Platelet-activating Factor Receptor Binding Research Articles

Magnone A and B, novel anti-PAF tetrahydrofuran lignans from the flower buds of Magnolia fargesii.

In a search for platelet-activating-factor (PAF) antagonists, two new lignan compounds were isolated from the Chinese crude drug shin-i, the flower buds of Magnolia fargesii. Their structures were elucidated as (2S,3R,4R)-tetrahydro-2-(3,4-dimethoxyphenyl)-4-(3, 4-dimethoxybenzoyl)-3-(hydroxymethyl)furan (magnone A, 1) and (2S,3R, 4R)-tetrahydro-2-(3,4,5-trimethoxyphenyl)-4-(3, 4-dimethoxybenzoyl)-3-(hydroxymethyl)furan (magnone B, 2). Magnones A and B showed antagonistic activity against PAF in the [3H]PAF receptor binding assay with the IC50 values of 3.8 x 10(-5) M and 2.7 x 10(-5) M, respectively.

(+/-)-trans-2-[3-methoxy-4-(4-chlorophenylthioethoxy)-5-(N-methyl-N- hydroxyureidyl)methylphenyl]-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran (CMI-392), a potent dual 5-lipoxygenase inhibitor and platelet-activating factor receptor antagonist.

By incorporating an N-hydroxyurea functionality onto diaryltetrahydrofurans, a novel series of compounds was investigated as dual 5-lipoxygenese (5-LO) inhibitor and platelet-activating factor (PAF) receptor antagonist. These dual functional compounds were evaluated in vitro for 5-LO inhibition in RBL cell extracts and human whole blood, and PAF receptor antagonism in a receptor binding assay. PAF-induced hemoconcentration and arachidonic acid- and TPA-induced ear edema in mice were used to determine in vivo activities. The structure-activity relationship analysis to define a preclinical lead is presented. (+/-)-trans-2-[3-methoxy-4-(4-chlorophenylthioethoxy)-5-(N-methyl- N-h ydroxyureidyl)methylphenyl]-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran (40, CMI-392) was selected for further study. In the arachidonic acid-induced mouse ear edema model, 40 was more potent than either zileuton (a 5-LO inhibitor) or BN 50739 (a PAF receptor antagonist), and it demonstrated the same inhibitory effect as a physical combination of the latter two agents. These results suggest that a single compound which both inhibits leukotriene synthesis and blocks PAF receptor binding may provide therapeutic advantages over single-acting agents. The clinical development of compound 40 is in progress.

Bioassays Using the Phospholipid Mediator PAF in the Search for Anti-Inflammatory Natural Products

<p> This review summarises the recent developments concerning natural products with platelet activating factor (PAF)-related activity. Four in vitro models based on the interaction of PAF with different cell types are discussed with respect to their possibilities and limitations. These assays are suitable for studies of bioassay directed isolation of anti-inflammatory natural products and the identified active compounds represent a variety of biosynthetic origins. </p> <p> The most common method used is thrombocyte aggregation induced by PAF and this review comprises some 50 different compounds studied in the model. The inhibition of PAF biosynthesis can be investigated at two levels and the activity of some 30 natural substances in corresponding assays is presented. Exocytosis induced by PAF (20 compounds) and PAF receptor binding (36 compounds) are summarised using neutrophil models. </p> <p> An application of some of these methods is given for bioactive directed isolation of three different types of compounds originating from the fern Polypodium decumanum Willd. used against psoriasis. </p>

Species differences in PAF receptor binding in the lungs between hamster and guinea pig

Platelet-activating factor (PAF) receptor in normal Golden Syrian hamster lung was characterized using radioligand binding studies and compared with guinea pig lung PAF receptor. [ 3H]WEB2086, a potent and specific PAF antagonist, was used as a radioligand for equilibrium binding, kinetic studies, competitive binding in receptor preparation (0–110 000 g fraction of lung homogenate) from hamster and guinea pig lungs. Binding of [ 3H]WEB 2086 to the receptor preparation was saturable, reversible and specific in both hamster and guinea pig lungs. Scatchard plot analysis of equilibrium binding data indicates a single binding site in hamster lung with the equilibrium dissociation constant ( K D) of 66.1 ± 36.7 nM ( n = 4) and maximal binding ( B max) of 135.4 ± 63.1 fmol/mg, but two binding sites in guinea pig lung with a high affinity site ( K D = 1.7 ± 0.6 nM; B max = 48.6 ± 2.6 fmol/mg) and a low affinity site ( K D = 83.8 ± 32 nM; B max = 480.8 ± 158 fmol/mg). The heterogeneity of [ 3H]WEB2086 binding to guinea pig lung but not to hamster lung was also confirmed by dissociation kinetic studies, in which biphasic dissociation kinetic was shown in guinea pig and monophasic kinetic in hamster lung. Although the specific [ 3H]WEB 2086 binding to lungs of both species was displaced by PAF-C18 and antagonists L659989 and CL 184005 in a dose-dependent manner and not by lyso-PAF (a biologically inactive form of PAF), the potencies of the competitive inhibition were significantly different between the two species. The relative potencies ranked WEB2086 ≈ L659989 > PAF > CL184005 in hamster lung, whereas in guinea pig lung the potencies ranked PAF > WEB2086 ≈ L659 989 ≈ CL184005. The present study demonstrates for the first time the existence of PAF receptor in the hamster lung and its binding characteristics different from guinea pig lung suggest the possible existence of different PAF receptor subtypes in hamster lung.

