Inhibition of the metabolism of platelet activating factor (PAF-acether) by three specific antagonists from Ginkgo biloba

Washed rabbit platelet suspensions were incubated in the presence of 1-[3H]O-alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H] PAF-acether), which was metabolized into 1-[3H]O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) through the sequential action of cytosolic acetylhydrolase and membrane transacylase. Within 60 min at 37 degrees, percentage of [3H] PAF-acether metabolized was 50.3 +/- 5.2% (9 experiments). This conversion was inhibited in a dose-dependent manner by various concentrations of ginkgolides A, B and C (BN 52020, 52021, 52022) known as specific antagonists of PAF-acether. The three compounds displayed the following order of potency: BN 52021 (IC50 = 3.6 X 10(-6) M) greater than BN 52020 (IC50 = 9.7 X 10(-6) M) greater than BN 52022 (IC50 = 37.6 X 10(-6) M). As this order is the same as that previously defined for inhibition of platelet aggregation to PAF-acether or for inhibition of PAF-acether binding to platelets, our data bring further support to the view that PAF-acether metabolism in platelets involves in some way its binding to its membrane receptor.     

Effects of BN 52063 and other agents inhibiting platelet-activating factor-induced contractile responses in rat portal vein

Platelet-activating factor (PAF-acether) is a potent agonist (EC50: 3.2 x 10(-8) M) of isolated rat portal vein. BN 52063 (composed of BN 52020, BN 52021 and BN 52022; molar ratio 2:2:1) specifically inhibits PAF-acether (10(-7) M) induced tone (IC50: 3.9 x 10(-5) M). Salbutamol (IC50: 3.1 x 10(-7) M), forskolin (IC50: 3.1 x 10(-6) M) and theophylline (IC50: 2.25 x 10(-4) M) are also effective in inhibiting PAF-acether-induced contractile responses and all excepting forskolin, show a certain specificity in this action. The basal myogenic activity of the rat portal vein is dose-dependently decreased by salbutamol (IC50: 1.2 x 10(-7) M), forskolin (IC50: 2.6 x 10(-6) M) and theophylline (IC50: 2.3 x 10(-4) M) whereas BN 52063 has no effect. The data suggest that rat portal veins possess specific PAF-acether receptors sensitive to BN 52063 and that PAF-acether effects could be inhibited by compounds which can bypass these putative receptors and modulate cAMP levels.     

Specific inhibition of PAF-acether-induced platelet activation by BN 52021 and comparison with the PAF-acether inhibitors kadsurenone and CV 3988

BN 52021 is a chemically defined substance extracted from Ginkgo biloba leaves. Its inhibitory potency was tested on washed human platelets prepared so as to render them specifically sensitivity either to adenosine 5'-diphosphate (ADP), arachidonic acid (AA) or PAF-acether. Its activity and specificity were compared with those of two other reported inhibitors of PAF-acether effects: Kadsurenone and CV 3988. PAF-acether-induced aggregation of washed human platelets was concentration dependently inhibited by BN 52021 (IC50: 2.22 +/- 0.79 microM against 7.5 nM PAF-acether (n = 3)). Under the same experimental conditions the aggregation triggered by ADP was not modified and that induced by AA was marginally affected. The PAF-acether EC50 in platelet-rich plasma was increased 5- and 46-fold with 1 microM and 5 microM of BN 52021 respectively. This strongly suggested that the mechanism of action of BN 52021 is of the competitive type. Analysis of [3H]PAF-acether binding showed that BN 52021 as well as unlabelled PAF-acether prevented [3H]PAF-acether binding to intact washed platelets. In washed human platelets Kadsurenone affected only PAF-acether-induced aggregation (IC50: 0.8 +/- 0.4 microM (n = 3)), whereas CV 3988 inhibited the aggregation induced by ADP, AA and PAF-acether (IC50 were 10.2 +/- 2.3 microM; 2.2 +/- 0.1 microM; 1.0 +/- 0.1 microM respectively (n = 3). In contrast, up to 30 microM, CV 3988 was a specific antagonist of PAF-acether-induced platelet aggregation in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

