Thromboxane synthase inhibition potentiates washed platelet activation by endogenous and exogenous arachidonic acid

The effect of the thromboxane (TX) synthase inhibitors dazoxiben and imidazole on platelet activation by endogenous and exogenous arachidonic acid (AA) was tested with human washed platelets. Dazoxiben (1-20 microM) inhibited the formation of TXB2 and markedly enhanced the shape change, aggregation, and (3H)serotonin release induced by added AA or when prostaglandin synthesis from endogenous AA was triggered by collagen, hydrogen peroxide or methyl mercury chloride (methyl-Hg). Platelet activation by hydrogen peroxide (20-1200 microM) or methyl-Hg (1-5 microM) was entirely dependent on endogenous prostaglandin (PG) synthesis since acetylsalicylic acid (ASA), indomethacin or the cyclic endoperoxide/TXA2-antagonist BM 13.177 counteracted these stimulants with and without dazoxiben. Apparently, the potentiation is due to accumulating cyclic endoperoxides which during TX synthase inhibition reach greater platelet-activating potency than TXA2. Albumin or human platelet-poor plasma inhibited the platelet activation by hydrogen peroxide and methyl-Hg and suppressed the potentiation by dazoxiben. The latter effect of albumin may result from its PGD isomerase activity which redirects the cyclic endoperoxide metabolism to the platelet-inhibitory PGD2. The results show that non-platelet factors such as albumin are necessary to prevent a potentiating effect of TX synthase inhibitors on platelet activation.     

Thromboxane synthase inhibitors, thromboxane receptor antagonists and dual blockers in thrombotic disorders.

Thromboxane A2 (TXA2) plays a pivotal role in platelet activation and is involved in the development of thrombosis. Thromboxane synthase inhibitors suppress TXA2 formation and increase the synthesis of the antiaggregatory prostaglandins PGI2 and PGD2; however, accumulated PGH2 may interact with the platelet and vessel wall TXA2 receptor, thus reducing the antiplatelet effects of this class of drug. TXA2 receptor antagonists block the activity of both TXA2 and PGH2 on platelets and the vessel wall. Very recently, drugs possessing both thromboxane synthase-inhibitory and thromboxane receptor-antagonist properties have been developed. Paolo Gresele and colleagues explain here why these drugs can be more efficacious than traditional antiplatelet agents and review the available experimental studies involving these drugs.     

Binding characteristics of the new thromboxane A2/prostaglandin H2 receptor antagonist [3H]BAY U 3405 to washed human platelets and platelet membranes.

The new thromboxane A2 antagonist [3H]BAY U 3405 was characterized for its binding to washed human platelets and platelet membranes. In washed platelets the specific binding was reversible, selective and stereospecific, but not saturable. The dissociation constant (Kd) was 6 +/- 2.5 nM, the number of specific binding sites 1177 +/- 306 per platelet. Three structurally different thromboxane A2 (TXA2)/prostaglandin H2 (prostaglandin endoperoxide) (PGH2) receptor ligands completely inhibited the specific binding of [3H]BAY U 3405 in a concentration-dependent manner, indicating that the observed high affinity binding site is the TXA2/PGH2 receptor. In platelet membranes, however, specific [3H]BAY U 3405 binding showed saturability in addition to reversibility, selectivity, and stereospecifity. The Kd of the binding was 9.6 +/- 2.3 nM in kinetic studies and 8.7 +/- 3.7 nM in saturation studies, the inhibition constant (Ki) was 10 +/- 1.1 nM in displacement studies. The TXA2/PGH2 receptor agonists U 46619 and CTA2, and the antagonists Daltroban (BM 13505), I-PTA-OH and SQ 29548 all completely inhibited the specific binding of [3H]BAY U 3405 thus defining the observed binding site as the TXA2/PGH2 receptor. In conclusion, the data suggest that the previously reported TXA2 antagonism of BAY U 3405 is mediated by binding to a specific high affinity binding site of human platelets and platelet membranes that represents the TXA2/PGH2 receptor.     

