Potentiation by adrenaline of human platelet activation and the inhibition by the alpha-adrenergic antagonist nicergoline of platelet adhesion, secretion and aggregation

Adrenaline (1 to 10 microM) can induce the aggregation of human platelets suspended in citrated plasma but does not induce the aggregation of washed human platelets at doses as high as 1 mM, although these platelets respond normally to ADP, PAF-acether, collagen, arachidonic acid, thrombin, the endoperoxide analog U-46619 and the Ca2+ ionophore A23187. Adrenaline (0.5 microM) potentiates the aggregation and secretion induced by all the previous agonists in citrated platelet-rich plasma (cPRP) or in washed platelets. The activation by adrenaline of human platelets is mediated by alpha 2-adrenergic receptors, as demonstrated by inhibition with a series of adrenergic antagonists. The alpha-adrenergic antagonist nicergoline inhibits the activation of human platelets by adrenaline in the following situations: nicergoline inhibits the aggregation and secretion caused by adrenaline in cPRP (IC50 0.22 microM and 0.28 microM respectively); nicergoline inhibits the aggregation and secretion induced by the combination of adrenaline and each aggregating agent listed above in cPRP (IC50 ranging from 0.1 to 2.5 microM) or in washed platelets (IC50 ranging from 0.1 to 0.8 microM); nicergoline inhibits the binding of 3H-yohimbine to washed human platelets (IC50 0.26 microM); the intravenous administration of nicergoline (0.5 mg/kg per day) to patients inhibits significantly the ex vivo response of their platelets to adrenaline in cPRP. High concentrations of nicergoline also inhibit the aggregation and secretion induced by the aggregating agents listed above in cPRP (IC50 range 108 to 670 microM) and in washed platelets (IC50 range 27 to 140 microM) and the adhesion of platelets to collagen-coated surfaces. This latter effect is not mediated through blockade of alpha-adrenoceptors. A possible role of adrenaline in platelet activation in vivo could justify the use of nicergoline (Sermion), an alpha-adrenergic antagonist in combination therapy to prevent arterial thrombosis.     

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)     

Cilostazol and dipyridamole synergistically inhibit human platelet aggregation

It has been previously shown that cilostazol (Pletal), a drug for relief of symptoms of intermittent claudication, potently inhibits cyclic nucleotide phosphodiesterase type 3 (PDE3) and moderately inhibits adenosine uptake. It elevates extracellular adenosine concentration, by inhibiting adenosine uptake, and combines with PDE3 inhibition to augment inhibition of platelet aggregation and vasodilation while attenuating positive chronotropic and inotropic effects on the heart. In the present study, we tested the hypothesis that cilostazol combined with a more potent adenosine uptake inhibitor, dipyridamole, synergistically inhibited platelet aggregation in human blood. In the presence of exogenous adenosine (1 microM), the combination of cilostazol and dipyridamole synergistically increased intra-platelet cAMP. Furthermore, cilostazol inhibited platelet aggregation in a washed platelet assay concentration-dependently with IC50s of 0.17 +/- 0.04 microM (P < 0.05 versus plus adenosine alone of 0.38 +/- 0.05 microM), 0.11 +/- 0.06 microM (P < 0.05), and 0.01 +/- 0.01 microM (P < 0.005) when combined with 1, 3, or 10 microM dipyridamole, respectively (n = 5). In whole blood, cilostazol (0.3 to 3 microM) and dipyridamole (1 or 3 microM) synergistically inhibited collagen- and ADP-induced platelet aggregation in vitro. Furthermore, the synergism was confirmed in an open-label, sequential study in healthy human subjects using ex vivo whole-blood collagen-induced platelet aggregation. Four hours after oral co-administration of cilostazol (100 mg) and dipyridamole (200 mg), platelet aggregation was inhibited by 45 +/- 17%, while no significant inhibition was observed from subjects treated with either drug alone. The combination may provide a potential treatment of arterial thrombotic disorders.     