BN 52021 displaces [3H]paf-acether from, and inhibits its binding to intact human platelets

Intact platelets incubated at 20 degrees C in the presence of 0.25% (w/v) bovine serum albumin (BSA) bound [3H]paf-acether in a concentration-dependent (0-6.5 nM) manner. Specific [3H]paf-acether binding in the presence of unlabelled paf-acether or BN 52021, a chemically defined ginkgolide, reached a plateau of 14.5 +/- 5 or 17.5 +/- 7.0 fmol at concentrations higher than 0.65 nM [3H]paf-acether. Unlabelled paf-acether or BN 52021 inhibited and displaced [3H]paf-acether binding in a concentration- and time-dependent manner reaching a plateau at 5 nM or 5 microM. Unlabelled paf-acether inhibited [3H]paf-acether binding (75.1 +/- 8%) more strongly if labelled and unlabelled ligands were incubated together than after 15 min preincubation with [3H]paf-acether alone (32.2 +/- 8.6%). The enantiomer of paf-acether (50 nM) failed to displace platelet-bound [3H]paf-acether and the possibility of degradation of [3H]paf-acether by platelets or BN 52021 was excluded under binding conditions. The results demonstrate the presence of specific binding sites for paf-acether in human platelets and the direct competition by BN 52021 for these sites. Serum albumin inhibited total and enhanced specific [3H]paf-acether binding. Radiolabelled albumin itself did not bind to human platelets at 20 degrees C and preincubation of platelets with antibodies to human serum albumin did not inhibit specific paf-acether binding but increased total [3H]paf-acether binding slightly. Serum albumin appears as a necessary phospholipid carrier for specific [3H]paf-acether binding.     

PAF antagonism in vitro and in vivo by aglafoline from Aglaia elliptifolia Merr.

Aglafoline, isolated from Aglaia elliptifolia Merr, inhibited in a selective and concentration-dependent manner the aggregation and ATP release reaction induced in washed rabbit platelets by PAF (platelet-activating factor). The IC50 values of aglafoline, BN52021 and kadsurenone on PAF (3.6 nM)-induced platelet aggregation were about 50, 12 and 18 microM, respectively. Aglafoline also inhibited [3H]PAF (3.6 nM) binding to washed rabbit platelets with an IC50 value of 17.8 +/- 2.6 microM. The concentration-response curve of PAF-induced platelet aggregation was shifted to the right by aglafoline with pA2 and pA10 values of 5.97 and 5.04, respectively. Although thromboxane B2 formation caused by collagen and thrombin was partially suppressed by aglafoline, thromboxane B2 formation caused by ionophore A23187 and arachidonic acid was not affected. Aglafoline inhibited the [3H]inositol monophosphate formation caused by PAF but not that caused by collagen or thrombin in the presence of indomethacin (20 microM). The cAMP content of washed rabbit platelets was not affected by aglafoline. Rat femoral intravenous administration of aglafoline (10 mg/kg) did not affect blood pressure. However, aglafoline (10 mg/kg) both prophylactically and therapeutically antagonized PAF (2.5 micrograms/kg)-induced hypotensive shock in rats. Intravenous PAF (30 ng/kg) caused severe bronchoconstriction in guinea pigs. This effect was completely blocked by aglafoline. This implies aglafoline is an effective PAF antagonist not only in vitro, but also in vivo.     

Interaction of the Paf antagonist WEB 2086 and its hetrazepine analogues with human platelets and endothelial cells.

