Inhibition of human platelet cyclic AMP phosphodiesterase and of platelet aggregation by a hemisynthetic flavonoid, amentoflavone hexaacetate

Amentoflavone hexaacetate (AmAc) was synthesized from natural amentoflavone (Am), a biflavonoid extracted from Viburnum lantana L. Am does not inhibit aggregation of intact platelets up to a concentration of 100 microM but inhibits human platelet cAMP phosphodiesterase (IC50 = 22.0 microM). AmAc is a potent inhibitor of the aggregation of washed human platelets induced by ADP (IC50 = 2.3 microM) or collagen (IC50 = 4.7 microM). AmAc inhibits crude (IC50 = 8.6 microM) or partially purified (IC50 = 42.2 microM) human platelet cAMP phosphodiesterase. In the presence of prostaglandin E1, AmAc (10 microM) induces a 3.7-fold increase in total platelet cAMP. The characteristics of this action suggest a role for cAMP in the mechanism of action of AmAc. The incubation of AmAc with intact platelets for 5 min is necessary for its activity.     

Inhibition of aggregation of rabbit and human platelets induced by adrenaline and 5-hydroxytryptamine by KB-R7943, a Na(+)/Ca(2+) exchange inhibitor

1. We investigated the effect of KB-R7943, a Na(+)/Ca(2+) exchange inhibitor, on the aggregation response induced by adrenaline and 5-hydroxytryptamine (5-HT), alone or in combination in human and rabbit platelets in the presence or absence of ouabain. 2. KB-R7943 inhibited aggregation induced by the combination of adrenaline and 5-HT in a concentration-dependent manner. The IC(50) values of KB-R7943 were 4.2+/-2.0 or 3.0+/-0.7 microM with washed rabbit platelets with or without ouabain pretreatment, respectively. 3. In platelet-rich human plasma, the aggregation was biphasic. The IC(50) value of KB-R7943 was 17.2+/-4.4 microM for the first phase aggregation. 4. KB-R7943 did not inhibit the first phase of aggregation induced by adrenaline alone, or the monophasic aggregation induced by 5-HT alone. 5. The aggregation of rabbit platelets depended on the presence of K(+) in the medium, and K(+)-dependent and K(+)-independent Ca(2+) influx were observed in resting platelets. Ouabain treatment increased only the K(+)-dependent but not the K(+)-independent Ca(2+) influx. 6. KB-R7943 inhibited K(+)-dependent Ca(2+) influx with or without ouabain pretreatment, but not K(+)-independent Ca(2+) influx. 7. From these results, we conclude that KB-R7943 inhibits the adrenaline plus 5-HT induced aggregation of rabbit and human platelets by inhibiting K(+)-dependent Na(+)/Ca(2+) exchange (NCKX). Our results suggest that NCKX plays an important role in platelet aggregation.     

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)     

Effects of imidazoline and non-imidazoline alpha-adrenergic agents on canine platelet aggregation

Aggregatory and antiaggregatory effects of imidazol(in)e and non-imidazol(in)e alpha-adrenergic agents on canine platelets were examined turbidimetrically in citrated platelet-rich plasma or washed platelet solution. Each alpha-adrenoceptor agonist alone did not induce aggregation, but adrenaline and noradrenaline potentiated dose-dependently aggregation stimulated by ADP, collagen or thrombin. Small potentiation of ADP- or collagen-stimulated aggregation was also observed in response to oxymetazoline. The alpha2-adrenoceptor antagonists and/or imidazol(in)e alpha-adrenergic agents inhibit dose-dependently adrenaline-potentiated aggregation, whereas alpha1-adrenoceptor antagonists, a beta-adrenoceptor antagonist and non-imidazol(in)e alpha-adrenergic agents were no or less effective in inhibiting adrenaline-potentiated aggregation. The alpha2-adrenoceptor agonists did not reduce inhibitory effect of alpha2-adrenoceptor antagonists for adrenaline-potentiated aggregation. The alpha2-adrenoceptor antagonists and/or imidazol(in)es were no or less effective in inhibiting aggregation induced by ADP or thrombin alone. These results demonstrated that alpha2-adrenoceptor-blocking agents and/or imidazol(in)e alpha-adrenergic agents inhibit effectively the adrenaline-potentiated platelet aggregation.     

Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro

1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP.     

Pharmacology of the thromboxane receptor antagonist and thromboxane synthase inhibitor BM-531

BM-531 (N-tert-butyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, is a novel noncarboxylic thromboxane receptor antagonist and thromboxane synthase inhibitor. Indeed, its affinity for human washed platelet TXA2 receptors labeled with [3H]SQ-29548 (IC50 = 0.0078 microM) is higher than sulotroban (IC50 = 0.93 microM) and SQ-29548 (IC50 = 0.021 microM). Moreover, BM-531 is characterized by a potent antiaggregatory property. Indeed, on one hand, in human citrated platelet-rich plasma BM-531 prevents platelet aggregation induced by arachidonic acid (600 microM) (ED100 = 0.125 microM), U-46619, a stable TXA2 agonist (1 microM) (ED50 = 0.482 microM) or collagen (1 microgram/mL) (percentage of inhibition: 42.9% at 10 microM) and inhibits the second wave of ADP (2 microM)-induced aggregation. On the other hand, when BM-531 is incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100) is significantly prolonged. In addition, at the concentrations of 10 and 1 microM, BM-531 totally prevents the production of TXB2 by human platelets activated by arachidonic acid. Finally, at 10 microM, BM-531 significantly prevents rat fundus contractions induced by U-46619 but not by prostacyclin. These results suggest that BM-531, which is devoid of the diuretic property of torasemide, can be regarded as a promising antiplatelet agent.     

YD-3, a novel inhibitor of protease-induced platelet activation

1. In the present study, the antiplatelet effects and mechanisms of a new synthetic compound YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole] were examined. 2. YD-3 inhibited the aggregation of washed rabbit platelets caused by thrombin (IC(50)=28.3 microM), but had no or little inhibitory effect on that induced by arachidonic acid, collagen, platelet-activating factor (PAF) or U46619. YD-3 also suppressed generation of inositol phosphates caused by thrombin. On the other hand, thrombin-induced fibrin formation was not affected by YD-3, indicating YD-3 does not inhibit the proteolytic activity of thrombin. 3. In washed human platelets, however, YD-3 had only mild inhibitory effect on the low concentration (0.05 u ml(-1)) of thrombin-induced human platelet aggregation, and did not affect that induced by higher concentrations (> or =0.1 u ml(-1)) of thrombin or SFLLRN, the protease-activated receptor 1 (PAR1) agonist peptide. By contrast, YD-3 inhibited both human and rabbit platelet aggregation elicited by trypsin with IC(50) values of 38.1 microM and 5.7 microM, respectively. 4. YD-3, at 100 microM, had no effect on ristocetin-induced glycoprotein Ib (GPIb)-dependent aggregation of human platelets. In addition, platelets treated with chymotrypsin, which cleaves GPIb, enhanced rather than attenuated the inhibition of YD-3 on thrombin-induced human platelet aggregation. These data indicate that GPIb plays no role in the antiplatelet effect of YD-3. 5. In SFLLRN-desensitized human platelets, high concentration of thrombin (1 u ml(-1)) could still elicit intracellular Ca(2+) mobilization, and the rise of [Ca(2+)](i) was prevented by either leupeptin or YD-3. 6. Our results suggest that YD-3 inhibits a non-PAR1 thrombin receptor which mediates the major effect of thrombin in rabbit platelets, but in human platelets, this receptor function becomes significant only when the function of PAR1 has been blocked or attenuated.     

Triggering by Paf-acether and adrenaline of cyclo-oxygenase-independent platelet aggregation.