Synthesis of substituted cinnamoyl-tyramine derivatives and their platelet anti-aggregatory activities

Substituted cinnamoyl-tyramine derivatives were synthesized by DCC-coupling of substituted cinnamic acid with tyramine or tyramine methyl-1-ether to evaluate PAF-receptor binding antagonistic activities and inhibitory activities on PAF-induced platelet aggregation with interest on structure-activity relations. The results show that 3,4-dimethoxy-cinnamoyl tyramine-amide or its methyl ether have significant PAF-receptor binding antagonistic activity and platelet anti-aggregatory activities.

Enhanced expression of platelet-activating factor receptor on human eosinophils by interleukin-3, interleukin-5 and granulocyte-macrophage colony-stimulating factor.

The effects of interleukin-3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the surface expression of platelet-activating factor (PAF) receptor on eosinophils was examined using a PAF receptor binding assay. Specific binding of [3H]WEB 2086, a specific ligand for PAF receptor, to peripheral blood eosinophils obtained from healthy individuals was significantly increased when the cells were cultured with IL-3, IL-5, or GM-CSF for 12 h, compared with untreated cells. The PAF-induced increase in intracellular calcium ion concentration in eosinophils was markedly augmented by exposure to IL-5 for 12 h. These results suggest that these cytokines modulate the responsiveness of eosinophils to PAF through enhancement of PAF receptor expression on the cell surface.

The Isolation and Structural Elucidation of Three New Neolignans, Puberulins A, B, and C, as Platelet Activating Factor Receptor Antagonists for Piper puberulum

Three new neolignans, piperulins [corrected] A [1], B [2], and C [3], were isolated from Piper puberulum. Their structures and relative stereochemistries were determined from spectral data and the X-ray crystallographic analysis of 1. Compounds 1 and 3 inhibit specific platelet activating factor receptor binding with IC50 values of 7.3 and 5.7 microM, respectively.

Cmi-206: A potent dual platelet activating factor antagonist and 5-lipoxygenase inhibitor

CMI-206, trans-2[3-methoxy-4-propoxy-5(N′-butyl-N′-hydroxyureidyl)phenyl]-5-(3,4,5-trimethoxyphenyl) tetrahydrofuran, was synthesized and was found to antagonize PAF receptor binding and inhibit 5-lipoxygenase activity both in vitro and in vivo.

Isolation and characterization of platelet-activating factor receptor binding antagonists from Biota orientalis.

The leaf extract of Biota orientalis showed potent PAF receptor binding antagonistic activity in our previous screening studies on 234 Korean medicinal plants using rabbit platelet receptor binding tests. The activity-guided purification of the plant extract resulted in the isolation of six compounds, including two active substances. The chemical structures of the compounds isolated were established by chemical and spectrometric analyses as dotriacontane, totarol, 8 beta-hydroxy-3-oxopimar-15-ene, cedrol (IC50 = 1.3 x 10(-5) M), pinusolide (IC50 = 2.52 x 10(-7) M), and 5-hydroxy-7,4'-dimethoxyflavone.

Characterization of platelet-activating factor binding to human airway epithelial cells: modulation by fatty acids and ion-channel blockers.

