Potential Role Of Platelet-activating Factor Research Articles

Platelet activating factor leads to initiation and promotion of breast cancer

Bioactive molecules present in the tumor milieu are known to contribute substantially to tumor progression. Phospholipid mediators are a group of molecules that have roles in normal physiology as well as in pathological conditions. Platelet activating factor (PAF), a phospholipd mediator, secreted by cells present in tumor microenvironment has been implicated to have a possible role in cancer progression. Here, we highlight our study of the potential role of PAF in inducing transformation of breast epithelial cells grown as three dimensional cultures. We have also attempted to dissect the motility related molecular pathway activated upon PAF stimulation in MDA-MB 231 cells. This study further calls for detailed analysis of pathways downstream of PAF signalling which would aid in identification of targets and designing of treatment strategies.

Platelet activating factor (PAF) and activity of its biosynthetic and catabolic enzymes in blood and leukocytes of male patients with newly diagnosed heart failure

Objectives To evaluate platelet activating factor (PAF) levels, its metabolic enzymes activity and its associations with other inflammatory markers in heart failure (HF) patients. Design and methods PAF, and two of its key biosynthetic enzymes [lyso-PAF acetyltransferase (lyso-PAF-AT) and DTT-insensitive CDP-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT)] along with its catabolic enzymes [PAF-acetylhydrolase (PAF-AH) and lipoprotein-associated phospholipase-A 2 (Lp-PLA 2)] were measured in serum and leukocytes of twelve newly diagnosed male HF patients. Serum CRP, TNF-α, IL-6, sCD14 and CD40L were also determined. Results PAF ranged from 0.03 to 5.6 pmol/mL. Median lyso-PAF-AT, PAF-CPT, PAF-AH and Lp-PLA 2 activities were 4.1, 68.42, 644.44 pmol/min/mg protein and 51.42 pmol/min/μL correspondingly. Lyso-PAF-AT and PAF-CPT activities positively correlated with CRP, IL-6 and with each other, whereas PAF-CPT activity correlated with sCD14 and CD40L ( P < 0.05). Conclusions PAF's biosynthetic enzyme activities correlated with inflammatory and immunologic molecules, which are activated in HF. Our study indicates a potential role of PAF in HF patients.

HIV drug nevirapine (Viramune): risk of severe hepatotoxicity.

Platelet-activating factor (PAF) has recently been demonstrated to be metabolized by B lymphocytes and to cause enhancement of Ig synthesis by Ig-secreting B lymphoblastoid cell lines. We have now examined some of the early activation events triggered by PAF binding to three Ig-secreting B cell lines, LA350 (IgM secreting), HSCE- (IgG secreting), and U266 (IgE secreting). After addition of 10(-7) to 10(-11) M PAF, but not equimolar concentrations of the inactive metabolite lyso-PAF, all three cell lines demonstrated rapid dose-dependent increases in free cytosolic Ca2+ concentrations ([Ca2+]i). The increases in [Ca2+]i resulted from both the release of Ca2+ from internal stores as well as transmembrane Ca2+ uptake. Addition of PAF triggered the rapid hydrolysis of phosphatidylinositol bisphosphate and accumulation of inositol phosphates. PAF also increased expression of the cell cycle-active genes c-fos and EGR2 in a dose-dependent fashion. The stimulated increases in [Ca2+]i and phosphatidylinositol bisphosphate hydrolysis and the increases in gene expression were all inhibited by the specific PAF receptor antagonist Web 2086. The LA350 cell line (which expresses surface IgM) was also shown to increase [Ca2+]i after addition of anti-IgM antibodies. Sequential addition of PAF or anti-IgM antibody in either order failed to reveal any evidence for heterologous desensitization. Furthermore, the PAF receptor antagonist did not affect anti-IgM induced changes in [Ca2+]i. These data provide evidence for the presence of functional PAF receptors on B lymphoblastoid cells and indicate a potential role for PAF in the regulation of B cell activation.

The macrophage response to endotoxin requires platelet activating factor.