Synthesis and thromboxane A2 antagonist activity of N-benzyltrimetoquinol analogues

It is currently believed that the platelet thromboxane A2 (TXA2/PGH2) receptor is different from the vascular TXA2/PGH2 receptor. While the majority of TXA2 receptor antagonists are structurally related to the prostaglandins, trimetoquinol (TMQ) represents a unique nonprostanoid antagonist. TMQ also possesses beta-adrenergic activity; however, an N-benzyl substituent on TMQ has been shown to impart some selectivity for platelet antiaggregatory activity versus beta-adrenergic activity. In this study, we examined the synthesis and TXA2 antagonist activity of a series of substituted N-benzyl analogues of TMQ. While these analogues showed an apparent direct correlation between platelet antiaggregatory activity and electron-donating ability of the N-benzyl substituents, no such correlation could be demonstrated for the inhibition of contractile responses. Thus, nonprostanoid TXA2 antagonists can be used to demonstrate differences between platelet and vascular TXA2/PGH2 responses.     

Binding of thromboxane A2/prostaglandin H2 agonists to human platelets

The competition of [125I]-9, 11 dimethylmethano-11, 12 methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13-aza 15 alpha beta-omega-tetranor-thromboxane A2 ([125I]-PTA-OH), a thromboxane A2/prostaglandin H2 receptor antagonist, with a series of thromboxane A2/prostaglandin H2 (TXA2/PGH2) mimetics for binding to the putative TXA2/PGH2 receptor in washed human platelets was studied. The rank order potency for the series of mimetics to compete with [125I]-PTA-OH for binding was compared with their rank order potency for induction of platelet aggregation. The rank order potency for the mimetics to compete with [125I]-PTA-OH for binding was ONO-11113 greater than SQ-26655 greater than U44069 greater than U46619 = 9, 11-azo PGH2 greater than MB28767. This rank order potency was highly correlated with their rank order potency for inducing platelet aggregation (r = 0.992). Changes in the intra or extracellular concentrations of Na+ did not have a significant effect on the competition between U46619 and [125I]-PTA-OH for binding to the putative receptor. In summary, it appears that these TXA2/PGH2 mimetics activate human platelets through the putative TXA2/PGH2 receptor.     

Regulation of activator protein-1 by 8-iso-prostaglandin E2 in a thromboxane A2 receptor-dependent and -independent manner

The thromboxane (TX) A(2) receptor (TP) encompasses two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP activity may be modulated by novel ligands, termed the isoprostanes, that paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes 8-iso-prostaglandin (PG)F(2 alpha) and 8-iso-PGE(2) regulate the activity of TP isoforms expressed in Chinese hamster ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist [9,11-dideoxy-9 alpha,11 alpha-methanoepoxy PGF(2 alpha) (U46619)] in CHO cells transfected with the human TP-P and TP-E receptors, and this response was fully inhibited by TP antagonists [1S-[1 alpha,2 beta(Z),3 alpha,5 alpha]]-7-[3-[[4-iodophenyl)sulfonyl]amino]-6,6-dimethylbicyclo[3.1.1]hept-2-yl]-5-heptenoic acid (I-SAP) and [1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-7-[[2-[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-5-heptenoic acid (SQ 29,548)]. AP-1-luciferase activity was potently (nanomolar concentrations) increased by 8-iso-PGE(2) in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, whereas 8-iso-PGF(2 alpha) was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE(2) mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA(2) biosynthesis. Interestingly, 8-iso-PGE(2)-mediated AP-1-luciferase activity was near maximal in naive cells between 1 and 10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP(1)/EP(3) receptors. These observations 1) support a role for novel ligands in the regulation of TP-dependent signaling, 2) indicate that TP-P and TP-E couple to AP-1, 3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and 4) indicate that additional targets regulated by 8-iso-PGE(2) couple to AP-1.     

