Deletions involving both KCNQ2 and CHRNA4 present with benign familial neonatal seizures.

OBJECTIVE: Mutations of the genes encoding subunits of potassium voltage-gated channel, KCNQ2 and KCNQ3, have been identified in patients with benign familial neonatal seizures (BFNS). This study set out to determine the frequency of microchromosomal deletions of KCNQ2 or KCNQ3 associated with BFNS. METHODS: The study subjects were patients with BFNS (n = 22). Microdeletions were sought by multiplex ligation-dependent probe amplification and then confirmed by fluorescence in situ hybridization and characterized by array-based comparative genomic hybridization. RESULTS: Heterozygous multiple exonic deletions of KCNQ2 were identified in 4 of 22 patients with BFNS. Concomitant deletions of adjacent genes, including nicotinic cholinergic receptor alpha4 (CHRNA4), were detected in 2 of the 4 cases. The clinical courses of patients with deletions of both KCNQ2 and CHRNA4 were those of typical BFNS, and none presented with the phenotype of autosomal dominant nocturnal frontal lobe epilepsy, some of which are caused by mutations of CHRNA4. CONCLUSIONS: Our findings indicate that the clinical courses of patients with deletions of both KCNQ2 and CHRNA4 are indistinguishable from those of patients with deletions of KCNQ2 only.

Potent and selective ET-A antagonists. 1. Syntheses and structure-activity relationships of N-(6-(2-(aryloxy)ethoxy)-4-pyrimidinyl)sulfonamide derivatives.

Modifications to the ET(A/B) mixed type compounds 1 (Ro. 46-2005) and 2 (bosentan) were performed. Introduction of a pyrimidine group into 1 resulted in a dramatic increase in affinity for the ET(A) receptor, and the subsequent optimization of substituents on the pyrimidine ring led us to the discovery of N-(6-(2-((5-bromo-2-pyrimidinyl)oxy)ethoxy)-5-(4-methylphenyl)-4-pyrimidinyl)-4-tert-butylbenzenesulfonamide (7k), which showed an extremely high affinity for the human cloned ET(A) receptor (K(i) = 0.0042 +/- 0.0038 nM) and an ET(A/B) receptor selectivity up to 29 000 (K(i) = 130 +/- 50 nM for the human cloned ET(B) receptor). The compound was designed on the hypothesis that the hydrogen atom of the hydroxyl group in 1 and 2 played a role not as a proton donor but as an acceptor in the possible hydrogen bonding with Tyr129. Since the incorporation of a pyrimidinyl group into the hydroxyethoxy side chain of the nonselective antagonist (1) dramatically enhanced both the ET(A) receptor affinity and selectivity, and since similar results were obtained from the benzene analogues, we put forward the hypothesis that a "pyrimidine binding pocket" might exist in the ET(A) receptor.

Potent and selective ET-A antagonists. 2. Discovery and evaluation of potent and water soluble N-(6-(2-(aryloxy)ethoxy)-4-pyrimidinyl)sulfonamide derivatives.

In the preceding article,(1) we outlined the discovery and structure-activity relationship of a potent and selective ET(A) receptor antagonist 1 and its related compounds. Metabolites of 1 having potent selective ET(A) receptor antagonist activity were identified. This study suggested the metabolic pathways of 1 were considerably affected by species. Consequently, structural modification of 1 intended to improve the complexity of the metabolic pathway, and water solubility was performed. The subsequent introduction of a hydroxyl group into the tert-butyl moiety of 1 led to the discovery of our new clinical candidate, 6b, which showed a higher water solubility, a uniform metabolic pathway among species, and very high affinity and selectivity for the human ET(A) receptor (K(i) for ET(A) receptor: 0.015 +/- 0.004 nM; for ET(B) receptor: 41 +/- 21 nM).

Role of adenosine and P2 receptors in the penile tumescence in anesthetized dogs.