Radioligand-binding studies were performed in primary cultured human airway epithelial cells with [3H]PAF to determine whether these cells express platelet-activating factor (PAF) receptors. Scatchard analysis of PAF binding data revealed a single class of PAF binding sites with Kd 1.8 +/- 0.2 nM and Bmax. 21.0 +/- 2.1 fmol/10(6) cells (13,000 receptors/cell). PAF binding increased the intracellular free Ca2+ concentration ([Ca2+]i), indicating functional PAF receptors. Palmitate (C16:0), linoleic acid (C18:2 omega 6) or eicosapentaenoic acid (C20:5 omega 3) was incubated with the cells to test the effect on PAF binding. Incorporation of each fatty acid into cellular phospholipid occurred. [3H]PAF (1 nM) binding decreased in cells supplemented with C20:5 omega 3, but increased in the cells supplemented with C16:0. Scatchard analysis revealed that the inhibition of PAF binding by supplementation with C20:5 omega 3 was due to a decrease in both affinity and number of PAF receptors. PAF-stimulated increase in [Ca2+]i was also decreased by 60% in cells supplemented with C20:5 omega 3. Verapamil, a Ca(2+)-channel blocker, and amiloride, a Na(+)-channel blocker, inhibited specific binding of [3H]PAF to the cells, with IC50 4-5 microM and 0.2 mM respectively. Diphenylamine-2-carboxylate (DPC), a Cl(-)-channel blocker, dramatically increased PAF binding to the cell in a dose-dependent manner. Scatchard analysis revealed that verapamil and amiloride decreased both binding affinity and number of PAF receptors, whereas DPC increased PAF binding sites without affecting binding affinity. These results demonstrate that human airway epithelial cells have a functional receptor for PAF and that PAF receptor binding can be modulated by exogenous fatty acids and by ion-channel blockers.

Cyclic ether acetal platelet activating factor (PAF) receptor antagonists I: 3-pyridyl derivatives

A novel series of 2-(3-pyridyl)alkoxy-5-aryltetrahydrofuran PAF antagonists has been identified and the effect of variation of the alkoxy chain length, aryl substitution and stereochemistry about the tetrahydrofuran ring studied. The optimal compound, cis-(±)-2-(3-(3-pyridyl)propanoxy)-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran ( 27a), inhibits 3H]-PAF receptor binding to washed human platelet membranes with an IC 50 value of 100 nM.

Cyclic ether acetal platelet activating factor (PAF) receptor antagonists II: imidazo[4,5-c]pyridyl derivatives

The 1H-2-methylimidazo[4,5-c]pyridyl group has been found to be the optimal heterocycle for a series of cyclic ether acetal PAF antagonists. A lead compound 8 inhibits [ 3H]-PAF receptor binding to washed human platelets with an IC 50 value of 15 nM, and both PAF-induced hypotension and endotoxin-induced hypotension in anaesthetised rats with ED 50 values of 1.4 μg/kg i.v. and 19 μg/kg i.v. respectively.

Patent Evaluation : WO9302080-: British Bio-technology Ltd: lmidazo[4,5-c]pyridine derivatives as PAF antagonists

AbstractNovelty: Novel imidazo[4,5-c]pyridine derivatives are claimed to be PAF antagonists. They are potentially useful for the treatment of inflammatory and allergic disorders, asthma, endotoxin shock, respiratory distress syndrome and transplant rejection.Biology: Inhibition of [3H]-PAF receptor binding was determined using washed human platelets. IC50 values are reported for seven compounds and are in the range 2-22 nM.Chemistry: A total of twenty-three compounds are disclosed. Detailed, stepwise syntheses are given for all compounds. Yields, physical appearance of products, elemental analyses, IR and 1H nmr data are given. All twenty-three compounds are specifically claimed including N-methyl-N-4-(5H-2-methylimidazo[4,5-c]pyridinylmethyl)-3-chlorophenylsulphonyl-L-leucine ethyl ester.

Patent Evaluation: Potent dual Acting PAF/Histamine Antagonists

SummarySummaryNovelty: Novel thazolidine carboxylic acid amide derivatives are disclosed. They are PAF antagonists and are potentially useful as anti-allergic and anti-asthmatic agents.Biology: The compounds are evaluated for PCA inhibition in rats. At a dose of 6.4 mg/kg po, 56% inhibition is observed. The compounds are also evaluated in vitro for inhibition of PAF-induced, blood platelet aggregation (IC50 = 1.8×10-6g/ml) and PAF-receptor binding (IC50 = 6.7 × 10–7 g/ml).Chemistry: 3-[4-(Diphenylmethylene)-1-piperidyl]propylcarbamoyl-2-(3-pyridyl)thiazolidine is one of fourteen specifically claimed compounds.

Two different sites of action for platelet activating factor and 1-O-alkyl-2-O-methyl-sn-glycero-3-phosphocholine on platelets and leukemic cells.