Platelet-activating factor (Paf-acether, 1-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) induced full aggregation and a limited release reaction of human platelets in plasma or in blood. Cyclo-oxygenase inhibition with aspirin only reduced aggregation when induced by threshold amounts of Paf-acether, whereas higher concentrations surmounted inhibition whether tested in citrated or in heparinized platelet-rich plasma or blood. Aspirin-induced inhibition of platelet secretion by Paf-acether was insurmountable and independent of the anti-coagulant used. Paf-acether and adrenaline acted synergistically in inducing aggregation in citrate and heparin. Aspirin in vitro or after oral ingestion at doses that suppressed aggregation induced by arachidonic acid alone, failed to reduce significantly the synergized aggregation induced by Paf-acether alone or combined with adrenaline. Twenty-four hours after the oral ingestion of aspirin, when aggregation by arachidonic acid remained blocked, a slight inhibitory activity on the effect of Paf-acether noted 4 h after aspirin, had ceased. This was probably accounted for by the synthesis of thromboxane A2 by newly formed platelets, since the in vitro addition of aspirin, or of the thromboxane/endoperoxide receptor inhibitor 13-azaprostanoic acid caused the 24 h platelets to behave in a manner similar to platelets collected 4 h after aspirin. The alpha 2-adrenoceptor inhibitor, yohimbine, blocked the direct effect of adrenaline as well as its synergism with Paf-acether. Since the synergistic effect of Paf-acether and adrenaline was maintained when thrombin-degranulated platelets were used, and aspirin remained ineffective against it, it is clear that the augmented platelet responsiveness is not accounted for by the platelet release reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of intravascular platelet aggregation in the rat by adrenaline

Intravascular platelet aggregation to adenosine diphosphate (ADP) was measured in anesthetized rats using 111indium-labelled platelets. Acute adrenalectomy increased the aggregatory effect of ADP in vivo, whereas infusions of low concentrations of adrenaline into adrenalectomized rats suppressed ADP-induced platelet aggregation. Similar antiaggregatory effects were seen with the alpha 2 agonist B-HT 933 and the alpha 1 agonist methoxamine, but not with isoprenaline. The effect of adrenaline was inhibited by phentolamine, yohimbine, the selective alpha 2 adrenoceptor antagonist WY 26392 and by indomethacin, but not by propranolol or prazosin. Adrenaline thus inhibits ADP-induced aggregation in vivo by a mechanism that may involve stimulation of an alpha 2 adrenoreceptor and may be dependent on activation of cyclooxygenase enzyme.     

The effect of SK&F 95654, a novel phosphodiesterase inhibitor, on cardiovascular, respiratory and platelet function.

1. SK&F 95654 inhibited the guanosine 3':5'-cyclic monophosphate (cyclic GMP)-inhibited phosphodiesterase (cGI-PDE) with an IC50 value of 0.7 microM. The IC50 values were greater than 100 microM for the other four phosphodiesterase isoenzymes tested. The R-enantiomer of SK&F 95654 (IC50 = 0.35 microM) was a more potent inhibitor of cGI-PDE than was the S-enantiomer (IC50 = 5.3 microM). 2. In the guinea-pig working heart, SK&F 95654 produced a positive inotropic response without altering heart rate. 3. Oral administration of SK&F 95654 to conscious dogs caused dose-dependent increases in left ventricular dp/dtmax in the range 10-50 micrograms kg-1. These positive inotropic responses were maintained for 3 h without simultaneous changes in heart rate or blood pressure. The peak effects on left ventricular dp/dtmax were similar for orally and intravenously administered compound, indicating good oral bioavailability. 4. SK&F 95654 caused a potent inhibition of U46619-induced aggregation in both a human washed platelet suspension (WPS) (IC50 = 70 nM) and in human platelet-rich plasma (PRP) (IC50 = 60 nM), indicating that the compound shows negligible plasma binding. 5. The R-enantiomer of SK&F 95654 was twenty fold more potent as an inhibitor of platelet aggregation than was the S-enantiomer. The similarity of this ratio to that obtained on the cGI-PDE suggests that SK&F 95654 inhibits platelet aggregation via its effects on cGI-PDE. This was also indicated by studies which showed that SK&F 95654 increased adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels and activated cyclic AMP-dependent protein kinase in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and Antithrombotic Effect of Xanthone Derivatives

