PAF-induced Aggregation Of Human Platelets Research Articles

Inhibition of human platelet aggregation by amides and ester of salicylic acid with platelet-activating factor analogs.

The influence of acetyl salicylic acid (ASA) derivatives with platelet-activating factor (PAF) lipid analogs on PAF-induced human platelet aggregation has been studied. It was found that the ASA amide with an ethanolamine plasmalogen PAF analog (1-0-alk-1'-enyl-2-acetyl-sn-glycero-3-phospho-(N-2'-acetoxybenzoyl)ethanolamine) and the ASA ester with a choline plasmalogen PAF analog (1-0-alk-1'-enyl-2-(2'-acetoxybenzoyl)-sn-glycero-3-phosphocholine) at concentrations of 10-7-10-6 M effectively inhibit PAF-induced aggregation of human platelets. In contrast to these compounds, the ASA amide with an alkyl PAF analog (1-0-alkyl-2-acetyl-sn-glycero-3-phospho-(N-2'-acetoxybenzoyl)ethanolamine) did not inhibit PAF-induced platelet aggregation. As possible mechanisms of action of the studied compounds, the blockade of PAF-receptor and cyclooxygenase inhibition are proposed.

Pharmacological evidence for the putative existence of two different subtypes of PAF receptors on platelets and leukocytes; studies with yangambin

Yangambin, a new naturally-occuring platelet activating receptor (PAF) receptor antagonist competitively displaced [ 3H]-PAF from its high affinity binding sites on washed human platelets with a Ki value of 1.1 ± 0.3 μM ( n = 3). Studies carried out in parallel demonstrated that SR 27417, a newly-developed PAF receptor antagonist also antagonized [ 3H]-PAF binding to these cells with a Ki value of 51 ± 2 pM. SR 27 417 ( N-(2-dimethylamino ethyl)- N-(3-pyridinyl methyl) [4-(2,4,6-triisopropyl phenyl) thiazol-2-yl] amine) selectively and competitively inhibited the specific binding of [ 3H]-PAF on human polymorphonuclear leukocytes (Ki = 65 ± 5.2 pM) whereas high doses of yangambin remained ineffective. Yangambin inhibited PAF-induced aggregation of human platelets in vitro (IC 50 = 1.0 ± 0.2 μM) but had no effect PAF-induced oxidative burst in human polymorphonuclear leukocytes. In guinea pigs, yangambin inhibited PAF-induced thrombocytopenia but did not affect leukocytopenia whereas SR 27 417 afforded complete protection against both PAF-induced thrombocytopenia and leukocytopenia. In conclusion, yangambin discriminates between two different types of PAF receptors on platelets and polymorphonuclear leukocytes and can be considered as the first PAF receptor antagonist described to date exhibiting such an effect.

Exogenous diacylglycerols synergize with PAF with human platelets, but inhibit PAF-induced responses of rabbit platelets

To investigate whether diacylglycerol (DAG) has a role in reversible platelet aggregation induced by low concentrations of platelet-activating factor (PAF), we attempted to use the DAG kinase inhibitor, R59022, to prevent rapid conversion of DAG to phosphatidic acid. However, we found that R59022 inhibited the binding of [ 3H]PAF to human and rabbit platelets and to rabbit platelet membranes. We then investigated whether exogenous, cell-penetrating DAGs (1,2-dihexanoyl- sn-glycerol (DHG) and 1-oleoyl-2-acetyl- sn-glycerol (OAG)) act synergistically with low concentrations of PAF that alone induce only reversible aggregation. Platelets were isolated and labeled with [ 14C]serotonin. DHG (25–75 μM) caused slow, weak aggregation and some release of [ 14C]serotonin with human, but not rabbit, platelets. OAG (25–75 μM) did not aggregate either species' platelets. Phosphorylation of pleckstrin by DHG was more transient in rabbit platelets than previously observed with human platelets. Both DHG and OAG synergistically potentiated PAF-induced aggregation of human platelets, but, paradoxically, concurrently inhibited the PAF-induced increase in intracellular Ca 2+ ([Ca 2+] i); potentiation decreased upon incubation with DAGs before PAF addition. In contrast, DHG strongly inhibited PAF-induced aggregation of rabbit platlets; inhibition decreased upon preincubation. OAG, added with PAF, slightly potentiated aggregation of rabbit platelets; upon preincubation, OAG progressively inhibited. Effects of DHG and OAG on PAF-induced increases in [Ca 2+] i in rabbit platlets followed a similar pattern; thus, with rabbit platelets, inhibition of the [Ca 2+] i increase may at least partially account for inhibition of PAF-induced aggregation by exogenous DAGs. Results with human platelets are consistent with stimulation of protein kinase C by DAGs, and then metabolism of DAGs and/or negative feedback by DAGs, but results with rabbit platelets indicate both an unexpected species difference and a difference between the effects of DHG and OAG on PAF-induced platelet aggregation.

