in the gastric mucosa. At 30 min after the completion of RES, increased platelet-activating factor (PAF) content and myeloperoxidase (MPO) activity were observed in the gastric mucosa. Luminol-dependent chemiluminescence activity in blood samples taken from a gastric vein was also elevated after the stimulation. Oral administration of Rikkunshi-To (50 mg/kg) attenuated the increases in luminol-dependent chemiluminescence activity, mucosal PAF content and MPO activity, and the decrease in mucosal blood flow, and prevented mucosal injury induced by the electrical stimulation. These results support the concept that PAF is involved in granulocyte-mediated oxidative stress and gastric mucosal injury, and suggest that Rikkunshi-To prevents mucosal microvascular injury and formation of hemorrhagic lesion by modulating PAF production and oxidative granulocyte activation.

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.

Verotoxin induces hemorrhagic lesions in rat small intestine. Temporal alteration of vasoactive substances.

E. coli O157:H7 produces a cytotoxin active against Vero cells that has been termed verotoxin. In this study, we demonstrated that local intraarterial injection of verotoxin induced a decrease in blood flow and an increase in hemorrhagic lesions in rat small intestine. Significant increases in the area of hemorrhagic lesions were observed at 120 min after verotoxin injection. These lesions were produced by either verotoxin 1 or 2, but verotoxin 2 produced more extensive lesions. The temporal alteration of vasoactive substances in microcirculatory beds was determined after the administration of culture filtrate of E. coli O157:H7. Tissue-type plasminogen activator activity in regional plasma was significantly elevated as early as 30 min, suggesting that local fibrinolytic activation mediated by microvascular endothelium occurred. There was also early elevation of platelet-activating factor content in the ileal mucosa and its level remained significantly elevated thereafter. Intestinal blood flow, as determined by a laser Doppler flowmeter, started to decrease at about 45 min. The platelet-activating factor antagonist CV6209 was shown to attenuate the decrease in blood flow as well as the development of hemorrhagic lesions, demonstrating that platelet-activating factor is an important mediator for the microcirculatory damage. Accumulation of neutrophils demonstrated by myeloperoxidase activity in the intestinal mucosa and overproduction of oxygen-radicals from neutrophils of the mesenteric veins determined by the luminol-dependent chemiluminescence assay were observed at 60 min, corresponding with the decreased blood flow. Platelet-activating factor may be closely involved in the process of leukocyte accumulation and increased oxygen radical generation, because CV6209 also significantly attenuated these changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiulcer effect of flavonoids. Role of endogenous PAF

AbstractQuercetin, rutin and kaempferol, administered intraperitoneally (25–100 mg/kg) inhibited dose‐dependently gastric damage produced by acidified ethanol in rats. Flavone was inactive while naringin was active at higher dose (200 mg/kg). The flavonols tested inhibited dose‐dependently the mucosal content of platelet‐activating factor (PAF) suggesting that the protective effects of these substances may be mediated by endogenous PAF.

Release of platelet-activating factor in acute experimental pancreatitis.

This study demonstrates the formation of platelet-activating factor (PAF) in rats with acute experimental pancreatitis (AEP). The AEP was induced by infusing sodium taurodeoxycholate and trypsin into the bile-pancreatic duct. The PAF content was increased in blood samples and in pulmonary and pancreatic tissue as compared with control animals. Significant amounts of PAF were also found in peritoneal fluid. The PAF content did increase in blood samples and in pulmonary tissue after administration of endotoxin intravenously. The effects of intraperitoneal PAF administration were also studied and showed an increase of polymorphonuclear cells in blood samples. These findings suggest that acute pancreatitis might generate and release PAF. Whether PAF release is associated with the pathophysiology of complications in acute pancreatitis remains to be elucidated.

Attenuation of endotoxin-induced intestinal microcirculatory damage by eicosapentanoic acid