1. Intact platelets and confluent human umbilical vein endothelial cells bound [3H]-Paf-acether (platelet activating factor, [3H]-Paf) at 20 degrees C in the presence of 0.25% (w/v) bovine serum albumin (BSA). 2. [3H]-Paf binding to platelets was inhibited in a concentration-dependent manner by WEB 2086. An excess of WEB 2086 indicated the presence of specific, saturable Paf binding which reached a maximum of 28.3 +/- 3.7 fmol [3H]-Paf per 5 x 10(7) platelets. In platelets, different hetrazepines (WEB 2098, 2105, but not 2118) also inhibited [3H]-Paf binding in a concentration-dependent manner. 3. WEB 2086 partially displaced platelet-bound [3H]-Paf in a concentration-dependent manner reaching a plateau at 400 nM WEB 2086. No further displacement was observed when WEB 2086 and an excess of unlabelled Paf were added together. 4. The hetrazepines inhibited platelet aggregation. Platelet aggregation IC50 values correlated well with the IC50 values of the hetrazepines against [3H]-Paf binding (r2 = 0.99). WEB 2086 shifted the Paf dose-response curve rightwards in a parallel manner. Tested against platelet aggregation the pA2 obtained for WEB 2086 was 7.9. 5. WEB 2086 inhibited [3H]-Paf binding to endothelial cells in a concentration-dependent manner. WEB 2086 also inhibited the Paf-mediated cytosolic calcium increase in endothelial cells with an IC50 value of 23.1 +/- 10.4 nM as compared with an IC50 of 21.6 +/- 10.4 nM WEB 2086 for platelet aggregation. 6. These results demonstrate an inhibition of [3H]-Paf binding to platelets and endothelial cells by different hetrazepines, most probably at the Paf receptor level.     

High affinity specific binding sites for tritiated platelet-activating factor in canine platelet membranes: counterparts of platelet-activating factor receptors mediating platelet aggregation

In canine platelet membranes, tritiated platelet activating factor (PAF) labels in a saturable and reversible manner a single population (nH = 0.97) of binding sites. The affinity of this binding was high (Kd = 0.23 +/- 0.02 nM, n = 4, and 0.21 +/- 0.05, n = 8, determined by kinetics or saturation experiments, respectively), and the density of binding sites (Bmax) was 911 +/- 31 fmol/mg of protein (n = 8). [3H]PAF binding was entirely reversed by unlabeled PAF (10 microM). [3H]PAF exhibited stereoselective discrimination inasmuch as it was poorly displaced by enantio-PAF, the PAF enantiomer that does not occur naturally. Furthermore, the displacing potency of the (+)-enantiomer of the PAF antagonist 52770 RP against [3H]PAF was 45 times higher than that of the (-)-enantiomer. [3H]PAF binding displayed a remarkable specificity in that it was not affected by a variety of classical pharmacological agents. However, this binding was displaced by several PAF receptor antagonists such as 59227 RP, CV-6209, Ro 19-3704, 52770 RP, brotizolam, WEB 2086, SRI 63-441, L-652,731, alprazolam, triazolam, and BN 52021. The Ki of the 16 studied antagonists ranged from 7.9 nM (59227 RP, most potent) to 16.8 microM (BN 52021, least potent). The possible biological significance of our binding procedure was assessed by correlating the potencies of 16 PAF antagonists as [3H]PAF displacers in dog platelet membranes and as inhibitors of PAF-induced platelet aggregation in washed canine platelets. This analysis revealed the existence of a highly significant correlation (r = 0.82, p less than 0.001) between biochemical and functional tests. However, two compounds (Ro 19-3704 and BN 52021) were found to be located outside the confidence limits when the probability level of belonging to the regression line was set at 0.01. In conclusion, this study provides evidence that [3H]PAF binding in canine platelet membranes exhibits the required properties for a valid binding procedure. Furthermore, the labeled sites are likely to be the counterparts of platelet receptors that, when activated by PAF, induce aggregation.     

Effects of a PAF-antagonist (BN 52063) on bronchoconstriction and platelet activation during exercise induced asthma.