Decrease in agonist affinity for human platelet thromboxane A2/prostaglandin H2 receptors induced by a platelet-derived supernatant

Platelets possess membrane receptors which mediate the aggregatory response to thromboxane A2 (TXA2) and prostaglandin H2 (PGH2). It has been observed recently that the affinities for a series of TXA2/PGH2 mimetics are decreased in crude human platelet membranes and solubilized membranes compared to intact washed platelets. The present study investigated the notion that platelets contain a substance that is released during platelet lysis that reduces the affinity of the TXA2/PGH2 receptor for agonists. The displacement of 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13 - aza-15 alpha beta-omega-tetranor-TXA2 ([125I]PTA-OH), a TXA2/PGH2 receptor antagonist, from its binding site in intact washed platelets by TXA2/PGH2 mimetics and antagonists was characterized in the presence or absence of the supernatant (50,000 g) obtained from sonicated platelets. In the presence of the supernatant, there was a significant (P less than 0.025) increase in the IC50 values for the TXA2/PGH2 mimetics U46619, SQ26655, and ONO11113. The increase in the IC50 for U46619 induced by the supernatant was abolished by either boiling or treating the supernatant with trypsin. The supernatant did not affect the Kd or Bmax of [125I]PTA-OH or the IC50 of the TXA2/PGH2 antagonist, SQ29548. Pretreatment of the platelets with the supernatant resulted in a significant (P less than 0.02) reduction in the aggregation response induced by U46619. Gel filtration (Sephacryl S200) of the supernatant revealed a fraction (molecular weight approximately 100,000 daltons) which significantly increased the IC50 for U46619 to displace [125I]PTA-OH from its binding site. Thus, human platelets appear to possess a protein(s) that is released into the supernatant upon sonication and inhibits the binding of TXA2/PGH2 agonists but not antagonists to their receptor. This protein may play a role in the regulation of platelet responses to the aggregatory stimuli TXA2/PGH2.     

Mediation of bradykinin-induced contraction in canine veins via thromboxane/prostaglandin endoperoxide receptor activation

1. Canine jugular and femoral veins were studied to determine the possible importance of thromboxane (TXA2) and prostaglandin endoperoxides (prostaglandin H2, PGH2) in mediating bradykinin(BK)-induced contraction. 2. Isolated vein rings incubated in modified Krebs solution contracted to TXA2/PGH2 analogs SQ26655 and U44069 with potency of contraction exceeding that for BK. The potency ranking for both veins was SQ26655 greater than U44069 greater than BK greater than PGF2 alpha greater than TXB2 much greater than PGD2. 3. The cyclo-oxygenase inhibitors indomethacin (3 x 10(-7) M) and flufenamic acid (10(-5) M) reduced BK contractions without affecting those induced by noradrenaline (NA). 4. TXA2/PGH2 receptor antagonists SQ29548 (10(-8) M) and BM13177 (10(-6) M) strongly inhibited BK-induced tension. The action of antagonists was reversible with negligible influence on NA-elicited contraction. Selective removal of endothelium had no effect on BK-induced contraction or the action of the antagonists. 5. The thromboxane synthase inhibitors dazoxiben (10(-4) M) and CGS 12970 (10(-5) M) had no significant inhibitory effect on BK-induced tension. 6. These results suggest that in canine jugular and femoral vein, the action of BK is largely dependent upon stimulation of the cyclo-oxygenase pathway to produce PGH2 and possibly TXA2, which can activate a smooth muscle TXA2/PGH2 receptor to elicit vasoconstriction.     

Ligand binding to thromboxane receptors on human platelets: correlation with biological activity.