Mechanisms involved in the antiplatelet activity of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H-1-benzopyran-2-one (RC414)

The effect on human platelets of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H-1-benzopyran-2-one (RC414) was tested in vitro by measuring aggregation induced by several agonists, cAMP and cGMP levels, cAMP phosphodiesterase and PKC activities and [Ca2+]i. The RC414 effect on nitric oxide production was also evaluated. RC414 in a dose-dependent manner inhibited aggregation both in platelet rich plasma and in washed platelets. It was particularly effective in platelets challenged by collagen, ADP and thrombin: IC50 values are 0.51 +/- 0.12 microM, 0.98 +/- 0.36 microM and 1.00 +/- 0.15 microM, respectively. RC414 increased cAMP levels, through the specific inhibition of the cAMP high affinity phosphodiesterase (IC50 = 1.73 +/- 0.35 microM). RC414 reduced [Ca2+]i transients and PKC activation induced by thrombin. In addition RC414 was able to increase nitric oxide formation involving the stimulation of constitutive nitric oxide synthase enzyme. In conclusion, RC414 exerts its powerful anti-platelet activity by increasing cAMP intracellular levels and nitric oxide formation.     

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 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     

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.     

Effects of SK&F 94120, an inhibitor of cyclic nucleotide phosphodiesterase type III, on human platelets

Elevation of cyclic AMP concentrations in platelets inhibits agonist-induced responses. Pharmacological interventions which could increase the levels of platelet cyclic AMP include activation of the synthesis of cyclic AMP or inhibition of its breakdown. In this study we have investigated the effects of SK&F 94120 on human platelet phosphodiesterase (PDE) activities separated by ion-exchange chromatography, and studied the effects of this agent on platelet responses caused by the agonists collagen, U44069 and ADP. Four PDE activities were identified from human platelet preparations. The PDE activities found comprised a cyclic GMP selective PDE, a Ca2+/calmodulin stimulated PDE, a cyclic GMP stimulated PDE and a "low Km" PDE activity called PDE III by analogy with activities described in other tissues. SK&F 94120 was found selectively to inhibit the "low Km" PDE III activity with an IC50 of 10.8 microM, which is consistent with the effects of this compound on cardiac ventricle PDE activities. Exposure of human platelets to SK&F 94120 produced concentration dependent increases in cyclic AMP, showing that inhibition of PDE III activity alone can cause an increase in the level of platelet cyclic AMP. SK&F 94120 also caused an inhibition of platelet responses to collagen, U44069 and ADP. However, SK&F 94120 was much less effective as an inhibitor of aggregation induced by ADP (IC50 greater than 100 microM) than by collagen (IC50 = 24.1 microM) or by U44069 (IC50 = 1.7 microM). Isobutylmethylxanthine (IBMX), a non-selective PDE inhibitor, was less effective than SK&F 94120 as an inhibitor of platelet responses for the same measured increase in cyclic AMP levels. M&B 22948 and rolipram, inhibitors of PDE I and PDE IV respectively, had no significant effect on platelet responses. These data suggest that selective inhibition of PDE III is the primary mechanism of action of SK&F 94120 as an inhibitor of agonist-induced platelet responses, and that increased cyclic AMP in the pool controlled by PDE III has important consequences on platelet responses. Moreover, these data suggest that some form of compartmentalization of cyclic AMP and/or PDE activity exists in human platelets.     

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.     

3,4-Dihydroquinolin-2(1H)-ones as combined inhibitors of thromboxane A2 synthase and cAMP phosphodiesterase.

A series of 1H-imidazol-1-yl- and 3-pyridyl-substituted 3,4-dihydroquinolin-2(1H)-ones was designed and synthesized as combined inhibitors of thromboxane (TXA2) synthase and cAMP phosphodiesterase (PDE) in human blood platelets. A number of structures, e.g. 4b, 7a, 7e, 13a, and 21-25, were superior to dazoxiben 26 as inhibitors of TXA2 synthase in in vitro ADP-induced aggregation experiments with human blood platelets. The TXA2 synthase inhibitory activity was confirmed by measurement of the prostanoid metabolites derived from 14C-labeled arachidonic acid. Three compounds (7a, 7e, and 25) demonstrated in vitro inhibition of human platelet cAMP PDE at micromolar concentrations in conjunction with their TXA2 synthase inhibitory activity. Synergistic enhancement of antiaggregatory and antithrombotic actions was expected when simultaneous stimulation of adenylate cyclase (through increased PGI2 production) and inhibition of platelet cAMP PDE were possible from the same compound. Ex vivo and in vivo experiments were conducted in rats and mice, respectively, to evaluate the effects of compounds 7e and 23 on platelet aggregation and thrombotic events within these animals. Compound 7e, which has a comparable level of TXA2 synthase (IC50 1.2 microM) and human platelet cAMP PDE (IC50 6.4 microM) inhibitory activities, was found to be orally bioavailable with a long duration of action and offered effective protection against mortality in a collagen-epinephrine-induced pulmonary thromboembolism model in mice. Significant blood pressure and heart rate effects were observed for several compounds, e.g. 7e, 9e, 13a, 13d, 18, 20, 21, and 23, when dosed orally in conscious spontaneously hypertensive rats.     