Inhibition of PAF-induced human platelet aggregation by antithrombotic nipecotamides

Nipecotamides (piperidine-3-carboxamides) are potent inhibitors of platelet aggregation induced by a variety of agonists in vitro and in vivo. The inhibitory effects of six structural types of nipecotamides on human platelet aggregation induced by platelet-activating factor (PAF) in vitro, are studied. Evaluation of 15 racemates and stereoisomers of two nipecotamides showed that bis-nipecotoyl alkanes were more active than their mono congeners. Mono- and bis-nipecotoyl decanes were more potent than the corresponding hexanes. Lipophilicity was found to play a significant role in the antiplatelet activity of these compounds. The stereoselectivity in the PAF-antagonist potential of nipecotamides was less pronounced than that resulting from their action on ADP- or collagen-induced aggregation. Oxidation of the two benzylic carbon atoms of α,α′-bis[3-( N,N-diethylcarbamoyl)piperidino]- p-xylene·2HBr (A-1) to form 1,4-bis[3- N,N-diethylcarbamoyl)piperidino]benzenedicarboxamide (A-40K), which has a second set of carbonyl oxygens but lacks basic N atoms, resulted in a remarkable loss of ADP-antagonist potency while retaining PAF-antagonist activity. It is suggested that in addition to their membrane effects, nipecotamides act at other sites, including the PAF receptor. Double reciprocal plots of PAF binding to gel-filtered platelets (GFP) in the presence and absence of a typical nipecotamide (A-1C) were indicative of competitive inhibition ( K i = 19.28 μM). Scatchard analysis of 3H-PAF binding to GFP suggested the presence of high, intermediate ( I) and low affinity binding sites, of which the I site gave a K D app of 0.332 nM with an estimated 564 sites/platelet. Key interactions of nipecotamides with the PAF receptor appear to be the following (i) electrostatic interactions of the two amide oxygens with a primary set of electropositive areas spaced at 5–7 Å, (ii) in the case of appropriate compounds, electrostatic interactions of the two amide oxygens spaced at 10–12 Å with corresponding secondary receptor sites carrying positive electrostatic potential, (iii) a hydrophobic moiety fitting into a hydrophobic pocket in the receptor, and (iv) the cationic piperidine N + (at pH 7.4) interacting with a counterion, probably aspartic acid.

A novel class of platelet activating factor antagonists from Phoma sp.

Four novel platelet activating factor (PAF) antagonists, Sch 47918, Sch 49026, Sch 49027 and Sch 49028, were isolated from the fermentation broth of the fungal culture, Phoma sp. (ATCC 74077). The structures of these compounds were elucidated by spectroscopic methods. The structure and stereochemistry of the first isolated component, Sch 47918, were confirmed by single crystal X-ray diffraction analysis. Sch 49028, the most active component, was found to inhibit PAF-induced human platelet aggregation in vitro with an IC50 of 1.26 microM. However, this compound was inactive in vivo at 5 mg/kg, iv against PAF-induced bronchospasm in guinea pigs.

Pharmacological actions of Y-24180, a new specific antagonist of Platelet Activating Factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC 50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC 50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC 50 0.84 nM) was more potent than WEB 2086 (IC 50 4.21 nM) and etizolam (IC 50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC 50 value of 3.50, 9.35 and 29.5 nM, respective- ly. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180(1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 μ M. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.

Effect of rat phosphorylcholine-binding protein on platelet aggregation

Rat serum phosphorylcholine-binding protein (PCBP), also referred to as rat C-reactive protein, is present in serum under normal physiological conditions. In the present study, PCBP is shown to inhibit, in a dose-dependent manner, ADP- and platelet-activating factor (PAF)-induced platelet aggregation, using either washed rat platelets or platelet-rich plasma from rats or humans. Rat platelets, as expected, were refractory to aggregation by PAF. However, when rabbit antiserum against PCBP was added to rat platelet-rich plasma, it resulsted in aggregation of platelets by PAF. PCBP also inhibited the PAF-induced aggregation of human platelets in a dose-dependent manner. The inhibition of ADP-induced platelet aggregation by PCBP was reversed by adding phosphorylcholine. Our results suggest that PCBP present in rat serum could be the cause of the refractory response of rat plasma to PAF. It is possible that PCBP inhibits platelet aggregation by binding to the platelet surface phospholipids.

Effect of a Paf antagonist, WEB 2086, on airway microvascular leakage in the guinea-pig and platelet aggregation in man.

1. The triazolodiazepine WEB 2086 has been evaluated as an antagonist of platelet-activating factor (Paf) by studying its effects on Paf-induced human platelet aggregation and microvascular leakage in guinea-pigs. 2. WEB 2086 inhibited Paf-induced platelet aggregation in platelet-rich plasma in vitro (IC50 = 117 +/- 35 nM, mean +/- s.d.) but had no effect on adenosine 3',5'-diphosphate-induced aggregation. 3. Paf-induced microvascular leakage, measured by the extravasation of intravenously-injected Evans blue dye, was inhibited in a dose-related fashion in the airways and other tissues by WEB 2086, achieving a maximal inhibitory effect at 10 micrograms kg-1, i.v. 4. However, WEB 2086 (10 micrograms kg-1, i.v.) did not inhibit a comparable increase in vascular permeability induced by ovalbumin in sensitized guinea-pigs. 5. We conclude that WEB 2086 is a potent antagonist of Paf and that Paf does not appear to be responsible for antigen-induced microvascular leakage.