The preparation of enantiomerically pure [3H]-15 (S) 9, 11-epoxymethano PGH2 (a thromboxane A2-like agonist) has enabled the binding of ligands to the thromboxane receptor of the human platelet to be studied. The binding of the radio-ligand to washed human platelets has 3 components. One component is not displaceable by 'cold' 9, 11-epoxymethano PGH2 and its concentration-binding plot is roughly linear. The other 2 components are displaceable and saturable, and the larger of the two, which is sensitive to the stereochemistry of the C15 secondary alcohol, appears to represent the thromboxane receptor. About 1700 15(S)9, 11-epoxymethano PGH2 molecules are specifically bound to a single platelet and 50% of this binding is achieved with a concentration of 75 nM. Displacement of [3H]-15(S)9, 11-epoxymethano PGH2 is effected by (a) TXA2 and PGH2 and a number of bicyclic stable analogues (e.g. 9,11-azo PGH2), all of which produce irreversible aggregation of human platelets; (b) analogues of PGF2 alpha with potent thromboxane-like activity (e.g. ICI 79939); (c) compounds with partial agonist activity on the platelet thromboxane system (e.g. CTA2); (d) Thromboxane/endoperoxide analogues which specifically antagonize thromboxane-like actions on the human platelet (e.g. PTA2 and EP 045). Displacement is not achieved with the natural prostaglandins PGE2, PGD2 and PGF2 alpha. Neither the thromboxane-synthetase inhibitor dazoxiben nor R(+)-trimethoquinol have high displacing activity. The correlation of radio-ligand displacement with the biological activity of the competing ligands is discussed in relation to the nature of the thromboxane receptor on the human platelet.     

Different pharmacologic activities for 13-azapinane thromboxane A2 analogs in platelets and blood vessels

A series of 16 13-azapinane thromboxane A2 analogs were synthesized and their pharmacological dissociation constants (Kd) determined for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor in both washed human platelets and canine saphenous veins. Twelve of the analogs were antagonists of both U46619-induced platelet aggregation and saphenous vein contraction. The rank order potencies of these analogs were significantly different in platelets compared to saphenous veins. Four of the derivatives were antagonists in the platelets but possessed agonist activity in the vessels. The potency of the analogs and the type of activity (agonist or antagonist) were found to be sensitive to the substitution pattern of the aromatic ring on the bottom side chain. These results further support the notion that platelet TXA2/PGH2 receptors are distinct from that of vascular receptors.     

Irreversible inhibition of thromboxane (TX) A2 synthesis by Y-20811, a selective TX synthetase inhibitor.

As Y-20811, sodium (+-)-4-[alpha-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylb+ ++ enzoic acid, has been reported to inhibit serum thromboxane (TX) A2 production with a long duration of action, its mechanism of action was investigated. When [3H]Y-20811 (3 mg/kg) was administered orally to rats, the peak platelet concentration of Y-20811 was obtained 1 hr after the administration, and the T1/2 was 43 hr. The peak plasma concentration of Y-20811 was also obtained 1 hr after administration, but the elimination of Y-20811 from plasma was faster (T1/2 alpha = 1.5 hr, T1/2 beta = 15 hr) than that observed in platelets. Serum TXA2 (estimated as TXB2) production was inhibited significantly from 1 to 72 hr after the oral administration of unlabeled Y-20811 (3 mg/kg), which temporally resembled the change of the platelet Y-20811 concentration. In platelet-rich plasma, [3H]Y-20811 completely inhibited TXA2 production at about 1500 pg/10(9) platelets, and the IC50 level was about 600 pg/10(9) platelets, which was similar to values obtained in ex vivo studies. In addition, inhibition of TXA2 production by Y-20811 still remained after washing the drug-pretreated microsomes, whereas that of dazoxiben completely disappeared. A similar irreversible inhibition of TXA2 production was observed with aspirin. These results suggest that Y-20811 may firmly combine with platelet TX synthetase and may irreversibly inhibit TXA2 production.     