1. The effects of a specific PAF acether antagonist (BN 52063) on the response to isocapnic hyperventilation with dry cold air (ISH study) and exercise (EIA study) were assessed in a single dose and short term treatment study in 10 patients with exercise induced asthma. 2. ISH challenge was performed twice within 1 h after administration of either placebo, 240 mg BN 52063 p.o. or inhalation of 2.4 mg BN 52063. Hyperventilation increased Raw from 0.30 +/- 0.02 to 0.89 kPa s l-1 (P less than 0.001) after the first challenge and from 0.28 +/- 0.04 to 0.84 +/- 0.06 kPa s l-1 (P less than 0.001) after the second challenge. Oral pretreatment with BN 52063 did not result in a reduction of bronchoconstriction during both challenges. A significant increase of Raw was noted immediately after inhalation of BN 52063. An inhibition of PAF induced platelet aggregation (by a factor of 2) occurred after oral administration of BN 52063 after both ISH challenges (P less than 0.05). No significant inhibition of PAF induced platelet aggregation was seen after inhalation of BN 52063. At concentrations up to 30 microM in vitro, BN 52063 inhibited PAF induced platelet aggregation in a dose dependent manner. The IC50 of BN 52063 against the aggregating effect of 1 microM PAF was 7.0 +/- 2.1 microM. 3. In the EIA study the patients were challenged on the third day of treatment with either placebo or 240 mg BN 52063 p.o. or 5 mg BN 52063 by inhalation. Peak expiratory flow rates (PEFR) fell by 155 +/- 37 1 min-1 after exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

PAF binding sites. Characterization by [3H]52770 RP, a pyrrolo[1,2-c]thiazole derivative, in rabbit platelets

52770 RP, the N-(3-chlorophenyl)-3-(3-pyridinyl)-1H,3H-pyrrolo[1,2-c]thiazole -7-carboxamide, displaces in a potent, specific and competitive manner [3H]PAF from its binding sites on rabbit platelets. Since 52770 RP is not structurally related to PAF and has low liposolubility with respect to PAF, it was selected as a potential radioligand for PAF receptor sites. [3H]52770 RP displayed high-affinity, specificity, as well as saturable and displaceable binding to a single class of recognition sites in intact platelets and crude platelet membranes. In these preparations, the values of binding parameters were, respectively, 8.5 and 7.6 nM for Kd, 0.2 pmol/5 X 10(7) platelets and 3.66 pmol/mg protein for Bmax and 0.96 and 0.91 for nH. Inasmuch as the (+)-52770 RP was 300-fold more potent than the (-)-isomer at displacing [3H]52770 RP in intact platelets, the studied binding site manifested stereospecific discrimination. A variety of pharmacological agents including pro- and anti-aggregant compounds did not exhibit affinity for [3H]52770 RP binding sites. In contrast, PAF, some of its active analogues and several recognized PAF antagonists (BN 52021, brotizolam, L-652,731, triazolam), displaced the [3H]52770 RP binding. Studies carried out using [3H]PAF demonstrated that 52770 RP was approximately 4- and 200-fold more potent than L-652,731 and BN 52021 respectively, as a PAF-receptor antagonist. In washed rabbit platelets, the rank order of potency (Ki) for several analogues of 52770 RP, to displace [3H]PAF from its binding sites, was highly correlated (r = 0.96) to their ability to antagonize [3H]52770 RP binding. In functional studies, 52770 RP antagonized not only the PAF-induced aggregation in washed rabbit platelets but also the hypotension evoked by PAF in the anesthetized rat. In this respect, it was 26 and 2 times more potent than L-652,731, respectively. In conclusion, [3H]52770 RP might represent a novel interesting tool for furthering our understanding of the role of PAF binding sites in pathophysiological processes.     

Inhibition by CV-3988 of the binding of [3H]-platelet activating factor (PAF) to the platelet

The inhibitory effects of CV-3988, a specific antagonist of PAF, on the binding of [3H]-PAF to washed platelets of various species including human were examined. The dissociation constant (Kd), binding capacity (Bmax), and the number of receptor/platelet for the specific binding site of rabbit platelets were 2.2 +/- 0.2 nM, 93.7 +/- 8.3 fmoles/10(8) platelets, and 568 +/- 50, respectively. CV-3988 selectively inhibited the specific binding of [3H]-PAF to rabbit platelets with an IC50 of 7.9 X 10(-8) M, and it slightly increased the Kd value (2.5 +/- 0.8 nM) and decreased the binding capacity for PAF (Bmax: 54.3 +/- 16.3 fmoles/10(8) platelets). The Ki value of CV-3988 for the specific binding of [3H]-PAF to rabbit platelets was 1.2 X 10(-7) M. CV-3988 had no effects on the binding of [3H]-5-hydroxytryptamine (5-HT) to rabbit platelets and on the shape change of the platelet induced by 5-HT. CV-3988 also inhibited the specific binding of [3H]-PAF to human and guinea-pig platelets with IC50 values of 1.6 X 10(-7) and 1.8 X 10(-7) M, respectively. CV-3988 inhibited the PAF-induced aggregation in rabbit, guinea-pig, and human platelets. These findings show that CV-3988 is a specific antagonist of PAF at the receptor site(s) of platelets and, in these species, inhibits PAF-induced platelet aggregation by inhibiting the binding of PAF to the "PAF receptor". No specific binding of [3H]-PAF to the platelet of rats and mice was observed, indicating that these species lack a PAF receptor.     