A series of xanthone derivatives was synthesized and tested in-vitro for their ability to inhibit aggregation of rabbit washed platelets and human platelet-rich plasma (PRP) induced by various inducers. 2-Prenyloxyxanthone showed the most potent inhibition of rabbit washed platelet aggregation induced by arachidonic acid (IC50 = 10.2 μM). Of the compounds tested in human PRP, 2-[3 (propylamino)-2-hydroxypropoxy]xanthone (4) hydrochloride salt exhibited the most potent inhibition of platelet aggregation induced by adrenaline (IC50 = 4.4 μM), whereas in evaluation of mouse antithrombotic activity, compound 4 exhibited the most potent protection of mice from thrombotic challenge. Compound 4, 2-[3-(isopropylamino)-2-hydroxypropoxylxanthone hydrochloride salt and 2,5 dihydroxyxanthone suppressed the secondary aggregation induced by adrenaline in human PRP. We conclude that the antiplatelet effects of these compounds are mainly due to an inhibitory effect on thromboxane formation.     

Effects of plumbagin on platelet aggregation and platelet-neutrophil interactions

The effects of plumbagin were investigated on platelet aggregation in vitro and ex vivo, on the binding of thrombin-stimulated platelets to neutrophils, and platelet aggregation induced by intact neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or platelet activating factor (PAF) activated neutrophils, by use of the methods of Hamburger, McEver and Born, respectively. The results showed that plumbagin in vitro significantly inhibited adenosine diphosphate (ADP)-, arachidonic acid (AA)-, or platelet activating factor (PAF)-induced platelet aggregation, in a concentration-dependent manner. The medium inhibitory concentrations (IC 50 ) were 39.4, 82.7 and 38.1 microM, respectively. Intragastric plumbagin at 10 mg/kg markedly suppressed platelet aggregation induced by ADP, AA, or PAF. Plumbagin decreased the binding between thrombin-stimulated platelets and neutrophils with an IC 50 of 62.9 microM. Plumbagin significantly inhibited washed platelet aggregation stimulated by fMLP- or PAF-activated neutrophils. The IC 50 values were 54.3 and 47.6 microM, respectively. On the other hand, plumbagin and aspirin increased the inhibition of intact neutrophils on AA-induced platelet aggregation. It is suggested that plumbagin inhibited platelet aggregation in vitro and ex vivo, suppressed the binding of activated platelets to neutrophils, inhibited platelet aggregation induced by activated neutrophils, and increased inhibition of intact neutrophils on platelet reactivity. Abbreviations. DMSO:dimethyl sulphoxide fMLP: N-formyl-methionyl-leucyl-phenylalanine ADP:adenosine diphosphate AA:arachidonic acid PAF:platelet activating factor     

An antagonistic activity of etizolam on platelet-activating factor (PAF). In vitro effects on platelet aggregation and PAF receptor binding

The antagonistic effect of etizolam, an anti-anxiety drug, on platelet-activating factor (PAF) was investigated in rabbit platelets in vitro. Etizolam inhibited PAF-induced aggregation in a dose-dependent manner, with an IC50 of 3.8 microM, about one tenth that of triazolam (IC50 = 30 microM). At 300 microM, it inhibited both ADP and arachidonic acid-induced aggregation only slightly, while the other anti-anxiety drugs tested had no effect on PAF-induced aggregation even at this concentration. Etizolam and triazolam inhibited the specific binding of 3H-PAF to PAF receptor sites on washed rabbit platelets with IC50 values of 22 nM and 320 nM, respectively. Diazepam and estazolam were inactive even at 1 microM. These results indicate that etizolam is a specific antagonist of PAF.     