Preparative HPLC purification of prostaglandin endoperoxides and isolation of novel cyclooxygenase-derived arachidonic acid metabolites

A preparative HPLC purification scheme for the isolation of prostaglandin endoperoxides prepared by short-time incubation of [1-14C]-labelled arachidonic acid (AA) with sheep seminal vesicle microsomes was developed. Milligram quantities of prostaglandin G2 (PGG2) and prostaglandin H2 (PGH2) were obtained in greater than or equal to 95% purity within shortest time. Furthermore, careful application of this HPLC technique led to the isolation of two minor [1-14C]-labelled fractions which according to their spectral and chromatographic characteristics, were identical with 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Another HETE substituted at either C11 or C12 was also present. The formation of these products was mediated by cyclooxygenase as evidenced by aspirin (100 microM) and indomethacin (10 microM) inhibition. Sulfhydryl-blocking agents such as p-hydroxymercuribenzoate (1 mM) and/or the 12-lipoxygenase inhibitor esculetin (100 microM) were without effect. In addition to these AA metabolites four other fractions contained arachidonate-derived endoperoxides with antiaggregatory properties, all of which released malondialdehyde upon incubation with thromboxane A2 synthase. No thromboxane formation was observed although turnover numbers were comparable to those of PGG2 and PGH2. The formation of these endoperoxides did not occur via enzymatic or non-enzymatic degradation of PGG2 or PGH2. The exact chemical nature of these endoperoxides remains to be established.     

Design, synthesis, and SAR study of a series of N-alkyl-N'-[2-(aryloxy)-5-nitrobenzenesulfonyl]ureas and -cyanoguanidine as selective antagonists of the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor

The prostanoid thromboxane (TX)A2 exerts its proaggregant and constrictive actions upon binding to the specific TXA2 receptor (TP), a member of the G-protein coupled receptor superfamily. In humans, TXA2 signals through two distinct TP isoforms, TPalpha and TPbeta. Herein, we describe the design, synthesis, and SAR study of a series of original N-alkyl-N'-[2-(aryloxy)-5-nitrobenzenesulfonyl]ureas and -cyanoguanidine. The SAR study was based on the results of a functional assay, TP-mediated intracellular calcium ([Ca2+]i) mobilization performed on the two separate isoforms. Optimal nature and position of several structural moieties was defined for both activity and selectivity toward TPalpha and TPbeta isoforms. Three compounds (9h, 9af, and 9ag), showing increased selectivity for TPbeta relative to TPalpha (23.2:1, 18.1:1, 19.9:1, respectively), were selected for further experiments, and their activity was confirmed in a platelet aggregation assay. This study represents the first extended SAR study dealing with the identification of isoform selective antagonists for the human TXA2 receptor.     

Photoaffinity receptor antagonist for human platelet thromboxane A2/prostaglandin H2 receptors

9,11-Dimethylmethano -11,12-methano-16-(3-azido-4-iodophenoxy)-13,14- dihydro-13-aza-15 alpha beta-omega-tetranor TXA2 (I-PTA-PON3) was synthesized and evaluated as a potential photoaffinity probe of the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. I-PTA-PON3 inhibited the aggregation of washed human platelets induced by the TXA2 mimetic U46619 [(15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid]. Schild analysis of the data revealed a Kd of 9.5 nM and a slope not significantly different from -1. Equilibrium binding studies using [125I]PTA-OH, a TXA2/PGH2 receptor antagonist, showed that I-PTA-PON3 plus photolysis resulted in a 52% reduction in the number of binding sites (1252 +/- 202/platelet) compared to the nonphotolyzed group (2557 +/- 293/platelet) (N = 5, P less than 0.05) with no significant change in the Kd. Repetition of the incubation with I-PTA-PON3 and photolysis a second time resulted in a further 77% (578 +/- 163 binding sites/platelet) reduction in the number of binding sites. Incubation of washed human platelets with I-PTA-PON3 (163 nM) followed by photolysis and removal of the non-covalently bound I-PTA-PON3 resulted in no change in the EC50 value for the TXA2 mimetic, U46619, when compared to controls that were either exposed to I-PTA-PON3 and not photolyzed or exposed only to photolysis. The second photolysis of I-PTA-PON3 resulted in a significant 42% increase in the EC50 value of U46619-induced aggregation compared to the non-photolyzed group (N = 4, P less than 0.05). These results suggest that I-PTA-PON3 is a useful probe for the study of TXA2/PGH2 receptors and that spare TXA2/PGH2 receptors may exist in the platelet.     