2-O-Methyl analogs of platelet activating factor (PAF) are potent anticancer agents. The sites of action and mechanisms of cell toxicity of these agents are as yet unknown. To better understand the mode of action of this class of anticancer agents, we examined the ability of 1-O-hexadecyl-2-acetylglycero-3-phosphocholine with the S or R configuration at C2 ((R)-PAF and (S)-PAF) and 1-O-hexadecyl-2-methoxyglycero-3-phosphocholine with the S or R configuration at C2 ((R)-ET-16-OCH3-GPC and (S)-ET-16-OCH3-GPC) to induce rabbit platelet aggregation and to inhibit [3H]thymidine uptake into WEHI-3B cells, HL-60 cells, and normal blood lymphocytes. The four chiral ether-linked lipids caused aggregation of rabbit platelets with the following order of potency: (R)-PAF greater than (S)-PAF greater than (R)-ET-16-OCH3-GPC greater than (S)-ET-16-OCH3-GPC; the EC50 values were 1 pM, 50 nM, 1 microM, and 50 microM, respectively. The cytotoxic effects of these ether lipids in leukemic cells was in reverse order to that observed for aggregation of platelets. The order of potency for inhibition of [3H]thymidine uptake by WEHI-3B and HL-60 cells was (R)-ET-16-OCH3-GPC = (S)-ET-16-OCH3-GPC greater than (S)-PAF greater than (R)-PAF; the EC50 values were 2, 2, 15, and greater than 40 microM, respectively. PAF antagonists (WEB 2086, CV 3988, triazolam, and SRI 63,441) blocked the action of the four ether lipids on platelets, while SRI 63,441 blocked the antineoplastic activity of the ether lipids on WEHI-3B and HL-60 cells. None of the four lipids was able to kill normal lymphocytes significantly. Scatchard analysis of PAF receptor binding revealed that HL-60 and WEHI-3B cells, which are sensitive to the cytotoxic action of ether-linked lipids, do not possess PAF receptors, whereas both normal lymphocytes and platelets do possess a PAF receptor. The present data indicate that the cytotoxic action of antineoplastic ether-linked lipids does not involve the PAF receptor. The protective role of SRI 63,441 in blocking the proaggregatory activity of the ether lipids in rabbit platelets involves PAF receptor, but cytotoxic activity against WEHI-3B and HL-60 cells does not result from its ability to act as a PAF antagonist.

Solubilization of a functionally active platelet-activating factor receptor from rabbit platelets.

Binding of platelet-activating factor (PAF) to a specific high-affinity membrane receptor has been demonstrated in numerous cell types, but very little is known about the molecular nature of this receptor. The receptor from rabbit platelets was solubilized using CHAPS, digitonin, octyl glucoside, Nonidet P-40 or sodium cholate, either with pre-bound [3H]PAF or in the absence of ligand. We have been able to demonstrate for the first time that the receptor solubilized with CHAPS, in the absence of ligand, could retain its binding activity. It migrated as a high molecular mass complex (greater than 350 kDa) on a Bio-Gel A-0.5 m gel filtration column. Binding to solubilized receptor rapidly reached an equilibrium at room temperature, but was much slower at 0 degrees C. Scatchard plots were used to calculate the number (approx. 100 per cell) and the affinity (Kd 2.5 +/- 1.4 nM) of the solubilized receptors. These values were comparable with those obtained from whole-cell binding experiments. Competition by PAF antagonists also verified that the assay was measuring PAF receptor binding activity. The presence of a protein in the receptor complex was demonstrated by heat and trypsin inactivation of binding activity. Trypsin had no effect on binding of PAF to whole cells, but was able to decrease binding activity in solubilized receptor preparations. Attempts to demonstrate the involvement of a glycoprotein by use of various lectin columns proved unsuccessful. The latter results are consistent with findings suggesting that the binding site of the PAF receptor may not be exposed at the cell surface.

The Role of Platelet-Activating Factor in the Pulmonary Response to Inhaled Bacterial Endotoxin

Quantitative morphometric analyses were carried out on animals subjected to aerosols of bacterial endotoxin (LPS) to further define the role of platelet-activating factor (PAF) in the development of pulmonary injury. Hamsters were exposed to either saline aerosol or dilute aerosols of LPS (4 micrograms/m3) for standard lengths of time. Within each aerosol exposure group, animals were further subdivided into groups receiving either the PAF receptor binding antagonist, RP 48740, or saline injections. LPS inhalation resulted in decreased fixed lung volume, increased sequestration of polymorphonuclear leukocytes and platelets in pulmonary capillaries, increased type I epithelial and endothelial cellular volumes, increased cellular interstitium, and increased endothelial pinocytotic vesicles. Treatment with RP 48740 either attenuated or abolished the ability of inhaled LPS to induce these structural alterations. The PAF antagonist also inhibited LPS-induced increases in pulmonary capillary permeability. It is concluded that PAF is one of the major injury-promoting mediators released upon inhalation exposure to environmentally realistic concentrations of bacterial endotoxin. A major but not exclusive target of this mediator is the pulmonary vascular endothelium.