Antiplatelet effect of 2-chloro-3-(4-acetophenyl)-amino-1,4-naphthoquinone (NQ301): a possible mechanism through inhibition of intracellular Ca2+ mobilization.

The effects of 2-chloro-3-(4-acetophenyl)-amino-1,4-naphthoquinone (NQ301), an antithrombotic agent, on aggregation, binding of fibrinogen to glycoprotein (GP)IIb/IIIa complex and intracellular signals were investigated using human platelets. NQ301 significantly inhibited the collagen-, thrombin-, arachidonic acid-, thapsigargin- and calcium ionophore A23187-induced aggregation of washed human platelets with IC50 values of 13.0+/-0.1, 11.2+/-0.5, 21.0+/-0.9, 3.8+/-0.1 and 46.2+/-0.8 microM, respectively. NQ301 also significantly inhibited FITC-conjugated fibrinogen binding to human platelet surface GPIIb/IIIa complex, but failed to inhibit the fibrinogen binding to purified GPIIb/IIIa complex. These data demonstrate that NQ301 inhibits platelet aggregation by suppression of the intracellular pathway, rather than by direct inhibition of fibrinogen-GPIIb/IIIa complex binding. NQ301 significantly inhibited the increase of cytosolic Ca2+ concentration and ATP secretion, and also significantly increased platelet cAMP levels in the activated platelets. These results suggest that the antiplatelet activity of NQ301 may be mediated by inhibition of cytosolic Ca2+ mobilization, enhancement of cAMP production and inhibition of ATP secretion in activated platelets.     

Effects of the P2-purinoceptor antagonist, suramin, on human platelet aggregation induced by adenosine 5'-diphosphate.

1. The effects of suramin, a trypanocidal drug which has been reported to be a P2-purinoceptor antagonist on smooth muscle, were investigated in human platelets, where adenosine 5'-diphosphate (ADP) induces aggregation by acting on a subtype of purinoceptors which has been called P2T. 2. Suramin (100 microM) had no inhibitory effect on ADP-induced platelet aggregation in plasma, even after 40 min incubation in the presence of bacitracin, a peptidase inhibitor, and did not affect the ability of adenosine 5'-triphosphate (ATP) (40 microM) to inhibit competitively ADP-induced aggregation. This lack of effect of suramin on platelets in plasma is probably due to its extensive binding to plasma proteins. 3. In washed platelets, suramin (50-400 microM) acted as an apparently competitive antagonist, causing parallel shifts to the right of the log concentration-response curve to ADP. No depression of the maximal response to ADP was observed at concentrations of suramin (50-150 microM) for which full log concentration-response curves to ADP could be obtained, but the slope of the Schild plot was around 2, indicating that this antagonism was not simply competitive. The apparent pA2 value for suramin, taken from this Schild plot, was 4.6. 4. Suramin (200-400 microM) also noncompetitively inhibited aggregation induced by U46619 (a thromboxane receptor agonist) or by 5-hydroxytryptamine in the presence of adrenaline (100 microM), and caused a depression of the maximal response to these agonists. This nonspecific effect of suramin may explain the high Schild plot slope obtained against ADP.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Structure-activity relationship studies on chalcone derivatives: potent inhibition of platelet aggregation