Antiplatelet effect of NQ12: a possible mechanism through the arachidonic acid cascade

NQ12, an antithrombotic agent, has been reported to display a potent antiplatelet activity. This study was undertaken to reveal the effect of NQ12 on rabbit platelet aggregation and signal transduction involved in the arachidonic acid (AA) cascade. NQ12 concentration-dependently suppressed collagen-, AA-, and U46619-induced rabbit platelet aggregation, with IC(50) values of 0.71 +/- 0.2, 0.82 +/- 0.3, and 0.45 +/- 0.1 microM, respectively. In addition, the concentration-response curve of U46619 was shifted to the right after NQ12 treatment, indicating an antagonism on thromboxane (TX) A(2) receptors. The collagen-stimulated AA liberation was inhibited by NQ12 in the same pattern as its inhibition of platelet aggregation. Further study revealed that NQ12 potently suppressed AA-mediated TXA(2) formation, but had no effect on the PGD(2) production, indicating an inhibitory effect on TXA(2) synthase, which was supported by a TXA(2) synthase activity assay indicating that NQ12 concentration-dependently inhibited TXA(2) formation converted from PGH(2). On the other hand, the AA-stimulated 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formation was also suppressed by NQ12. Taken together, these results suggest that NQ12 has a potential to inhibit TXA(2) synthase activity and TXA(2) receptors, and it can modulate AA liberation as well as 12-HETE formation in platelets. This may be a convincing mechanism for the antithrombotic action of NQ12.     

Synthesis and thromboxane A2/prostaglandin H2 receptor antagonistic activity of phenol derivatives.

Consideration of possible structural similarities between thromboxane A2 and the hydroquinone form of (R)-(+)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7- phenylheptanoic acid (R-(+)-AA-2414) led to the development of a new series of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor antagonists, namely 7-(4-fluorophenyl)-7-(2-hydroxyphenyl)heptanoic acids (I). These compounds were found to be potent TXA2/PGH2 receptor antagonists. Compounds having either a carbonyl or a hydroxymethyl group at the para-position of the phenolic hydroxy group exhibited most potent activities in this series. Compounds 14, 15, 18, and 26 inhibited the specific binding of [3H]U-46619 to guinea pig platelet membranes (IC50 = 4.4, 80, 32, and 13 nM, respectively), and also inhibited U-46619-induced human platelet aggregation (IC50 = 310, 69, 79, and 78 nM, respectively). Comparison of the UV spectra of the compounds with a carbonyl group at the para-position of phenolic hydroxy group revealed that the activity tended to increase in accordance with a decrease in the torsional angle between the carbonyl group and the phenol ring. These results suggested that the spacial location of the carbonyl and hydroxymethyl oxygen are important for significant increase in activity and that the carbonyl and hydroxymethyl oxygen at the para-position of the phenolic hydroxy group might interact with one of the TXA2/PGH2 receptor sites.     

Non-prostanoid thromboxane A2 receptor antagonists with a dibenzoxepin ring system. 2.