Platelet-activating factor (PAF) inhibitory profile of KO-286011 on blood platelets in vitro and in vivo

Summary A newly synthesised structural analogue of PAF, coded KO-286011 (1-O-hexadecyl-2-O-ethyl-racglycero-3-phosphoric acid 4-(N,N-dimethylamino)pyridinium butylester), was proved for its ability to inhibit PAF-mediated platelet responses in vitro and in vivo. The compound inhibited effectively the PAF-induced aggregation and secretion of human and rabbit platelets. In contrast, there was little influence on ADP-, collagen-, and arachidonic acid-triggered platelet responses. Schild-analysis of aggregation data ascertained in human platelet-rich plasma was consistent with a simple competitive antagonism and yielded a pA₂, of 6.44. Pro-aggregatory activity of KO-286011 was excluded turbidimetrically as well as by means of a single cell counting technique. [³H]PAF binding studies provided evidence that KO-286011 exerts its inhibitory action at the PAF-receptor level. A significant inhibition of the ex vivo PAF-induced platelet aggregation was found after i.v. administration of 0.5 mg/kg KO-286011 to rabbits. The effect was most pronounced 5 min after dosing the inhibitor and detectable over a period of 30 min. Intravenous administration of 10 and 25 μg/kg KO-286011 to guinea pigs prevented dose-dependently the PAF-induced formation of thromboxane A₂. The PAF-inhibitory action of KO-286011 was more potent than that of the ginkgolide BN 52021. Send offprint requests to G. Ostermann at the above address     

Selective inhibition of adrenaline-induced human platelet aggregation by the structurally related Paf antagonist Ro 19-3704.

1. Two non-lipid antagonists of platelet-activating factor acether (Paf), BN 52021 and WEB 2086, at concentrations which completely blocked Paf-induced platelet aggregation, failed to interfere with aggregation by adrenaline. In contrast, Ro 19-3704, a structurally related antagonist of Paf, inhibited concentration-dependently aggregation induced by adrenaline or by the simultaneous addition of submaximal concentrations of adrenaline and Paf. Reversal of aggregation was obtained when Ro 19-3704 was added to the platelet suspension after adrenaline. 2. Ro 19-3704 was selective for Paf and adrenaline since it failed to interfere with platelet aggregation induced by arachidonic acid or ADP. CV-3988, an antagonist of Paf structurally similar to Ro 19-3704, also inhibited adrenaline-induced aggregation. However, a morpholine analogue (MA) of Paf, which has no anti-Paf activity, failed to interfere with the aggregation induced by adrenaline. This suggests that the effect of Ro 19-3704 and CV-3988 on adrenaline is not simply due to their lipid structure. 3. Experiments on plasma membrane preparations showed that Ro 19-3704 inhibited [3H]-yohimbine binding with an inhibition constant (Ki) of 7 +/- 3 microM. In contrast, BN 52021 and MA did not interfere with [3H]-yohimbine binding. Equilibrium binding experiments showed that Ro 19-3704 increased the apparent KD of [3H]-yohimbine binding from 2.02 +/- 0.15 to 7.3 +/- 0.4 nM. The Paf antagonist Ro 19-3704 interacts specifically with the alpha 2-adrenoceptor and may thus prevent the early steps involved in the mechanism of adrenaline-induced platelet activation.     

Comparison of the actions of some platelet-activating factor antagonists on platelets and aortic smooth muscles.