Effects of fibrinogen receptor antagonist GR144053F and aurintricarboxylic acid on platelet activation and degranulation.

Activated blood platelets play crucial role in restenosis due to their fundamental significance in thrombus formation. Therefore, platelets are attractive targets for the inhibition with a variety of antagonists. In this study, we present direct evidence that GR144053F [non-peptide antagonist of glycoprotein IIb-IIIa complex (GPIIb-IIIa)] inhibits activation and degranulation of human platelets, and opposes the action of aurintricarboxylic acid (ATA), the antagonist of von Willebrand factor, which augments platelet secretion. The effects of both drugs on platelet function were monitored by using various instrumental methods. Platelet-rich plasma and whole-blood aggregation was measured by using ADP and collagen as agonists. Platelet degranulation was assessed based on the expression of surface membrane activation markers: P-selectin, glycoprotein Ib, and activated GPIIb-IIIa complex. Measurements of closure time with platelet function analyzer PFA-100 enabled us to reason on primary hemostatic capacity and reflected both aggregability and adhesiveness. GR144053F markedly reduced primary hemostatic platelet response (IC(50) = 114.0 +/- 9.6 nM) under conditions that closely mimicked natural blood flow in circulation, and inhibited aggregation in platelet-rich plasma (IC(50) = 17.7 +/- 7.0 nM). It was equally potent inhibitor of platelet activation, degranulation, fibrinogen binding, platelet consumption, and aggregate formation. Also, ATA was efficient in inhibition of platelet aggregation and adhesion (by up to 50% at 100 microM), but the combined action of both drugs on primary haemostatic capacity was not additive. GR144053F suppressed the activating effects of ATA on platelet degranulation and secretion. Overall, our data indicate that GR144053F is not only the efficient blocker of fibrinogen binding to GPIIb-IIIa, but also hampers platelet degranulation and may attenuate the activating effects of ATA.     

New mechanism of action for cilostazol: interplay between adenosine and cilostazol in inhibiting platelet activation

Cilostazol, a potent phosphodiesterase 3 inhibitor and anti-thrombotic agent, was recently shown to inhibit adenosine uptake into cardiac myocytes and vascular cells. In the present studies, cilostazol inhibited [ H]-adenosine uptake in both platelets and erythrocytes with a median inhibitory concentration (IC ) of 7 micro M. Next collagen-induced platelet aggregation was studied and it was found that adenosine (1 micro M ), having no effect by itself, shifted the IC of cilostazol from 2.66 micro M to 0.38 micro M (p < 0.01). This shifting was due to an enhanced accumulation of cAMP in platelets and was significantly larger than that by the combination of adenosine and milrinone, which has no effect on adenosine uptake. Similarly, cilostazol, by blocking adenosine uptake, enhanced the adenosine-mediated cAMP increase in Chinese hamster ovary cells that overexpress human A receptor. Furthermore, the inhibitory effect of cilostazol on platelet aggregation in whole blood was significantly reversed by ZM241385 (100 n ), an A adenosine receptor antagonist, and by adenosine deaminase (2 U/ml). These data suggest that the inhibitory effects of cilostazol on adenosine uptake and phosphodiesterase 3 together elevate intracellular cAMP, resulting in greater inhibition of agonist-induced platelet activation.     

Inhibitory effect of curcumin, a food spice from turmeric, on platelet-activating factor- and arachidonic acid-mediated platelet aggregation through inhibition of thromboxane formation and Ca2+ signaling.