We studied the role of adenosine and P2 receptors in the pelvic nerve stimulation-induced penile tumescence in anesthetized dogs. A local intracavernous injection of adenosine induced the tumescence, which was abolished by intracavernous 8-(p-sulfophenyl)theophylline (8-SPT), an unspecific adenosine receptor antagonist, and by 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol (ZM241385), an adenosine A(2A) receptor antagonist. ATP also induced the tumescence, which was diminished by 8-SPT, but not by reactive blue-2, a P2 receptor antagonist. Neither intracavernous beta, gamma-meATP nor ADP(beta)S, P2X and P2Y receptor agonists, induced tumescence. N(G)-nitro-L-arginine (L-NAME), a nitric oxide synthase inhibitor, and T-1032, a phosphodiesterase type V inhibitor, had no effects on the tumescence induced by adenosine. 8-SPT and reactive blue-2 had no effects on the tumescence induced by pelvic nerve stimulation. These results show that although exogenous adenosine and ATP induce tumescence, neither the adenosine nor the P2 receptor is involved in the tumescence induced by pelvic nerve stimulation in anesthetized dogs.

T-1032, a novel specific phosphodiesterase type 5 inhibitor, increases venous compliance in anesthetized rats.

Nitric oxide (NO) donors including organic nitrates dilate capacitance vessels. As inhibition of phosphodiesterase type 5 results in the accumulation of guanosine 3'5'-cyclic monophosphate (cGMP), specific phosphodiesterase type 5 inhibitors are expected to have a vasodilator property similar to that of NO donors. To test this hypothesis, we examined the effect of methyl2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel specific phosphodiesterase type 5 inhibitor, on mean arterial pressure and mean circulatory filling pressure (an index of venodilation) compared with that of nitroglycerin and diltiazem in mecamylamine- and noradrenaline-treated anesthetized rats. Intravenous infusion of T-1032 (0.1, 1, 10 microg/kg/min) dose-dependently decreased mean arterial pressure (-3.8+/-0.3%, -9.1+/-0.8%, -16.8+/-1.5% at doses of 0.1, 1 and 10 microg/kg/min, respectively) and mean circulatory filling pressure (-6.1+/-0.9%, -12.5+/-0.7%, -18.6+/-3.0% at doses of 0.1, 1 and 10 microg/kg/min, respectively). The mean circulatory filling pressure-mean arterial pressure relationship revealed that T-1032 had a selective action on the mean circulatory filling pressure compared with diltiazem (10, 100 microg/kg/min) and a similar or more selective effect than nitroglycerin (0.3, 3 and 30 microg/kg/min). In the next study, we calculated venous compliance and unstressed volume from the mean circulatory filling pressure-volume relationship. Intravenous infusion of T-1032 (3 microg/kg/min) increased venous compliance (3.35+/-0.40 in T-1032 vs. 2.31+/-0.15 ml/kg/mm Hg in vehicle, P<0.05) without changing the unstressed volume (37.2+/-2.80 in T-1032 vs. 42.6+/-2.37 ml/kg in vehicle, P>0.05). It was concluded that T-1032 increased venous capacitance by increasing venous compliance, and that this selective phosphodiesterase type 5 inhibitor appeared to have a different vasodilator action from that of an NO donor and a Ca(2+) channel antagonist in that it had a selective action on the mean circulatory filling pressure.

Novel, potent, and selective phosphodiesterase 5 inhibitors: synthesis and biological activities of a series of 4-aryl-1-isoquinolinone derivatives.

A novel class of potent and selective phosphodiesterase 5 (PDE5) inhibitors, 4-aryl-1-isoquinolinone derivatives, which have been designed by the comparison of the structure of cGMP and a previously reported 1-arylnaphthalene lignan, was disclosed. Among these compounds, methyl 2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate dihydrochloride (36a) exhibited potent PDE5 inhibitory activity (IC(50) = 1.0 nM) with high isozyme selectivities (IC(50) ratio: PDE1/PDE5 = 1300, PDE2/PDE5 > 10 000, PDE3/PDE5 > 10 000, PDE4/PDE5 = 4700, PDE6/PDE5 = 28). Compound 36a also showed the most potent relaxant effect on isolated rabbit corpus cavernosum (EC(30) = 7.9 nM). Compound 63 (T-1032), the sulfate form of 36a, was selected for further biological and pharmacological evaluation of erectile dysfunction.

Design, syntheses, and structure-activity relationships of indan derivatives as endothelin antagonists; new lead generation of non-peptidic antagonist from peptidic leads.