The pharmacological actions of five platelet-activating factor (PAF) antagonists were compared in rabbit platelets and rat thoracic aorta. In PAF (2 ng/ml)-induced aggregation of washed rabbit platelets, WEB 2086 and WEB 2170 much were more potent inhibitors than BN 52021, kadsurenone and denudatin B, and the IC50 values were calculated to be 0.1, 0.3, 5, 8 and 10 micrograms/ml, respectively. WEB 2086, WEB 2170 and BN 52021 did not affect the platelet aggregation caused by collagen (10 micrograms/ml), ADP (20 microM), arachidonic acid (100 microM) or thrombin (0.1 U/ml). Kadsurenone and denudatin B suppressed ATP release, thromboxane B2 formation and the rise in intracellular calcium of washed rabbit platelets caused by collagen and thrombin, while WEB 2086, WEB 2170 and BN 52021 did not have an effect. Norepinephrine (3 microM) induced a sustained contraction in rat thoracic aorta. Pretreatment with these PAF antagonists (20-100 micrograms/ml) caused inhibition of the aortic contraction in the following order: kadsurenone greater than denudatin B greater than WEB 2086 greater than BN 52021 greater than WEB 2170. In high potassium (60 mM)-induced contraction of rat aorta, kadsurenone and denudatin B caused marked relaxation, while WEB 2086, WEB 2170 and BN 52021 had only a slight effect. It is concluded that WEB 2086, WEB 2170 and BN 52021 are specific PAF antagonists in rabbit platelets, and weak relaxants in rat aorta. Two other PAF antagonists, kadsurenone and denudatin B, may inhibit some aspects of signal transduction, e.g., thromboxane formation or intracellular Ca2+ mobilization in rabbit platelets, and cause vasorelaxation in rat aorta by inhibiting calcium influx.     

Triflavin potentiates the antiplatelet activity of platelet activating factor receptor antagonist on activated neutrophil-induced platelet aggregation

In this study, specific platelet activating factor (PAF) receptor antagonist ginkgolide B (BN52021) was tested for its antiplatelet activity in zymosan activated polymorphonuclear neutrophil-induced platelet aggregation. Triflavin was also tested for its antiplatelet activity compared with PAF receptor antagonist. Triflavin, an Arg–Gly–Asp-containing disintegrin purified from venom peptide inhibited platelet aggregation by interfering with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. Furthermore, we also report an efficient high resolution method for quantitative analysis of PAF using high-performance capillary electrophoresis (HPCE). The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed rabbit platelets. In rabbit platelets, BN52021 (100–1000 μM) only partially inhibited activated polymorphonuclear neutrophil-induced platelet aggregation, and its maximal inhibition was estimated to be about 79%. Triflavin also partially inhibited platelet aggregation about 82% induced by activated polymorphonuclear neutrophils. Furthermore, after treatment with a combination of triflavin (0.26 μM) with various concentrations of BN52021 (4–1000 μM), the inhibitory effect of platelet aggregation was almost completely. This inhibition was greater than that produced by the individual drugs alone. These results indicate that a combination of glycoprotein IIb/IIIa complex and PAF receptor antagonist could completely inhibit activated polymorphonuclear neutrophil-induced platelet aggregation. In addition, the amount of PAF released from zymosan (6 mg/ml)-activated polymorphonuclear neutrophils was accurately calculated about 11.8±1.5 ng/10⁶ cells, and did not further increase even at a high concentration of zymosan (10 mg/ml). These results suggest that PAF play a major role in the interaction between platelets and polymorphonuclear neutrophils. This interaction may be important in the pathogenesis of thrombosis and inflammatory diseases. Our present findings support the hypothesis that combination therapy with glycoprotein IIb/IIIa complex antagonists and PAF receptor antagonists may represent a new approach to the treatment of ischemic disorders.     