Triazolodiazepines are potent antagonists of platelet activating factor (PAF) in vitro and in vivo.

The triazolodiazepines brotizolam, triazolam and alprazolam inhibited PAF-induced human platelet aggregation in vitro (IC50 = 0.54, 7.6 and 13.7 microM, respectively) but showed only a weak or no effect against other aggregating agents (ADP, adrenaline, collagen, serotonin, arachidonic acid). In comparison, flunitrazepam and diazepam, two diazepines without the triazole ring, showed IC50-values of 42 and 260 microM, respectively. Flunitrazepam does not possess the specificity shown by the other compounds. Brotizolam and triazolam also inhibited PAF-induced human neutrophil aggregation in vitro, with IC50-values 0.21 and 6.6 microM, respectively. In anesthetized guinea pigs, brotizolam (2.5 to 10 mg/kg p.o. or 0.1 to 0.5 mg/kg i.v.) or triazolam (20 to 100 mg/kg p.o.) inhibited dose-dependently the intrathoracic accumulation and aggregation of 111Indium labelled platelets induced by an i.v. infusion of PAF (30 ng/kg X min). Brotizolam at doses of 1 to 10 mg/kg p.o. and 0.1 to 0.5 mg/kg i.v. inhibited dose-dependently the reduction in tidal volume (bronchoconstriction), the systemic hypotension and the lethal effect due to i.v. PAF in guinea pigs. Triazolam inhibited these effects of PAF at doses of 50 to 200 mg/kg p.o. PAF-induced systemic hypotension in rats can be reversed by cumulative i.v. doses (0.05 to 1.0 mg/kg) of brotizolam. In conclusion, these results show that triazolodiazepines, like brotizolam and triazolam, are potent inhibitors of PAF-induced effects in vitro and in vivo.

Triazolodiazepines: dissociation of their Paf (platelet activating factor) antagonistic and CNS activity.

The relationship between the activity of thieno- or benzo-triazolodiazepines on platelet-activating factor (Paf)-induced effects and on the CNS (central nervous system) was studied in vitro and in vivo. Brotizolam and triazolam inhibited Paf-induced human platelet aggregation. The IC50 -values were 0.54 and 7.6 microM, respectively. This inhibitory effect was not blocked by the specific central-type benzodiazepine (BDZ) antagonist, Ro 15-1788, or the specific peripheral-type BDZ ligand, Ro 5-4846. These BDZ ligands also showed an inhibitory effect on Paf-induced platelet aggregation (IC50 = 200 and 560 microM, respectively). Ro 15-1788 or Ro 5-4846 in combination with brotizolam or triazolam enhanced the Paf inhibitory effect of these triazolodiazepines. In guinea-pigs, Ro 15-1788, 100 mg kg-1 p.o. and 10 mg kg-1 i.v. completely inhibited the hypnogenic effect of 10 mg kg-1 p.o. and 1 mg kg-1 i.v. of brotizolam, respectively. Similar results were obtained with triazolam but at higher doses. In anaesthetized guinea-pigs, a dose of 100 mg kg-1 p.o. of Ro 15-1788 did not inhibit bronchoconstriction and hypotension caused by Paf (30 ng kg-1 min-1 i.v.). The combination of brotizolam (10 mg kg-1 p.o.) or triazolam (200 mg kg-1 p.o.) with this BDZ antagonist (100 and 400 mg kg-1 p.o., respectively) did not affect the Paf inhibitory activity of these triazolodiazepines. These results show that the Paf antagonistic properties of the triazolodiazepine can be dissociated from their CNS activity. It is conceivable that compounds of this structural type could be the forerunners of a novel series of potent Paf antagonists.

Cyclic platelet-activating factor analogues derived from 2-deoxy- d- erythro-pentose

A method for the synthesis of chiral cyclic analogues of platelet-activating factor (PAF) is reported. Treatment of suitably substituted derivatives of 2-deoxy- d- erythro-pentose with phosphorus oxychloride, followed by choline p-toluenesulfonate generates cyclic phospholipids in good yield. Further chemical modification produces other compounds including optically active γ-butyrolactones such as 2-deoxy-5- O-hexadecyl-3- O-phosphocholyl- d- erythro-pentono-1,4-lactone and 2-deoxy-3- O-hexadecyl-5- O-phosphocholyl- d- erythro-pentono-1,4-lactone. All phospholipids were poor antagonists of PAF-induced aggregation of human platelets, and two analogs were poor agonists. The chemistry presented should be useful for the syntheses of other conformationally restricted analogues of PAF.

PAF-Induced Human Platelet Aggregation is Selectivley Inhibited by Class II and Class III But Not Class I Calcium "Antagonists"

PAF-Induced Human Platelet Aggregation is Selectivley Inhibited by Class II and Class III But Not Class I Calcium "Antagonists"