Yoshikuni Tamao

Potent inhibition of thrombin by the newly synthesized arginine derivative No. 805. The importance of stereo-structure of its hydrophobic carboxamide portion

Abstract Four stereoisomers of 4-methyl-1-[N 2 -(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid were synthesized and examined for the inhibitory effect on thrombin. The inhibitory potency varied largely with the stereo-configuration of the 4-methyl-2-piperidinecarboxylic acid portion. The (2R, 4R)-isomer was the most potent inhibitor with a Ki of 0.019 μM, while the (2R, 4S) and (2S, 4R)-isomers showed the values of Ki 0.24 and 1.9 μM, respectively. The least potent inhibitor, (2S, 4S)-isomer, showed a Ki of 280 μM which is approximately 15,000 times that of (2R, 4R)-isomer.

A new series of synthetic thrombin-inhibitors (OM-inhibitors) having extremely potent and selective action.

Abstract The studies in searching for potent and highly selective synthetic inhibitors to thrombin led the authors to find a new series of the thrombin-inhibitors which belong to N α -naphthalenesulfonyl-L-arginine derivatives; values of I 50 of these potent inhibitors are found in the range from 0.03 μM to 2 μM, when fibrinogen (3 μM) or N α -benzoyl-L-phenylalanyl-L-valyl-L-arginine p-nitroanilide (100 μM) is used as substrate. These potent thrombin-inhibitors are tentatively called OM-inhibitors according to their code name in our laboratories.

Inhibition of factor Xa-induced platelet aggregation by a selective thrombin inhibitor, argatroban

In the present study, a direct selective thrombin inhibitor, argatroban, and an indirect non-selective inhibitor, heparin, were examined for the inhibitory effect on factor Xa-induced platelet aggregation. Platelet aggregation was induced by thrombin or factor Xa+prothrombin in the presence of Ca++ using rabbit gel-filtered platelets. IC50 of argatroban on factor Xa-induced aggregation was 5-7 times higher than that on thrombin-induced aggregation, while IC50 of heparin in the presence of antithrombin III on factor Xa-induced aggregation was 90-200 times higher than that on thrombin-induced aggregation. This finding suggests that argatroban inhibits thrombin generating on the platelet surface more efficiently than heparin, and this may be one of the reasons for the higher efficacy of argatroban in arterial thrombosis as compared with heparin.

In vitro and in vivo studies of a new series of synthetic thrombin-inhibitors (OM-inhibitors)

Abstract To evaluate the thrombin-inhibitors (OM-inhibitors) synthetized by the authors, their actions in vitro and in vivo have been investigated. Results obtained are summarized as follows: 1. (i) The inhibitory action of the OM-inhibitor (OM-205) is highly selective to thrombin when compared with trypsin, plasmin or reptilase. 2. (ii) The mode of inhibition of the OM-inhibitor (OM-46) to thrombin is competitive in the hydrolysis of Nα-benzoyl-DL-arginine p-nitroanilide. 3. (iii) The decreasing effects of thrombin infusion in rabbits on plasma fibrinogen content and platelets in number are remarkably blocked by the presence of the OM-inhibitor (OM-189) in the blood, which suggests that the OM-inhibitors, very effective in vitro, possess the anti-thrombin activity also in vivo.

SIMILARITY AND DISSIMILARITY IN THE STEREOGEOMETRY OF THE ACTIVE SITES OF THROMBIN, TRYPSIN, PLASMIN AND GLANDULAR KALLIKREIN

The relationship between chemical modifications of arginine derivatives and inhibitory activity to trypsin, plasmin and glandular kallikrein was investigated comparing with that of thrombin and concluded as follows: The hydrophobic binding pocket, which has been reported previously to be stereogeometrically very similar in trypsin and thrombin, corresponded to the length of ethylpiperidine. Concerning the site (termed the P site) next to the hydrophobic binding pocket, there were large differences in stereogeometry between trypsin and thrombin; the binding site of trypsin extended further to allow propyl and phenyl group attached to piperidine, while that of thrombin would be much narrower and unable to allow them. The P sites of plasmin and glandular kallikrein resembled that of trypsin in being able to allow phenyl group. To substantialize the hydrophobic binding pocket and the P site, a (2R, 4R)-MQPA-trypsin complex model was generated using the results of X-ray crystallography of (2R, 4R)-MQPA and BPTI-trypsin complex by calculation to minimize van der Waals contacts, and it was of great use for understanding the geometry of the active sites of trypsin, thrombin, plasmin and glandular kallikrein.

Beneficial effects of a new prostacyclin analogue, KP-10614, on acute myocardial infarction in rats.

Summary: The potential therapeutic value of a new prostacyclin analogue, (4z, 16s)-4,5,18,18,19,19-hexadehydro-16,20-dimethyl-Δ6(9a)-9-(O)-methano-PGI1 (KP-10614), was studied in acute myocardial infarction in rats. Myocardial infarction was induced by ligation of the left coronary artery and ischemic injury was followed up to 4 h. The infarct size, evaluated by the area unstained by 2,3,5-triphenyltetrazolium chloride, reached 41.1 ± 1.4% of the left ventricle at 4 h. KP-10614 (3 ng/kg/min × 4 h) reduced the infarct size at 4 h significantly (26.5 ± 2.9%). At the same dose, KP-10614 inhibited ADP-induced ex vivo platelet aggregation significantly (21.5 ± 4.0% of the control aggregation), but did not alter the arterial blood pressure or heart rate. To assess the role of platelets in myocardial infarction, circulating platelets were reduced by about 95% with rabbit antiserum to rat platelets. In platelet-depleted rats, the infarct size decreased significantly to 24.1 ± 4.6% of the left ventricle at 4 h. These results suggest that platelets play an important role in expression of myocardial ischemic injury resulting from coronary artery occlusion in rats, and the ability of KP-10614 to decrease the infarct size appeared to be attributable, at least in part, to the inhibition of platelet aggregation or cellular metabolic effects produced by platelets at the site of tissue injury.