The major objective of this study is to investigate whether oral administration of eicosapentanoic acid (EPA) has any preventive effect on endotoxin-induced microcirculatory damage of rat small intestine. EPA in a daily dose of 300 mg/kg was orally given to male Wistar rats for 3 wk. Submucosal microvessels of the ileum were observed by intravital microscopy equipped with a high-speed video camera system after the intra-arterial infusion of endotoxin at a dose of 2 mg.kg-1.h-1. The number of sticking leukocytes was significantly increased at 30 min after the treatment of endotoxin especially along the smaller branch of intestinal venules. It reached the maximal plateau at 45 min after treatment. The pretreatment of EPA significantly attenuated the increase in sticking leukocytes induced by endotoxin. A platelet-activating factor (PAF) antagonist 2-[N-acetyl-N-(2-methoxy-3-octadecylcarbamoyloxy propoxycarbonyl) aminomethyl]-1-ethylpyridinium chloride (CV-6209) significantly prevented the increased leukocyte sticking to the same extent as EPA treatment. Thirty minutes after endotoxin infusion, red blood cell (RBC) velocity was significantly decreased in both arterioles and venules. RBC velocity appeared to be continuously decreased thereafter and reached its minimum value at approximately 60 min. EPA treatment was revealed to prevent the decrease in RBC velocity of microvessels induced by endotoxin. CV-6209 also significantly attenuated the decreased RBC velocity. The remarkable elevation of PAF content in the ileal mucosa as observed by endotoxin infusion was also significantly attenuated by administration of EPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Raised concentrations of platelet activating factor in colonic mucosa of Crohn's disease patients.

Platelet activating factor (PAF-ACETHER or PAF) and precursors of platelet activating factor were investigated in 26 patients with acute Crohn's disease and in 10 healthy controls. Platelet activating factor, lyso platelet activating factor, and alkyl acyl glycerophosphocholine, were determined in colonic mucosal biopsies in patients with acute Crohn's disease, during prednisolone therapy, and in remission. Biopsy specimens were submitted to histopathology examination and to phospholipid extraction. Platelet activating factor, lyso platelet activating factor, and alkyl acyl glycerophosphocholine were found in patients with acute Crohn's disease and in remission as well as in controls. Whatever the site of the biopsy, the level of platelet activating factor in colonic mucosa was higher (p < 0.01) in Crohn's disease than in controls. There was no correlation between the level of colonic PAF-ACETHER and age, sex, Crohn's disease activity index, and biological parameters in sera. Although concentrations of colonic platelet activating factor content were higher (p < 0.01) when colonic mucosa displayed cell infiltration, they were neither related to the severity nor the type of inflammatory cells. Platelet activating factor decreases with prednisolone therapy and might return to normal concentrations in quiescent patients. Lyso platelet activating factor and alkyl acyl glycerophosphocholine were not significantly higher in Crohn's disease than in controls. These data suggest that platelet activating factor may be involved in the pathogenesis of Crohn's disease and that it could be used as a marker of the mucosal activity of the disease.

Effects of senna and its active compounds rhein and rhein-anthrone on PAF formation by rat colon.

A single or a prolonged oral administration of senna (60 mg kg-1) to rats did not increase either colonic PAF (platelet activating factor) content or intraluminal release of acid phosphatase. A similar result was observed in the colonic tissue of rats perfused in-vitro with rhein (1-300 micrograms mL-1) or rhein-anthrone (1-300 micrograms mL-1). A single or prolonged administration of castor oil (2 mL) to rats increased both colonic PAF content and intraluminal release of acid phosphatase. Colonic tissue of rats perfused in-vitro with calcium ionophore A23187 (1 and 10 micrograms mL-1) formed large amounts of PAF and acid phosphatase. Since PAF can mediate intestinal damage and acid phosphatase is a marker of cellular injury, we conclude that senna and its derivatives, rhein and rhein-anthrone, are well tolerated in rats.

Dexamethasone-induced gastric mucosal damage in the rat: possible role of platelet-activating factor.

1. The aim of the present experiments was to study the possible role of platelet-activating factor (PAF) in mediating gastric mucosal damage induced by dexamethasone in the rat by measuring gastric tissue levels of PAF during dexamethasone-treatment and by investigating the effects of specific PAF receptor antagonists on dexamethasone-induced gastric lesions. PAF-like bioactivity extracted from the rat glandular stomach was determined by a platelet aggregation assay. 2. Dexamethasone treatment (0.4-4 mg kg-1, daily for 1-6 days) produced time- and dose-dependent damage to the glandular mucosa of the stomach as characterized by extensive, uniform hyperaemia with multiple, focal petechiae and erosions. 3. These changes were accompanied by a time-, and dose-dependent increase in PAF content of the glandular stomach. Control rat stomach contained small amounts of PAF (0.14 +/- 0.04 ng per g wet weight), which increased over 40 fold in response to dexamethasone treatment (4 mg kg-1, daily for 6 consecutive days). The presence of PAF-like material in the stomach extract was ascertained by thin-layer chromatography, high performance liquid chromatography and by alkaline hydrolysis. 4. Pretreatment of the animals with one or other of the structurally unrelated PAF receptor antagonists, BN 52021 (10 mg kg-1, i.p.) or BN 50727 (1 mg kg-1, i.p.) significantly reduced dexamethasone-induced gastric damage. In these animals neither petechiae nor erosions were observed. 5. These observations suggest that PAF is a likely endogenous mediator of glucocorticoid-induced gastric mucosal damage in the rat.