Curcumin, a dietary spice from turmeric, is known to be anti-inflammatory, anticarcinogenic, and antithrombotic. Here, we studied the mechanism of the antiplatelet action of curcumin. We show that curcumin inhibited platelet aggregation mediated by the platelet agonists epinephrine (200 microM), ADP (4 microM), platelet-activating factor (PAF; 800 nM), collagen (20 microg/mL), and arachidonic acid (AA: 0.75 mM). Curcumin preferentially inhibited PAF- and AA-induced aggregation (IC50; 25-20 microM), whereas much higher concentrations of curcumin were required to inhibit aggregation induced by other platelet agonists. Pretreatment of platelets with curcumin resulted in inhibition of platelet aggregation induced by calcium ionophore A-23187 (IC50; 100 microM), but curcumin up to 250 microM had no inhibitory effect on aggregation induced by the protein kinase C (PKC) activator phorbol myrsitate acetate (1 microM). Curcumin (100 microM) inhibited the A-23187-induced mobilization of intracellular Ca2+ as determined by using fura-2 acetoxymethyl ester. Curcumin also inhibited the formation of thromboxane A2 (TXA2) by platelets (IC50; 70 microM). These results suggest that the curcumin-mediated preferential inhibition of PAF- and AA-induced platelet aggregation involves inhibitory effects on TXA2 synthesis and Ca2+ signaling, but without the involvement of PKC.     

Inhibitors of blood platelet cAMP phosphodiesterase. 3. 1,3-Dihydro-2H-imidazo[4,5-b]quinolin-2-one derivatives with enhanced aqueous solubility.

Two series of 1,3-dihydro-2H-imidazo[4,5-b]quinolin-2-one derivatives incorporating an additional site for acid salt formation were synthesized and evaluated as inhibitors of human blood platelet cAMP phosphodiesterase (PDE) and ADP-induced platelet aggregation. The objective of this study was to identify compounds that blended potent biological activity with a satisfactory level of aqueous solubility. From a series of 7-aminoimidazo[4,5-b]quinolin-2-ones, biological and physical properties were optimally combined in the 1-piperidinyl derivative 11c. However, this compound offered no significant advantage over earlier studied compounds as an antithrombotic agent in an animal model of small vessel thrombosis. A series of 7-alkoxy alkanoic piperazinamide derivatives, in which the additional basic nitrogen atom was remote from the heterocyclic nucleus and accommodated in a secondary binding region of the cAMP PDE enzyme, demonstrated greater intrinsic cAMP PDE inhibitory activity. Structural modifications of this series focused on variation of the piperazine substituent and side-chain length. The lipophilicity of the N-substituent influenced biological potency and aqueous solubility, with substituents of seven carbon atoms or less generally providing acceptable solubility properties. The N-(cyclohexylmethyl)piperazinamide 21h was identified from this series of compounds as a potent inhibitor of platelet cAMP PDE, IC50 = 0.4 nM, and ADP-induced platelet aggregation, IC50 = 0.51 microM after a 3-min exposure and 0.1 microM after a 15-min exposure of platelet-rich plasma to the drug. Evaluation of 21h and representative analogues in vivo using a rabbit model of small vessel thrombosis revealed significantly greater antithrombotic efficacy compared to that of previously studied compounds with similar intrinsic biological activity measured in vitro but inferior aqueous solubility.     

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.     

A potent and selective inhibitor of cyclic AMP phosphodiesterase with potential cardiotonic and antithrombotic properties

Some biochemical and pharmacological properties of a novel, potent inhibitor of cyclic AMP phosphodiesterase, N-cyclohexyl-N-methyl-4-(7-oxy-1,2,3,5-tetrahydroimidazo[2,1-b] quinazolin-2-one) butyramide (RS-82856), were investigated. RS-82856 selectively inhibits the high affinity form of cyclic AMP phosphodiesterase (type IV) isolated from human platelets (Ki = 0.5 nM) with only weak effects on both the nonspecific and cyclic GMP-sensitive phosphodiesterases. The inhibitor reduces both maximum velocity and substrate affinity of the type IV enzyme. This mixed pattern of partial competitive and noncompetitive inhibition was also obtained with one of the two high affinity forms of phosphodiesterase found in dog heart (Ki = 0.75 nM). Of several human and dog tissues examined, RS-82856 exhibits marked selectively for the platelet high affinity enzyme. It also has significant inhibitory effects on cardiac membrane-bound phosphodiesterase. RS-82856 inhibits the aggregation of human platelets in response to adenosine 5'-diphosphate (IC50 = 0.11 microM) in vitro and is active ex vivo for at least 2 hr following oral administration (10 mg/kg) to rhesus monkeys. Administration of RS-82856 to instrumented, anesthetized dogs by either intravenous or intraduodenal routes increases cardiac contractile force and reduces afterload. These data suggest that RS-82856 may be useful as an agent to increase cardiac output in the treatment of congestive heart failure.     