Protection by two ginkgolides, BN-52020 and BN-52021, against guinea-pig lung anaphylaxis

Interference between the ginkgolides BN-52020 and BN-52021 and the effects of PAF-acether on the cardiovascular and pulmonary functions of guinea-pigs has been studied. BN-52020 (ED50 = 1.1 mg/kg i.v.) and BN-52021 (ED50 = 0.78 mg/kg i.v.) inhibit bronchospasm, hypotension and concomitant generation of TXA2-like activity induced by PAF-acether in anaesthetized guinea-pigs. This protecting activity is specific against PAF-acether since the two ginkgolides do not affect bronchoconstriction, hypotension and TXA2-like activity in the circulating blood due to Histamine, Acetylcholine and LTC4. BN-52021 reduces in a concentration-dependent way the formation of TXB2 caused by PAF-acether in guinea-pig perfused lungs without interference with the effect of Histamine, LTC4 and Arachidonic acid on these tissues. Using actively sensitized (Ovalbumin) guinea-pigs BN-52020 (ED50 = 2.45 mg/kg i.v.) and BN-52021 (ED50 = 1.71 mg/kg i.v.) protect the animals from lethal immunological reaction suggesting that PAF-acether must play a role in the expression of anaphylactic bronchoconstriction and hypotension. The present results indicate that BN-52021 and in a lesser extent BN-52020, which are neither bronchodilators nor cyclooxygenase inhibitors, display a selective antagonistic activity against PAF-acether and may have potential therapeutical implication in asthma.     

Binding of a thromboxane A2/prostaglandin H2 receptor antagonist to guinea-pig platelets

The binding of [125I]9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxypheny l)-13,14- dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 [( 125I]PTA-OH), a thromboxane A2/prostaglandin H2 receptor antagonist, to washed guinea-pig platelets was studied. [125I]PTA-OH bound to guinea-pig platelets in a saturable and displaceable manner. The Kd for [125I]PTA-OH was 14.5 +/- 2 nM (n = 4) and the Bmax was 32 +/- 7 fmol/10(7) platelets or 1,927 +/- 422 binding sites/platelet. The IC50 value for a series of 13-azapinane TXA2 analogs to antagonize the TXA2/PGH2 mimetic U46619-induced platelet aggregation and displace [125I]PTA-OH from its binding site was determined. The IC50 values for the series of five antagonists were highly correlated (r = 0.99) in the binding assays and aggregation studies. The ability of a series of five agonists to displace [125I]PTA-OH from its binding site was compared to their ability to induce platelet aggregation. All the agonists completely displaced the ligand from its binding site but their rank order did not correlate well with their ability to induce aggregation (r = 0.37). Collectively, the data are consistent with the notion that [125I]PTA-OH binds to a putative TXA2/PGH2 receptor in guinea-pig platelets.     

Plasma components are required for platelet activation by the toxin of Loxosceles reclusa

We have used a partially purified toxin from the venom of the brown recluse spider, Loxosceles reclusa, to study its effects on human platelets isolated from plasma proteins. This toxin, which produced skin necrosis in rabbits, contained sphingomyelinase D activity. The toxin induced platelet aggregation and secretion of [3H]serotonin in human plasma but not in buffer or in human neonate plasma. Ca2+ was required for the interaction of toxin, platelets, and plasma factor(s). The addition of C-reactive protein restored aggregation and serotonin release of platelets incubated in human neonate plasma. The ADP-degrading enzyme, apyrase, and the non-steroidal, anti-inflammatory drug, indomethacin, inhibited platelet aggregation, suggesting that ADP secreted from platelet storage granules and indomethacin-sensitive pathway(s) are involve in the toxin-induced human platelet activation (aggregation and serotonin release). Generation of platelet activating factor (PAF) from the platelet by brown recluse toxin is not likely since the PAF receptor antagonist, BN 52021, did not inhibit platelet aggregation induced by the brown recluse toxin.     