Presence of PAF-acether in stool of patients with pouch ileoanal anastomosis and pouchitis

Platelet-activating factor is an endogenous phospholipid produced by a wide variety of inflammatory cells. Platelet-activating factor induces severe pathological changes in various organs and, among numerous potent effects, causes bowel necrosis. Pouchitis is a poorly understood complication of ileoanal pouch anastomosis which occurs in patients who undergo surgery for ulcerative colitis. The aim of this study was to measure ileal or fecal platelet-activating factor and lyso platelet-activating factor contents in normal volunteers (n = 12), in patients with terminal ileostomy (n = 7), and in patients with ileoanal anastomosis (n = 15) (8 patients have pouchitis defined by the presence of ulcerations on the reservoir). Fecal samples were processed and assessed for platelet-activating factor by platelet aggregation assay. The aggregating material was further characterized as platelet-activating factor by the following: inhibition of the platelet aggregation it induced by specific platelet-activating factor receptor antagonist (BN 52021; IHB, Le Plessis Robinson, France); abolition of platelet aggregation after incubation with phospholipase A2 but not with lipase A1; and retention time on high-performance liquid chromatography. Stool platelet-activating factor content (in nanograms per gram of stool, mean ± 1SD) was significantly increased in patients with pouchitis (22.2 ± 16 ng/g) compared with patients with normal reservoir (1.59 ± 0.63 ng/g, P < 0.01), terminal ileostomy (0.59 ± 0.43 ng/g, P < 0.01), and healthy controls (0 ± 0 ng/g of stool, P < 0.001). Lyso platelet-activating factor (nanograms per gram of stool) was increased in patients with pouchitis (10,704 ± 5499 ng/g) compared with patients with normal reservoir (4721 ± 4549 ng/g of stool, P < 0.05), terminal ileostomy (3042 ± 4019 ng/g, P < 0.02), and healthy volunteers (128 ± 107 ng/g, P < 0.001). In patients with ileoanal anastomosis and pouchitis, increased platelet-activating factor production could be implicated in the inflammation and ulcerations observed in the reservoir.

The presence of platelet-activating factor-like activity in human spermatozoa

Evidence is provided for the existence of platelet-activating factor (PAF)-like activity in the lipid extracts of human spermatozoa. The PAF content of human spermatozoa based on [ 3 H]-serotonin release from washed rabbit platelets was noted to be 1.45 pmol/10 8 sperm cells in highly purified motile spermatozoa. No PAF activity was associated with the seminal fluid. Platelet-activating factor content of spermatozoa may be related to its fertility potential.

Platelet-Activating Factor Mediates Glycerol-Induced Acute Renal Failure in Rats

1. We have studied the effect of inhibiting the interaction of platelet-activating factor with its receptor by using two structurally different antagonists BN-52021 (1 mg/kg, intraperitoneally) and alprazolam (1 and 5 mg/kg, intraperitoneally) on the evolution of glomerular filtration and renal blood flow, in the experimental model of acute renal failure induced by the intramuscular injection of glycerol in rats. 2. We have also measured arteriovenous differences in platelet-activating factor concentration, as well as platelet-activating factor content in glomeruli from rats with glycerol-induced acute renal failure. 3. After glycerol injection, untreated rats showed a marked reduction in inulin clearance which reached 77% in the first 30 min. The reduction was only 27% in the rats treated with BN-52021 and 38% in the rats treated with alprazolam (5 mg/kg), with statistically significant differences between treated and untreated groups. 4. Clearance of p-aminohippuric acid was also improved with BN-52021 or alprazolam treatment. 5. This protective effect of BN-52021 was observed in a more prolonged follow-up (3 days). 6. Glomeruli from rats with acute renal failure, treated or untreated with BN-52021, showed similar amounts of platelet-activating factor, whereas it was undetectable in glomeruli from normal rats. Furthermore, rats with glycerol-induced acute renal failure released greater amounts of platelet-activating factor. 7. These results provide evidence of a role for platelet-activating factor in the genesis of this model of experimental acute renal failure.