Renal effects and mesangial cell contraction induced by endothelin are mediated by PAF

The recently discovered vasoactive peptide endothelin and platelet activating factor (PAF) share similar renal effects. The purpose of the present series of experiments has been to analyze the functional relations between the effect of endothelin on renal function and glomerular and mesangial cell contraction and the production and actions of PAF in kidney. Endothelin 1 nmol/kg body wt induced a transient decrease of glomerular filtration rate (from 1.99 +/- 0.17 to 0.56 +/- 0.18 ml/min) and renal blood flow (from 10.8 +/- 1.3 to 2.7 +/- 0.3 ml/min). Endothelin also induced a marked reduction of planar cell surface area of cultured mesangial cells (30 +/- 5%) and of cross sectional area of isolated glomeruli (from 1.51 +/- 0.02 to 1.31 +/- 0.02 m2 x 10(-8]. BN-52021 or WEB-2170, two potent specific inhibitors of PAF receptor binding, blocked the effects of endothelin on renal function and on the contraction of isolated glomeruli and mesangial cells. In addition, endothelin induced a significant increase in the production of PAF by isolated glomeruli (Basal, 81 +/- 10 pg/mg protein; endothelin, 10(-7) M, 140 +/- 18 pg/mg protein). Endothelin also stimulated the incorporation of [3H]acetate into PAF, both in glomeruli (264.27 +/- 27.7%) and mesangial cells (251 +/- 41%). These effects were blocked by EGTA and by verapamil. Our results suggest that PAF may be a mediator of the effects of endothelin on renal function and glomerular and mesangial cell contraction.

Competitive inhibition of tritium-labeled platelet-activating factor binding to rabbit platelet membranes by amiloride and amiloride analogs

Amiloride and its structural analogs, ethylisopropyl amiloride, benzamil, and dichlorobenzamil, inhibit both the specific [ 3H]C 18-PAF binding to rabbit platelet membranes and PAF-induced aggregation of gel-filtered rabbit platelets. Detailed analysis of binding inhibitions demonstrate that ethylisopropyl amiloride is a competitive inhibitor with an equilibrium dissociation constant (K B) of 23 μM. The concentration of amiloride and its analogs needed to inhibit the PAF-induced aggregation is high and there exists no correlation between their inhibitory activities of platelet aggregation and those of Na +/H + antiporter. However, the inhibitory effects on the PAF-induced aggregation are parallel to those on the specific [ 3H]C 18-PAF binding. The inhibitory effects of amiloride and its analogs on the activation of platelets are at the PAF-receptor binding step, rather than at the Na +/H + antiporter.

Attenuation of platelet activating factor (PAF)-induced stimulation of rabbit platelet GTPase by phorbol ester, dibutyryl cAMP, and desensitization: Concomitant effects on PAF receptor binding characteristics

The GTPase activities of rabbit platelet membrane were stimulated by platelet activating factor (PAF) in a receptor-mediated manner. The activities of the GTPase were investigated in the platelets which had been pretreated with tetradecanoyl phorbol acetate (TPA), dibutyryl cAMP, and PAF. The specific binding of PAF to intact platelet cells was also determined in these treated cells. In platelets which had been pretreated with PAF and then specifically desensitized to PAF, higher concentrations were required for stimulation of the receptor-coupled GTPase. In addition the extent of stimulation of the GTPase by PAF was also decreased. By contrast thrombin stimulation of GTPase activity was unaffected by this process. In platelets pretreated with high levels of dibutyryl cAMP (>4 m m), the specific binding of PAF was reduced to nearly 50% of the control. Although this specific binding apparently was not inhibited by lower concentrations of dibutyryl cAMP (<2 m m), PAF could stimulate the receptor-coupled GTPase only to a much lower extent in these treated cells. TPA had virtually no effect on PAF specific binding. However, higher concentrations were needed for stimulation of the GTPase. On the other hand, the extent of PAF stimulation of the GTPase was not altered. Interestingly in the TPA-treated platelet membrane, thrombin stimulated GTPase activity to a higher level than that in untreated platelet membrane. Thus, TPA, dibutyryl cAMP, and desensitization affected the PAF receptor binding and the receptor-coupled GTPase activities in a characteristic fashion. The molecular mechanisms of these effects are discussed.