In an effort to develop potent antiplatelet agents with anti-inflammatory action, a novel series of anti-inflammatory chalcones was screened to evaluate their antiplatelet effects. Structure-activity relationships and mode of action were investigated and characterized. The antiplatelet effects of the chalcones on washed rabbit platelets and human platelet-rich plasma were evaluated. Arachidonic acid-induced platelet aggregation was potently inhibited by almost all the chalcone derivatives. Collagen-induced platelet aggregation was potently inhibited by all the chalcone derivatives at 300 microM, except for compound 4 at 100 microM. Compounds 6, 7 and 9 significantly inhibited the aggregation of washed rabbit platelets induced by platelet-activating factor at 300 microM. Of the compounds tested in human platelet-rich plasma, compounds 2, 8 and 9 showed significant inhibition of secondary aggregation induced by adrenaline. It is concluded that the antiplatelet effect of 2, 8 and 9 is mainly owing to an inhibitory effect on thromboxane formation. The inhibitory effect of 6, 7 and 9 on platelet aggregation induced by platelet-activating factor could be owing to a calcium antagonizing effect or inhibition of intracellular calcium mobilization.     

盐酸非洛普对人和兔血小板聚集的影响

用比浊法和放射免疫法分别测定盐酸非洛普对兔和人血小板聚集及兔血栓素Ａ２（ＴＸＡ２）和动脉壁前列环素（ＰＧＩ２）含量的影响。盐酸非洛普呈剂量依赖性抑制ＡＤＰ（ＩＣ５０＝５．８×１０－４ｍｏｌ·Ｌ－１）和ＡＡ诱导的兔血小板聚集。对ＡＤＰ和Ａｄｒ诱导的人血小板聚集亦有明显抑制作用且呈剂量依赖性，ＩＣ５０值分别为１．２和１．３×１０－４ｍｏｌ·Ｌ－１。盐酸非洛普短期应用（８ｍｇ·ｋｇ－１ｉｖｔｉｄ×２ｄ）明显抑制ＡＤＰ和ＡＡ诱导的兔血小板聚集及ＴＸＢ２的产生和释放，对兔动脉壁和血浆６－ｋｅｔｏ－ＰＧＦ１α含量无显著影响。研究结果表明，盐酸非洛普抗血小板聚集作用与其抑制血小板ＴＸＡ２合成和释放有关，亦可能与其α２受体阻断作用有关。     

Studies on inhibition of human platelet response by diltiazem

1. Aim of the present investigation was to investigate the effects of calcium blocking agent diltiazem on human platelet response to aggregating agents. 2. Results showed that diltiazem inhibits platelet aggregation induced by ADP, arginine vasopressin, adrenaline, collagen, Na arachidonate, thrombin and phorbol ester PMA in a dose-dependent way. 3. Diltiazem decreased also beta-thromboglobulin release and Thromboxane B2 production from stimulated platelets. 4. Intraplatelet cyclic AMP levels were not modified by the substance. 5. Data provide evidence that the modulation of human platelet function by diltiazem could be also related to inhibition of protein kinase C.     

Effect of trilinolein on cyclic nucleotide formation in human platelets: relationship with its antiplatelet effect and nitric oxide synthesis.

1. Trilinolein, a triacylglycerol with linoleic acid as the only fatty acid residue in all three esterified positions of glycerol, was recently reported to have an inhibitory effect on adrenaline-induced platelet aggregation. In the present study, we found that trilinolein at concentrations ranging from 0.01 to 1 microM increased cyclic GMP formation and decreased cyclic AMP formation in washed human platelets. Both NG-nitro-L-arginine methyl ester (L-NAME) and methylene blue attenuated the trilinolein-induced increase in cyclic GMP. 2. Adrenaline decreased not only the production of cyclic AMP but also that of cyclic GMP. Trilinolein antagonized the inhibitory effect of adrenaline on cyclic GMP formation, but potentiated the inhibitory effect of adrenaline on cyclic AMP accumulation. 3. Both trilinolein and adrenaline enhanced intracellular calcium but the increment of intracellular calcium induced by them was much less than that produced by thrombin. 4. We propose that the anti-platelet effect of trilinolein is mediated through an increase in cyclic GMP, and that the change in cyclic GMP results from stimulation of nitric oxide synthesis in platelets. 5. We also propose that reduction of both cyclic AMP and cyclic GMP are involved in adrenaline-induced platelet aggregation.