A new lead generation of non-peptidic ET(A) antagonists from two peptidic ET(A)-selective ones, BQ-123 and FR139317, was performed. Using computer assisted molecular modeling, a putative pharmacophore was constructed from the superposition of the reported three-dimensional structure of the cyclic peptide BQ-123 and a presumable beta-turn active conformation of the linear peptide FR139317 formed by an intramolecular hydrogen bond. According to this model, a new series of indan derivatives were designed and synthesized. Among these, 5-isobutyrylamino-6-(1-naphthylmethyloxy)-3-(2-thienyl)-1-indancarboxylic acid (1b) showed a moderate ET(A) antagonistic activity (IC50 = 28 microM).

Pharmacological profile of T-1032, a novel specific phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum.

This study was designed to examine the pharmacological properties of T-1032 (methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate), a novel phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum. T-1032 (3x10(-11) to 3x10(-7) M) caused an endothelium-dependent relaxation in the isolated rat aorta precontracted with phenylephrine, and the relaxation was accompanied by an increase in cGMP but not cAMP levels. The T-1032-induced relaxation was attenuated by N(G)-nitro-L-arginine methyl ester (L-NAME) (10(-3) M), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) (10(-5) M), a guanylyl cyclase inhibitor. T-1032 (10(-9), 10(-8) M) produced a potentiation of the relaxation induced by sodium nitroprusside, but not of the relaxation induced by isoproterenol. In the isolated rabbit corpus cavernosum precontracted with phenylephrine, the electrical field stimulation-induced relaxation was attenuated by treatment with tetrodotoxin (10(-6) M) as well as L-NAME (10(-4) M). The L-NAME-inhibited relaxation was restored by treatment with L-arginine (5x10(-4) M). T-1032 (10(-9) to 10(-6) M) and sildenafil (10(-9) to 10(-6) M) produced a potentiation of the electrical field stimulation-induced relaxation as well as a decrease in basal tension in a concentration-dependent manner. It was concluded that T-1032 had potentiating effects on the NO/cGMP signaling pathway in isolated tissues, probably through specific blockade of phosphodiesterase type 5. T-1032 would be a useful compound to examine the physiologic functions of phosphodiesterase type 5 in mammalian tissues.

Effect of wearing clothes on oxygen uptake and ratings of perceived exertion while swimming.

For a comparative study between swimming in swimwear (control-sw) and swimming in clothes (clothes-sw), oxygen uptake (VO2) and ratings of perceived exertion (RPE) were measured. The subjects were six male members of a university swimming team. Three swimming strokes--the breaststroke, the front crawl stroke and the elementary backstroke--were applied. With regards to clothes-sw, swimmers wore T-shirts, sportswear (shirt and pants) over swimwear and running shoes. In both cases of control-sw and clothes-sw, the VO2 was increased exponentially with increased swimming speed. The VO2 of the subjects during the clothed tests did not exceed 1.4 times of that in the case of control-sw at swimming speeds below 0.3 m/s. As swimming speeds increased, VO2 difference in both cases increased. Consequently, VO2 in the clothed tests was equal to 1.5-1.6 times and 1.5-1.8 times of that in the swimwear tests at speeds of 0.5 and 0.7 m/s, respectively. At speeds below 0.6 m/s in clothes-sw, the breaststroke showed lower VO2 than the front crawl stroke, and the elementary backstroke showed higher VO2 than the other two swimming strokes. RPE increased linearly with %peak VO2. In addition, any RPE differences among the three swimming strokes were not shown in the control-sw tests. At an exercise intensity above 60 %peak VO2, clothed swimmers showed slightly higher RPE in the front crawl stroke compared to that in the two other swimming strokes.

Potentiation of penile tumescence by T-1032, a new potent and specific phosphodiesterase type V inhibitor, in dogs.