PAF metabolism in resident and activated alveolar macrophages: Role of protein kinase C

Platelet-activating factor (PAF) metabolism was studied in resident and activated alveolar macrophages. Macrophages were obtained from normal Sprague-Dawley rats and from rats previously injected with complete Freund's adjuvant. Macrophages were attached and stimulated for 90 min. Then, cell PAF was extracted and quantitated by thin-layer chromatography. We found that in both resident and activated macrophages, calcium ionophore A23187 was a potent stimulus for PAF production while phorbol myristate acetate (PMA) was not. PMA and ionophore acted synergistically to increase PAF content in resident macrophages. This synergism was not observed in activated macrophages. To examine if this difference between resident and activated macrophages was due to a difference in PAF degradation, we assayed acetylhydrolase, the PAF-degrading enzyme. We found that ionophore stimulated acetylhydrolase activity in activated macrophages, but not in resident macrophages. Furthermore, PMA potentiated the ionophore effect in activated macrophages. This synergism was less obvious in resident cells. We conclude that PAF metabolism is different in activated and resident alveolar macrophages. Protein kinase C may play an important role in acetylhydrolase regulation in these cells.

Glomerular platelet-activating factor levels and origin in experimental glomerulonephritis

The content of platelet activating factor (PAF) in glomeruli isolated from rats with nephrotoxic serum glomerulonephritis (NSGN) was quantified at various stages of the disease and the role of complement, platelets and neutrophils in mediating changes in glomerular PAF levels was evaluated. PAF content was assessed following extraction, isolation and quantification of this alkyl ether lipid using a bioassay based on [3H]-serotonin release from labelled rabbit platelets. Following induction of NSGN using proteinuric doses of rabbit immune serum raised against rat glomerular basement membrane, enhanced glomerular PAF levels were observed at 3 hours, 24 hours and on day 15 following induction of the disease. In complement depleted rats and at three hours following induction of NSGN, glomerular PAF levels were significantly lower than in complement replete controls studied in parallel. At the same time point of the disease, platelet depleted rats with NSGN demonstrated significantly lower glomerular PAF levels than parallel controls, whereas in neutrophil depleted rats glomerular PAF levels were no different than controls. These observations indicate that in infiltrative and complement dependent forms of glomerular immune injury, glomerular PAF levels are increased via a complement mediated mechanism. Infiltrating platelets, but not neutrophils, partially account for the enhanced glomerular PAF levels. The observations are of potential importance in the pathophysiology of glomerulonephritis.

Novel Aspect of Crohn’s Disease: Increased Content of Platelet-Activating Factor in Ileal and Colonic Mucosa

Intestinal mucosal content of platelet-activating factor (PAF-acether) was investigated in Crohn's disease. The PAF-acether content was determined in mucosal biopsies from the ileum and colon in Crohn patients (n = 13), and in normal mucosa of control patients (n = 11). PAF-acether was found in both groups and was raised in Crohn patients, both in the ileum (6.3 +/- 4.7 vs. 0.6 +/- 0.4 pmol/g; p less than 0.01) and colon (6.2 +/- 4.2 vs. 0.7 +/- 0.4 pmol/g; p less than 0.01). Colonic PAF-acether content was raised irrespective of the presence of colonic inflammation as judged macroscopically. These findings add further support to the importance of bioactive lipids in inflammatory bowel disease and suggest a possible role for PAF-acether in Crohn's disease.

Occurrence of platelet-activating factor (PAF) in normal rat stomach and alteration of PAF level by water immersion stress.

We detected platelet-activating substance in gastrointestinal areas, which was confirmed to be platelet-activating factor (PAF) on the basis of the following findings: 1) it comigrated with authentic PAF on thin-layer chromatography; 2) it did not aggregate PAF-desensitized platelets; and 3) its activity was completely antagonized by the receptor antagonists CV3988 and L-652,731. The level of PAF was determined with a bioassay method based on the release of [3H]serotonin from washed rabbit platelets. In the normal rat stomach, the level of PAF was high in the antrum (940 +/- 200 nmol PAF/mol phosphorus of original phospholipids), especially in the antral mucosa (1801 +/- 426 nmol/mol phosphorus of original phospholipids). The stomach PAF level was significantly altered by water immersion stress. Stress for a period of 1 h was associated with a decrease in the antral PAF level to 39 +/- 7% of that of untreated controls. This low PAF level persisted during stress. On the other hand, in the corpus, stress for periods of 1 and 3 h was associated with decreases in the PAF content, and further stress (7 h) resulted in restoration of the PAF level to normal. Furthermore, 7 h of stress was associated with distinct hemorrhagic lesions, which were prevented by CV3988 infused i.v. before the stress. This is the first report of an association between a decrease of the endogenous PAF level in animal tissues and tissue damage.