YC-1 inhibited human platelet aggregation through NO-independent activation of soluble guanylate cyclase.

1. Our previous study demonstrated that YC-1, a derivative of benzylindazole, is a novel activator of soluble guanylate cyclase (sGC) in rabbit platelets. This work investigated whether the antiplatelet effect of YC-1 was mediated by a nitric oxide (NO)/sGC/cyclic GMP pathway in human platelets. 2. In human washed platelets, YC-1 inhibited platelet aggregation and ATP released induced by U46619 (2 microM), collagen (10 micro ml(-1)) and thrombin (0.1 u ml(-1)) in a concentration-dependent manner with IC50 values of (microM) 2.1 +/- 0.03, 11.7 +/- 2.1 and 59.3 +/- 7.1, respectively. 3. In a 30,000 g supernatant fraction from human platelet homogenate, YC-1 (5-100 microM) increased sGC activity in a concentration-dependent manner. At the same concentration-range, YC-1 elevated cyclic GMP levels markedly, but only slightly elevated cyclic AMP levels in the intact platelets. 4. MY-5445, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in cyclic GMP caused by YC-1, and shifted the concentration-anti-aggregation curve of YC-1 to the left. In contrast, HL-725, a selective inhibitor of cyclic AMP phosphodiesterase, did not affect either the increases in cyclic nucleotides or the anti-aggregatory effect caused by YC-1. 5. Methylene blue, an inhibitor of sGC, blocked the increases of cyclic GMP caused by YC-1, and attenuated markedly the anti-aggregatory effect of YC-1. The adenylate cyclase inhibitor, 2'',5''-dideoxyadenosine (DDA) did not affect YC-1-induced inhibition of platelet aggregation. 6. Haemoglobin, which binds NO, prevented the activation of sGC and anti-aggregatory effect caused by sodium nitroprusside, but did not affect YC-1 response. 7. These results would suggest that YC-1 activates sGC of human platelets by a NO-dependent mechanism, and exerts its antiplatelet effects through the sGC/cyclic GMP pathway.     

Morphine-potentiated agonist-induced platelet aggregation through alpha2-adrenoceptors in human platelets

Morphine dose-dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and ATP release stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Furthermore, morphine (1 and 5 microM) markedly potentiated collagen (1 microg/ml) evoked an increase of intracellular Ca2+ mobilization in fura 2-AM loading human platelets. Morphine (1 and 5 microM) did not influence the binding of fluorescein isothiocyanate-triflavin to platelet glycoprotein IIb/IIIa complex. Yohimbine (0.1 microM), a specific alpha2-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by collagen. Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E1 (10 microM)-induced cAMP formation in human platelets, and yohimbine (0.1 microM) significantly reversed the inhibition of cAMP by morphine (0.6 and 1 microM) in this study. Morphine (1 and 5 microM) significantly potentiated thromboxane B2 formation stimulated by collagen in human platelets, and yohimbine also reversed this effect of morphine in this study. In addition, morphine (1 and 5 microM) did not significantly affect nitrate production in human platelets. Morphine may exert its potentiation in platelet aggregation by binding to alpha2-adrenoceptors in human platelets, which leads to reduced cAMP formation and subsequently to increased intracellular Ca2+ mobilization; this, in turn, is followed by increased thromboxane A formation and finally potentiates platelet aggregation and ATP release.     

Inhibition of human platelet aggregation by dihydropyrano- and dihydrofuranocoumarins, a new class of cAMP-phosphodiesterase inhibitors

Certain esters of dihydropyranocoumarin and dihydrofuranocoumarin alcohols have previously been shown to inhibit the cAMP-phosphodiesterase from bovine heart. We now report that these naturally occurring coumarins inhibit the high affinity (Km = 1.1 microM) cAMP-phosphodiesterase from human platelets with activities that closely correlate with those obtained using phosphodiesterase from bovine heart tissue. Additionally the coumarins inhibit the aggregation of human platelets induced with ADP, adrenaline and collagen with activities comparable to those of dipyridamole. A lack of significant correlation between these metabolic and functional activities indicates that there exist, besides cAMP-phosphodiesterase inhibition, additional mechanisms of action for the platelet aggregation inhibitory effect of dihydropyrano- and dihydrofuranocoumarins.     

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.