We examined the mechanism underlying the potentiation of penile tumescence by methyl 2-(4-aminophenyl)-1, 2dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)3-isoquinoline carboxylate sulfate (T-1032), a new potent and selective phosphodiesterase type V inhibitor. In vivo, pelvic nerve stimulation induced a penile tumescence together with increase of total nitric oxide metabolite levels within the corpus cavernosa of anesthetized dogs. Intravenous (1-100 microg/kg) and intraduodenal (3, 30, 300 microg/kg) treatment with T-1032 dose dependently potentiated the tumescence. The potency of T-1032 was equivalent to that of sildenafil. T-1032 did not influence the intracavernous pressure when the pelvic nerve stimulation was absent. The potentiation of tumescence was more pronounced by intracavernous than i.v. injection. Intracavernous N(G)-nitro-L-arginine, a nitric-oxide synthase inhibitor, but not N(G)-nitro-D-arginine diminished the effects of T-1032 on the tumescence. Furthermore, i.v. T-1032 augmented the tumescence induced by sodium nitroprusside (SNP) but not by vasoactive intestinal polypeptide (VIP). In vitro, in isolated preparations of canine corpus cavernosum precontracted with phenylephrine, SNP (0. 01-100 microM) and VIP (0.01-1 microM) produced a dose-dependent relaxation accompanied by an increase in cGMP and cAMP levels, respectively. T-1032 augmented the relaxation induced by SNP but not by VIP. These data suggest that oral treatment with T-1032 has potential to improve erectile dysfunction through the inhibition of phosphodiesterase type V in the smooth muscles of corpus cavernosa.

Prevention of cerebral vasospasm by a novel endothelin receptor antagonist, TA-0201.

This study was designed to examine the preventive effect of a novel endothelin (ET)-receptor antagonist TA-0201 on the cerebral vasospasm in a canine double-hemorrhage model. TA-0201 (10(-9)-10(-7) M) inhibited ET-1-induced vasoconstriction in the isolated canine basilar artery without endothelium in a concentration-dependent manner. Its pA2 value was 9.2 (ET(A) antagonism). In a canine double-hemorrhage model, intravenous treatment with TA-0201 (3 mg/kg, twice a day for 7 days) ameliorated the basilar artery narrowing significantly on day 7 compared with that in nontreated dogs. The reductions of the basilar artery diameter were 26.1+/-3.9% and 40.5+/-4.1% with and without TA-0201 treatment, respectively (p<0.05). Histologic study on day 7 indicated that treatment with TA-0201 inhibited vessel-wall damage such as disintegration of endothelium architecture and degeneration of medial smooth-muscle cells. We conclude that intravenous treatment with TA-0201 prevents the development of cerebral vasospasm and accompanying pathologic changes of the vessel wall, probably through blockade of ET(A) receptors.

Characteristics of heterogeneity in the expression of vasoconstriction in response to N(G)-monomethyl-L-arginine in isolated canine arteries.

We characterized the contractile effect of a nitric oxide (NO) synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), in isolated canine arteries. L-NMMA induced a heterogenous response: potent vasoconstriction in the cerebral arteries, and weak or no vasoconstrictor responses in different peripheral arteries. The vasoconstriction of the cerebral artery was inhibited by L-arginine but not D-arginine. L-NMMA (1(-4) M) caused a 53% decrease in guanosine 3'5'-cyclic monophosphate (cGMP) levels in the cerebral artery, but it was not significant compared with that in peripheral arteries. The L-NMMA-induced vasoconstriction was inhibited by diltiazem and nicardipine, and the heterogeneity was mimicked by treatment with charybdotoxin, a Ca2+-activated K+ (BK(Ca)) channel blocker, channels which are regulated by NO/cGMP. Both L-NMMA and charybdotoxin caused a potent vasoconstriction in the mesenteric artery precontracted with 20 mM KCl. 1 H-[1,2,4]oxadiazolo[4,3-alpha] quinoxalin-1-one (ODQ) (10(-5) M), a selective guanylate cyclase inhibitor, caused vasoconstriction in the presence of nitroprusside in the endothelium-denuded basilar artery, but not in the endothelium-denuded mesenteric artery. In conclusion, LNMMA-induced heterogenous vasoconstriction was due to the different sensitivities of vascular smooth muscles to NO/cGMP. The heterogeneity may result from a difference in the basal state of ion channels such as the voltage-dependent Ca2+ channel and the BK(Ca) channel in vascular smooth muscles.