Platelet activating factor (PAF) is a key proinflammatory mediator of septic shock and is metabolized by PAF-acetylhydrolase (PAF-AH). Low circulating levels of PAF-AH have been associated with the development of autodestructive excessive inflammatory responses such as post-injury multiple organ failure, and recombinant PAF-AH is being studied for the prevention of acute respiratory distress syndrome (ARDS). However, the potential role of PAF as an autocrine mediator of macrophage activation is unclear. We wanted to examine the role of PAF in the endotoxin- (LPS) induced macrophage response using PAF-AH. Rabbit alveolar macrophages were stimulated with LPS (10 ng/mL) with or without PAF-AH (0.1-10 microg/mL). Supernatants were collected to measure the production of tumor necrosis factor (TNF), interleukin 8 (Il-8), and prostaglandin E2 (PGE2). Cell monolayers were assessed for procoagulant activity (PCA). TNF mRNA production was determined by Northern blot and RNA stability was assessed. Evaluation of intracellular signaling pathways for LPS included western blots for phosphorylated p38 and ERK kinases and gel shift for nuclear factor-kappaB. There was a dose-response inhibition of TNF, PCA, Il-8, and PGE2 production following pretreatment with PAF-AH. Time course studies revealed effective inhibition of TNF production with administration of PAF-AH up to 2 h after LPS challenge. TNF mRNA production was inhibited, while mRNA stability was not affected. There was no effect on the phosphorylation of p38 or ERK 1 kinases; however, the nuclear translocation of NF-kappaB was inhibited. Macrophage cytokine production in response to endotoxin is PAF dependent. This effect involves the inhibition of TNF gene transcription and concomitant inhibition of NF-kappaB.

P2B61 - Role of caspase-3 in apoptosis induced by glucocorticoids on lymphoid cells

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2. Several studies indicate an important role of platelet activating factor (PAF) and thromboxane A2 (TXA2) in glomerular pathophysiology. However, the potential role of PAF or TXA2 in renal tubular pathophysiology has received little attention, and the presence of functional receptors for these autacoids in renal tubular epithelium has not been previously studied. We examined the effects of PAF and the TXA2 analogue, ONO11113, on the cytosolic free calcium concentration ([Ca2+]i) in cultured LLC-PK1, cell line using a fluorescent probe, fura-2. In these cells, the addition of PAF or ONO11113 caused a significant increment in [Ca2+]i in a dose-dependent manner: both agonists (10-7 M) increased [Ca2+]i from 148 ± 16 to 288 ± 39 nM and from 130 ± 8 to 240 ± 18 nM, with the values of EC50 for PAF and ONO11113 being 17 ± 4 and 17 ± 2 nM, respectively. These effects were both rapid and transient, returning to baseline in two minutes. The effect of PAF was selectively blocked by PAF receptor antagonist BN50730, but not by TXA2 receptor antagonist L657925. Similarly ONO1113 response was abolished by L657925, but not by BN50730. PAF- or ONO11113-challenged cells did not respond to a second addition of the same agent and showed heterologous desensitization to the other agonist. The initial peaks of [Ca2+]i as well as the sustained elevations in [Ca2+]i induced by PAF or ONO11113 were reduced following the chelation of extracellular Ca2+ by 10 mM ethylene glycol-bis(β-aminomethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). In the absence of extracellular Ca2+ 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) (which has extensively been used to study the contribution of Ca2+ released from intracellular storage sites in the increase in [Ca2+]i), blocked the increment in [Ca2+]i induced by PAF or ONO1113. These results indicate that LLC-PKi cells express discrete receptors for PAF and TXA2 that are coupled to an increase in [Ca2+]i through mobilization of calcium from both intracellular storage sites and extracellular milieu, and suggest the possible importance of PAF and TXA2 in tubular pathophysiology.

25 Molecular mechanisms of angiogenesis associated to kaposi's sarcoma

Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS) a multifocal lesion characterized by a prominent angiogenesis. We demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a lipid mediator of inflammation and cell-to-cell communication. ILI, TNF and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells (EC) and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a PAF receptor antagonist. Since PAF activates EC we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in Matrigel model. These effects were inhibited by treating mice with WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic FGF, PIGF, VEGF and its specific receptor flk-1 , HGF, KC, and MIP2. Treatment with WEB 2170 abolished the expression of these transcripts within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development, in KS.

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2.

Several studies indicate an important role of platelet activating factor (PAF) and thromboxane A2 (TXA2) in glomerular pathophysiology. However, the potential role of PAF or TXA2 in renal tubular pathophysiology has received little attention, and the presence of functional receptors for these autacoids in renal tubular epithelium has not been previously studied. We examined the effects of PAF and the TXA2 analogue, ONO11113, on the cytosolic free calcium concentration [( Ca2+]i) in cultured LLC-PK1 cell line using a fluorescent probe, fura-2. In these cells, the addition of PAF or ONO11113 caused a significant increment in [Ca2+]i in a dose-dependent manner: both agonists (10(-7) M) increased [Ca2+]i from 148 +/- 16 to 288 +/- 39 nM and from 130 +/- 8 to 240 +/- 18 nM, with the values of EC50 for PAF and ONO11113 being 17 +/- 4 and 17 +/- 2 nM, respectively. These effects were both rapid and transient, returning to baseline in two minutes. The effect of PAF was selectively blocked by PAF receptor antagonist BN50730, but not by TXA2 receptor antagonist L657925. Similarly ONO11113 response was abolished by L657925, but not by BN50730. PAF- or ONO11113-challenged cells did not respond to a second addition of the same agent and showed heterologous desensitization to the other agonist. The initial peaks of [Ca2+]i as well as the sustained elevations in [Ca2+]i induced by PAF or ONO11113 were reduced following the chelation of extracellular Ca2+ by 10 mM ethylene glycol-bis(beta-aminomethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 250 WORDS)