A series of 11-[2-(1-benzimidazolyl)ethylidene]-6,11-dihydrodibenz[b,e]oxep in-2- carboxylic acid derivatives and related compounds were synthesized and found to be potent TXA2/PGH2 receptor antagonists. Each compound synthesized was tested for its ability to displace [3H]U-46619 binding from guinea pig platelet TXA2/PGH2 receptors. Structure-activity relationship studies revealed that the following key elements were required for enhanced activities: (1) an (E)-2-(1-benzimidazolyl)ethylidene side chain in the 11-position of the dibenzoxepin ring system and (2) a carboxyl group in the 2-position of the dibenzoxepin ring system. The studies also indicated that the TXA2/PGH2 receptor binding affinities of this series of compounds in guinea pig platelet were poorly correlated with those in human platelet. Introduction of substituent(s) to the benzimidazole moiety was effective and sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]- 6,11-dihydrodibenz[b,e]oxepin-2-carboxylate monohydrate (57) recorded the highest affinity for human platelet TXA2/PGH2 receptor with a K(i) value of 1.2 +/- 0.14 nM. It demonstrated potent inhibitory effects on U-46619-induced guinea pig platelet aggregation (in vitro and ex vivo) and human platelet aggregation (in vitro). Compound 57, now designated as KW-3635, is a novel, orally active, and specific TXA2/PGH2 receptor antagonist with neither TXA2/PGH2 receptor agonistic nor TXA2 synthase inhibitory effects. It is now under clinical evaluation.     

Characterization of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors of rat platelets and their interaction with TXA2/PGH2 receptor antagonists

Characterization of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors of rat platelets was performed on both intact platelets and crude membrane fractions. The binding of [3H]U46619, a stable TXA2 mimetic, to intact platelets was found to be saturable and displaceable. Scatchard analysis of equilibrium binding at 24 degrees revealed a single class of binding sites with a Kd of 37 nM and a Bmax of 160 fmol/10(8) platelets. The binding affinity of [3H]U46619 to the platelet membrane fractions was remarkably and specifically enhanced by addition of Mg2+ without alteration of the maximum density level. Kinetic analysis for [3H]U46619 binding to the membrane fractions in the presence of 20 mM MgCl2 gave a K1 of 6.9 x 10(6) M-1 min-1 and a K-1 of 0.25 min-1, yielding a Kd (K-1/K1) of 36 nM; the value corresponded well to Kd values from Scatchard analysis in both intact (37 nM) and crude membrane fractions (39 nM). A series of TXA2/PGH2 receptor antagonists completely suppressed U46619 binding to rat platelets as well as collagen-induced platelet aggregation. The rank order of binding affinities to rat platelets (intact platelets or crude membranes) among the respective antagonists correlated well with (a) that of human platelet membrane fraction and (b) the potencies for suppression of collagen-induced platelet aggregation in rat. These results may support our proposed mechanism of TXA2/PGH2 action in collagen-stimulated platelets [K. Hanasaki et al., Thromb. Res. 46, 425 (1987)] and also suggest that they may provide a simple technique for evaluating synthetic TXA2/PGH2 receptor antagonists.     

Antiplatelet activity of [5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl]guanidine (KR-32570), a novel sodium/hydrogen exchanger-1 and its mechanism of action

The antiplatelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen (10 microg/mL), thrombin (0.05 U/mL), arachidonic acid (100 microM), a thromboxane (TX) A2 mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F2, 1 microM) and a Ca2+ ATPase inhibitor thapsigargin (0.5 microM) (IC50 values: 13.8 +/- 1.8, 26.3 +/- 1.2, 8.5 +/- 0.9, 4.3 +/- 1.7 and 49.8 +/- 1.4 microM, respectively). KR-32570 inhibited the collagen-induced liberation of [3H]arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at 50 microM. The TXA2 synthase assay showed that KR-32570 also inhibited the conversion of the substrate PGH2 to TXB2 at all concentrations. Furthermore, KR-32570 significantly inhibited the [Ca2+]i mobilization induced by collagen at 50 microM, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen (10 microg/mL)-induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, TXA2 synthase, the mobilization of cytosolic Ca2+ and NHE-1.