Acceleration of widespread adenoviral gene transfer to intact rabbit hearts by coronary perfusion with low calcium and serotonin.

Previous attempts at adenoviral gene transfer to the intact heart have been limited by the requirement for prolonged exposure to high virus concentrations. In an ex vivo coronary perfusion model of intact adult rabbit hearts, we previously reported gene transfer to 96% of cardiac myocytes after a 60 min exposure to 1.6 x 10(9) p.f.u./ml Ad beta gal, a recombinant adenovirus encoding beta-galactosidase. Here we sought to decrease the virus exposure time by enhancing microvascular permeability to increase the efficiency of adenoviral gene transfer. Baseline perfusion with 1.0 x 10(8) p.f.u./ml Ad beta gal in normal Krebs solution (1 mM calcium) caused infection of 22% of myocytes at 30 min and 40% at 60 and 120 min. Increasing the virus concentration, decreasing perfusate calcium concentration, or pretreating with serotonin or bradykinin in Krebs solution or L-NAME in heparinized rabbit blood significantly decreased the necessary exposure time. Under optimal conditions of serotonin pretreatment, 50 mumol/l perfusate calcium, and a virus concentration of 1.6 x 10(9) p.f.u./ml, 2 min of coronary perfusion sufficed to produce near-total infection. This profound enhancement of infection parameters has important implications for in vivo myocardial gene transfer, where a similar strategy could facilitate gene therapy for common myocardial disorders.

Pharmacological profile of T-0201, a highly potent and orally active endothelin receptor antagonist.

The authors studied the pharmacological properties of N-(6-(2-(5-bromopyrimidin-4-yl)-4-(2-hydroxy-1, 1-dimethylethyl)benzensulfonamide sodium salt sesquihydrate (T-0201), a new nonpeptide endothelin (ET) receptor antagonist, in vitro and in vivo. In binding studies, T-0201 competitively antagonized the specific binding of [125I]-ET-1 to human cloned ETA receptors (the Ki value was 0.015 +/- 0.004 nM). T-0201 weakly inhibited [125I]-ET-1-binding to human cloned ETB receptors; the Ki value was 41 +/- 21 nM. T-0201 shifted the concentration-response curve of ET-1-induced contraction of the isolated rat aorta (ETA receptors) to the right (pA2 = 9.0 +/- 0.2). In the isolated rat trachea, a selective ETB agonist sarafotoxin S6c-induced contraction was inhibited by T-0201 (pA2 = 6.8 +/- 0.3). T-0201 also caused the inhibition of ET-1-induced contraction of the isolated rabbit pulmonary artery (pA2 = 5.7 +/- 0.3). In anesthetized rats, T-0201 (0.01-1 mg/kg) inhibited the pressor response to exogenous big ET-1 (1 nmol/kg i.v.), after both i.v. and p.o. administration, in a dose-dependent manner. The significant inhibitory effect of orally administered T-0201 on big ET-1-induced pressor response lasted for 4 hr at 0.1 mg/kg and for 8 hr at 1 mg/kg. Thus the present study demonstrates that T-0201 is a highly potent, long-lasting, orally active and selective ETA receptor antagonist.

45,X/46,X,idic(Yq) mosaicism: clinical, cytogenetic, and molecular studies in four individuals.

45,X/46,X,idic(Yq) mosaicism is associated with a variety of sex phenotypes, including Ullrich-Turner syndrome (UTS), intersexuality, and complete male. It remains unclear whether the phenotypic variability results from a dilutional effect by the 45,X cell line in the primordial gonad or an abnormality of the SRY gene (SRY). We conducted cytogenetic and molecular studies on four patients with such mosaicism, two of whom had a complete male phenotype and two who had UTS. Chromosome analyses showed that the frequency of cells carrying an idic(Yq) chromosome in peripheral blood lymphocytes and skin fibroblasts was not related to the given sex phenotype. The SRY, PABY, and ZFY genes were present in all four patients. A fluorescence in situ hybridization (FISH) study showed that both a patient with a complete male phenotype and another with UTS had duplicate copies of SRY in their idic(Yq) chromosomes, whereas a patient with UTS had a single copy of the gene. These findings suggested that the coexisting 45,X cell line is more influential on the determination of the sex phenotype in individuals with 45,X/ 46,X,idic(Yq) mosaicism.