Anticardiolipin antibody-positive serum enhances endothelial cell platelet-activating factor production

Circulating antiphospholipids have been linked to recurrent pregnancy loss by a mechanism involving placental and decidual thrombosis. We hypothesized that platelet-activating factor, an autacoid synthesized by vascular endothelium, might mediate this phenomenon through its ability to promote platelet aggregation and fibrin deposition. Alternatively, antiphospholipid antibodies might exert a procoagulant effect by inhibiting the synthesis of prostacyclin. To evaluate these theories, endothelial cells (harvested from human umbilical veins) were grown to confluence and incubated for 48 hours with 20% concentrations of anticardiolipin antibody-positive and -negative human sera as well as fetal bovine serum. After incubation culture wells were stimulated with 10 μmol/ml calcium ionophore A23187 (an agonist of platelet-activating factor and prostacyclin synthesis). Intracellular platelet-activating factor was measured by tritiated acetate incorporation, phospholipid extraction, thin-layer chromatography, and scintillation spectrophotometry. Enhanced platelet-activating factor synthesis was identified in cultures incubated with anticardiolipin antibody-positive serum (25,544 ± 2604 disintegrations per minute, mean ± SD) when compared with anticardiolipin antibody-negative serum (18,600 ± 3316 dpm) or fetal bovine serum (19,014 ± 4233 dpm; analysis of variance, p = 0.033). In similar experiments, prostacyclin synthesis was determined by measuring its primary metabolite, 6-keto-prostaglandin F1α in culture supernatants. No differences between anticardiolipin antibody-positive and control cultures were observed (analysis of variance, p = 0.90). We conclude that in this endothelial cell model, anticardiolipin antibody-positive serum enhances ionophore-mediated platelet-activating factor synthesis but has no apparent effect on the production of prostacyclin. These findings suggest a potential role for platelet-activating factor in anticardiolipin antibody-mediated vascular thrombosis. (AM J OBSTET GYNECOL 1991;165:1748-52.)

Activation of guinea pig eosinophils by human recombinant IL-5. Selective priming to platelet-activating factor-acether and interference of its antagonists.

The potential role of platelet-activating factor (PAF)-acether and of IL-5 as an eosinophil-proliferating, activating, and/or recruiting mediator in asthma led us to study the effects of human (h) rIL-5 (hrIL-5) and PAF-acether, alone or combined, on isolated guinea pig eosinophils. Two populations of eosinophils were separated from peritoneal lavages of polymyxin B-treated guinea pigs upon a discontinuous metrizamide gradient: one of low density (between 20 and 22% of metrizamide, purity: 63 +/- 3%, n = 27) and another of normal density (between 22 and 24% of metrizamide, purity: 87 +/- 2%, n = 16). Chemotactic activity was evaluated on a micro-Boyden chamber, results being expressed as the number of migrating eosinophils (mean +/- SEM) at 40 microns through a cellulose nitrate filter (3 microns pore size) in the presence of the agonist or of the solvent alone. hrIL-5 dose-dependently stimulated normodense eosinophil chemotaxis, reaching a peak at 500 ng/ml (98 +/- 21 migrating eosinophils, n = 5, p less than 0.05). These eosinophils also responded to PAF-acether and to LTB4 and not to FMLP, hrTNF alpha, and LPS. Eosinophil preincubation with hrIL-5 increased significantly the migration by PAF-acether (173 +/- 23 migrating eosinophils with PAF-acether 10 nM after preincubation with hrIL-5 500 ng/ml vs 69 +/- 10 after preincubation with buffer alone, p less than 0.01) and failed to enhance migration by LTB4 or to uncover an activity for FMLP. Migration by PAF-acether was antagonized when the cells were preincubated with the antagonists BN 52021 and WEB 2086, which also inhibited migration by hrIL-5. Eosinophils were auto-desensitized by and to PAF-acether or LTB4, but were not cross-desensitized to each other. Eosinophils desensitized to PAF-acether failed to migrate with hrIL-5, but those desensitized to LTB4 responded to hrIL-5 as controls. hrIL-5 failed to induce the elevation of intracellular free calcium concentration and superoxide anion generation from basal values, whereas preincubation of eosinophils with hrIL-5 induced a significant increase in the rise in intracellular free calcium concentration and in superoxide anion generation by 10 nM PAF-acether but not by LTB4. In conclusion, the in vivo eosinophil migration in allergy may involve hrIL-5, particularly associated to PAF-acether.