Characterization of receptors for platelet-activating factor on platelets, polymorphonuclear leukocytes and macrophages

1. We have compared the potency of the putative platelet-activating factor (Paf) receptor antagonists (WEB 2086, L-652,731 and BN 52021) against Paf-induced aggregation of rabbit and guinea-pig platelets, aggregation of rabbit polymorphonuclear leukocytes (PMNLs) and prostacyclin generation by guinea-pig resident peritoneal macrophages. 2. On rabbit washed platelets and PMNLs WEB 2086, L-652,731 and BN 52021 each antagonized competitively Paf-induced aggregation. The rank order of potency was WEB 2086 congruent to L-652,731 greater than BN 52021 and was the same for the two cell types. 3. The pA2 values for each of the three antagonists were similar on rabbit washed platelets and PMNLs. Moreover, the pA2 for WEB 2086 on rabbit platelets (7.58) did not differ significantly from that on guinea-pig platelets (7.69). 4. On guinea-pig resident peritoneal macrophages WEB 2086 was 10 fold less potent for receptors mediating increased generation of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) than for those mediating platelet aggregation. 5. The potencies of L-652,731 and BN 52021 were also markedly less (2 log units) for the macrophage receptors than for platelet or PMNL receptors and BN 52021 was more potent than L-652,731 in the macrophages. 6. WEB 2086 and L-652,731 significantly reduced basal 6-oxo-PGF1 alpha produced by macrophages, but none of the antagonists affected 6-oxo-PGF1 alpha production during stimulation by A23187. 7. These data raise the possibility that there may be a Paf receptor-subtype mediating prostacyclin generation in macrophages that is different from that on the platelet and PMNL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on antiplatelet effects of OP-41483, a prostaglandin I2 analog, in experimental animals. II. Mechanism of its antiplatelet effect

The mechanism for the inhibition of platelet functions by a prostaglandin I2 analog, OP-41483, was studied with guinea pig platelets. OP-41483, and PGI2 as well, inhibited aggregation, ADP release and thromboxane formation of platelets with IC50 values of 4.3-5.8 ng/ml and 0.6-0.9 ng/ml, respectively. The ligand binding study using [3H]-OP-41483 suggested that OP-41483 bound with different affinities to two classes of binding sites on platelets. The dissociation constant of OP-41483 for the higher affinity site corresponded to the IC50 values of its antiplatelet effect. PGI2 as well as OP-41483 displaced [3H]-OP-41483 previously bound to platelets, thus indicating that both agents exerted their antiplatelet effects by binding to the same site on platelets. OP-41483 and PGI2 activated adenylate cyclase and raised cyclic AMP levels in platelets. However, their inhibitory effect on platelet aggregation was not fully antagonized by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), at a concentration completely inhibiting the increase of cyclic AMP. Moreover, this DDA-resistant effect of OP-41483 disappeared in the presence of calcium chloride (10(-4)-10(-3) M). OP-41483 and PGI2 inhibited thrombin-induced Ca++ influx into platelets. The inhibition of Ca++ influx was not reversed by DDA. Based on these results, we speculate that the inhibitory effects of OP-41483 and PGI2 on platelet functions are produced through dual mechanisms: one mediated by activation of adenylate cyclase and the other by an inhibition of Ca++ influx; and these two mechanisms seem to be independent of each other.     

Effects of verapamil and nisoldipine on human platelets: in vivo and in vitro studies.

Inhibition of platelet aggregation was observed after 4 days of oral dosing with the calcium antagonists, verapamil (160 mg) or nisoldipine (20 mg) but not following acute dosing. These effects were observed at plasma concentrations that had no effect on platelet aggregation when investigated in vitro. Verapamil added in vitro inhibited adrenaline-induced platelet aggregation at relatively low concentrations (IC50 16 microM) but only inhibited aggregation to adenosine diphosphate at very high concentrations (IC50 700 microM). Nisoldipine, a dihydropyridine, added in vitro had no effect on platelet aggregation induced by adenosine diphosphate but inhibited by 67%, the secondary phase of platelet aggregation induced by adrenaline. Verapamil but not nisoldipine displaced [3H]-yohimbine from the specific binding sites on human platelets, suggesting an interaction with alpha 2-adrenoceptors. Inhibition of adrenaline-induced aggregation by verapamil in vitro may be a result of antagonism of alpha 2-adrenoceptors but long term treatment with both verapamil and nisoldipine also inhibits platelet aggregation mechanisms other than by alpha 2-adrenoceptor blockade.