Cloning of hamster preproendothelin-1 cDNA and its expression in the heart.

To elucidate the pathophysiologic roles of endothelin-1 (ET-1) in the heart, we first cloned and sequenced a part of hamster preproET-1 cDNA from the heart of the CHF146 hamsters. The amino acid sequence has 89% homology to that of rat preproET-1 in the cloned part. The deduced hamster 21-residue mature ET-1 is identical to human, rat, canine, and mouse ET-1. In the next step we investigated the expression of preproET-1 mRNA in the failing heart of CHF146 hamsters. For this purpose, we used 46-week-old CHF146 hamsters and age-matched control healthy hamsters. Left ventricular (LV) + dP/dtmax was significantly lower in CHF146 hamsters than in control hamsters. LV end-diastolic pressure was significantly higher in CHF146 hamsters than in control hamsters, as was central venous pressure. These results suggested that the CHF146 hamsters developed congestive heart failure. The expression of preproET-1 mRNA was greatly enhanced in the LV of the CHF146 hamsters. Because it has been reported that ET-1 induces cardiac hypertrophy and injury to cardiac myocytes in addition to its potent positive inotropic and chronotropic actions, the present findings suggest that endogenous ET-1 plays pathophysiologic roles in the failing heart of CHF146 hamsters.

Ultrarapid, highly efficient viral gene transfer to the heart.

Gene therapy for common myocardial diseases will require effective and homogeneous gene delivery throughout the intact heart. We created two experimental models to identify and optimize parameters important for adenovirus-mediated cardiac gene transfer. In cultured rabbit ventricular myocytes, the percentage of infected cells increased with higher absolute numbers of virus particles, longer durations of virus exposure, physiological temperatures, and specific culture media compositions. Simulating the in vitro conditions, we delivered adenovirus to intact rabbit hearts by intracoronary perfusion. The percentage of infected cells increased with higher coronary flow rates, longer virus exposure times, and higher virus concentrations. Under optimal conditions, nearly 100% of myocytes expressed the reporter gene beta-galactosidase after ex vivo infection. This novel delivery method, the first to demonstrate virtually complete transduction of any intact organ, could be adapted to achieve widespread gene transfer in vivo.

Inhibitory effects of TA-993, a new 1,5-benzothiazepine derivative, on platelet aggregation.

TA-993, an l-cis 4',8-dimethyl derivative of the Ca2+ antagonist diltiazem, and some of its metabolites inhibited platelet aggregation induced by collagen, ADP, epinephrine, platelet activating factor, arachidonic acid, and U-46619 in human platelets in vitro. Among the metabolites, MB3 was the most potent (IC50, <1 micromol/L; several hundred times more potent than the parent compound). The d isomer of MB3 was >100 times less potent than the l isomer. Unlike acetylsalicylic acid (ASA), TA-993 inhibited both primary and secondary phases of ADP-induced platelet aggregation and also exhibited a disaggregating effect on human platelet aggregates. The inhibitory effect of TA-993 was enhanced when used in combination with ASA. In ex vivo studies involving rats, TA-993 (approximately 0.3 to 100 mg/kg PO) dose-dependently inhibited collagen-induced platelet aggregation (ED50, 3 mg/kg PO). In the whole-blood platelet aggregation system in rats, orally administered TA-993 was also inhibitory in single (3 to 30 mg/kg) or repeated daily (10 mg/kg per day for 10 days) dosage. Orally administered TA-993 dose-dependently inhibited ADP-induced platelet aggregation ex vivo in dogs (0.3 to 10 mg/kg), significantly protected mice against collagen + epinephrine-induced thromboembolic death (10 mg/kg), and inhibited thrombus formation in an arteriovenous shunt in rats (30 mg/kg). The Ca2+-antagonistic action of TA-993 was very weak in depolarized canine basilar arteries: the potency was approximately 1/10 that of diltiazem (d-cis) and d-TA-993. These results suggest that antiplatelet action is more characteristic of the l-cis than the d-cis 1,5-benzothiazepine structure and that TA-993 may become a clinically useful antiplatelet agent of this structure series.