Superoxide Dismutase (SOD) and the Paf-Antagonist (BN 52021) Reduce Small Intestinal Damage Induced by Ischemia-Reperfusion

Oxygenated free-radicals appear to play a prominent role in mediating damage associated with gastrointestinal diseases. Production of reactive oxygen metabolites in ischemia-reperfusion involves oxidases found in resident phagocytic cells and microvascular and mucosal epithelial cells. Platelet activating factor (PAF), a phospholipid associated with inflammatory disorders, has been shown to both prime and amplify the release of superoxide anion and hydrogen peroxide from polymorphonuclear neutrophils and macrophages stimulated by FMLP or PMA. To further elucidate the involvement of free radicals in intestinal damage and the potential role of PAF in their production, we examined the effect of superoxide dismutase (SOD) and BN 52021 (ginkgolide B) on ischemia-reperfusion induced damage in the small intestine. The study involved 32 Sprague-Dawley rats (100-200 g) divided into four groups. Three of these groups were subjected to occlusion of the mesenteric artery 30 mins followed by 24 h reperfusion. On 2 groups SOD (15,000 U/kg/iv) and BN 52021 (20 mg/kg/po) were administered 45 mins before arterial occlusion. Following the 24 h reperfusion, the rats were sacrificed after overnight fasting. The jejunum and ileon were removed and fixed for morphological examination. Lesions in the small intestine were quantified. The results showed extensive necrosis, hemorrhage, oedema and neutrophil invasion in the jejunal and ileal mucosa. This injury was significantly reduced by SOD (15,000 U/kg/iv) and BN 52021 (20 mg/kg/po) pretreatment. In conclusion, free-oxygenated radicals appear to mediate reperfusion damage in the small intestine and PAF appears to be involved in the genesis of these toxic products. Thus, SOD and BN 52021 may be considered as protectors against ischemic disorders.

Evidence for platelet-activating factor as a mediator of endotoxin-induced gastrointestinal damage in the rat: Effects of three platelet-activating factor antagonists

The potential role of platelet-activating factor (PAF) as a mediator of gastrointestinal ulceration associated with septic shock was examined in the rat. The damaging effects of both PAF and Escherichia coli endotoxin in the stomach and small intestine were compared, as were their effects on plasma leakage into the lumen of the gastrointestinal tract. Intravenous administration of either endotoxin or PAF produced extensive necrosis and vascular congestion in the stomach and small intestine, but not the distal colon. With either agent, the duodenum and jejunum were the tissues most susceptible to damage and in which the greatest plasma leakage was observed. The prolonged hypotension and gastrointestinal damage induced by PAF or endotoxin were significantly inhibited by three structurally dissimilar PAF antagonists (CV-3988, BN-52021, and RO-193704J. CV-3988 (10 mg/kg) significantly (p < 0.05) reduced both endotoxin- and PAF-induced plasma leakage in the stomach and small intestine. Of the three antagonists, only CV-3988 significantly reduced ethanol-induced gastric mucosal damage, perhaps reflecting actions of this compound unrelated to antagonism of PAF receptors. These studies support the hypothesis that PAF is an important mediator of the hypotension and plasma leakage observed during endotoxic shock and its endogenous release may contribute to the gastrointestinal ulceration associated with this syndrome. Thus, PAF receptor antagonists may be useful for prevention of such ulceration.

Platelet-activating factor and the kidney.

Platelet-activating factor (PAF) represents a group of phospholipids with the basic structure of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine. A number of different cells are capable of producing PAF in response to various stimuli. The initial step of PAF formation is activation of phospholipase A2 in a calcium-dependent manner, yielding lyso-PAF. During this step arachidonic acid is also released and can be converted to its respective cyclooxygenase and lipoxygenase products. The lyso-PAF generated is then acetylated in position 2 of the glycerol backbone by a coenzyme A (CoA)-dependent acetyltransferase. An additional pathway may exist whereby PAF is generated de novo from 1-alkyl-2-acetyl-sn-glycerol by phosphocholine transferase. PAF inactivation in cells and blood is by specific acetylhydrolases. PAF exhibits a variety of biological activities including platelet and leukocyte aggregation and activation, increased vascular permeability, respiratory distress, decreased cardiac output, and hypotension. In the kidney PAF can produce decreases in blood flow, glomerular filtration, and fluid and electrolyte excretion. Intrarenal artery injection of PAF may also result in glomerular accumulation of platelets and leukocytes and mild proteinuria. PAF increases prostaglandin formation in the isolated kidney and in cultured glomerular mesangial cells. PAF also causes contraction of mesangial cells. Upon stimulation with calcium ionophore the isolated kidney, isolated glomeruli and medullary cells, and cultured mesangial cells are capable of producing PAF. The potential role for PAF in renal physiology and pathophysiology requires further investigation that may be complicated by 1) the multiple interactions of PAF, prostaglandins, and leukotrienes and 2) the autocoid nature of PAF, which may restrict its action to its site of generation.