Akiko Hijikata-Okunomiya

Synthetic selective inhibitors of thrombin.

Publisher Summary In circulatory blood, thrombin is found as the zymogen, prothrombin, which is activated by intrinsic and extrinsic pathways. Once activated, thrombin is neutralized by plasma antithrombins, e.g., antithrombin III (ATIII). The result is that active thrombin is rarely found in circulatory blood. However, the activation of thrombin occurs in pathological states and such activation is undoubtedly hazardous. The synthetic selective inhibitors discussed in this chapter are designed based on biochemical knowledge regarding the conversion of fibrinogen by thrombin to fibrin. Studies on the fibrinopeptides described in the chapter suggests that thrombin recognizes the L-arginine residue, in particular its biophysical and geometrical arrangements. Therefore, L-arginine is the most promising skeleton for designing thrombin inhibitors for studying the structure–function relationship between a number of arginine derivatives and their thrombin inhibitory activity. It is known that (1) argatroban accelerates fibrinolysis by t-PA, (2) argatroban inhibits the activation of FXIII, (3) argatroban may inhibit the activity of the remaining thrombin within the thrombi, and (4) argatroban does not require ATIII.

Development of plasma kallikrein selective inhibitors.

During the course of the development of active center-directed plasmin inhibitors, it was found that N-(trans-4-aminomethylcyclohexanecarbonyl)-lysine-4-methoxycarb onylanilide inhibited plasma kallikrein more potently than other enzymes such as plasmin, urokinase, and thrombin, although the inhibitory activity was not as potent and enzyme selectivity not as high. Based on studies of structure-activity relationship, we designed and synthesized the plasma kallikrein selective inhibitor, N-(trans-4-aminomethylcyclohexanecarbonyl)-phenylalanine-4-carboxy methyl- anilide (Tra-Phe-APAA). Tra-Phe-APAA inhibited plasma kallikrein with a Ki value of 0.81 microM, while it inhibited glandular kallikrein, plasmin, urokinase, tissue plasminogen activator, factor Xa, factor XIIa, and thrombin with Ki values of > 500, 390, 200, > 500, > 500 > 500, and > 500 microM, respectively. We designated Tra-Phe-APAA as PKSI-527. Using PKSI-527 as an affinity ligand, we synthesized a new affinity gel (PKSI-Toyopearl) and employed it for the rapid purification of plasma kallikrein from human plasma. Human plasma activated with kaolin after acid treatment was applied to a PKSI-527-Toyopearl column. Adsorbed protein was eluted with 50 mM glycinehydrochloric acid buffer (pH 3.0). Plasma kallikrein was purified 181-fold with a yield of 85% from the kaolin-activated plasma.

Development of plasmin and plasma kallikrein selective inhibitors and their effect on M1 (melanoma) and ht29 cell lines

trans-4-Aminomethylcyclohexanecarbonyl-Tyr(O-Pic)-octylamide (YO-2) inhibited plasmin (PL) selectively, while trans-4-aminomethylcyclohexanecarbonyl-Phe-4-carboxymethylanili de (YO-1) inhibited plasma kallikrein (PK). YO-2 induced apoptosis of M1 (melanoma) cell line and HT29 colon carcinoma cells during 24 h through activation of caspase-3, while YO-1 did not affect either cell line even during 48 h.

Effect of a synthetic thrombin-inhibitor MD805 on the reaction between thrombin and plasma antithrombin III

MD805, a synthetic thrombin-inhibitor, effectively retarded the time-dependent inactivation of thrombin which was generated endogeneously or added exogeneously in human plasma. The kinetical study of the time-dependent inactivation indicated that the type of inhibition was competitive and the obtained Ki of MD805 for thrombin was 3 x 10(-8)M. MD805 also inhibited the formation of thrombin-ATIII complex. These results indicated that the active site of thrombin was involved in the reaction between thrombin and ATIII, and that MD805 competed with ATIII for thrombin in exactly the same manner as it competed with fibrinogen or synthetic peptide substrates. As a result, MD805 would serve as a protective agent for ATIII from being consumed, in addition to its potent thrombin-inhibitory activity without the aid of ATIII. By contrast, heparin accelerated the time-dependent inactivation rate of thrombin and the formation of thrombin-ATIII complex, which indicates that heparin accelerates the consumption of ATIII.

Detection of PF3 availability in whole blood from volunteers and beta-thalassemia/HbE patients: a promising method for prediction of thrombotic tendency.

The platelet factor 3 (PF 3) plays a very important role in activation of coagulation factors and is regarded to be available during activation of platelets. However, membrane fraction of erythrocytes is also shown to have PF 3-like activity, suggesting that the abnormal erythrocytes may accelerate the activation of platelet by forming thrombin on their abnormal membrane or by way of other factors of the abnormal erythrocytes, and may increase the availability of PF 3 in whole blood (WB). To examine this hypothesis, we developed a method for determination of PF 3 activity, because the method now available for the PF3 determination could not detect changes in PF 3 activity with time. The principles of our method were as follows: 1) The reaction system was adjusted so that the amount of thrombin generated in a fixed reaction time correlates with the amount of PF 3. 2) To avoid inhibition of thrombin activity by antithrombin III, a synthetic thrombin inhibitor, MD 805, was added to the system and the activity of thrombin generated was measured by synthetic thrombin substrate S-2238 using A405 as an indicator of the availability of PF3. The results obtained by the method were the following: WB taken from volunteers showed A405 of 0.12 +/- 0.02 at 30 minutes after blood collection and then the A405 increased to 0.27 +/- 0.03 at 90 minutes. However, one volunteer showed the value of 0.59 at 90 minutes, though the value at 30 minutes was 0.16. The platelet number in his WB did not change during the study.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A new method for the determination of prothrombin in human plasma

A two stage method for the determination of prothrombin is described in which the potent synthetic thrombin-inhibitor MD805 is used to suppress the inactivation process of thrombin by antithrombins in plasma. In the activation stage, prothrombin was activated almost instantaneously after the addition of tissue thromboplastin in 20-times diluted plasma and then the generated thrombin was inactivated progressively. Addition of MD805 suppressed the inactivation process of thrombin in a dose-dependent manner, while it hardly affected the complete activation of prothrombin. Thus, the generated thrombin was maintained stable in the presence of 50 microM MD805, and was measured by using 0.5 mM S-2238 as substrate. The method exhibited a linear relationship to the dilution of plasma. The decreased level of the other extrinsic coagulation factors such as FVII, FV, FX and tissue thromboplastin did not cause any significant changes until their activities were decreased to 3, 12, 12 and 20% of their controls. In addition, the method is very simple and easy for standardization.

Use of an active center-directed plasmin inhibitor elucidates the multiplicity of plasmin actions

In our studies, designed to synthesize an active center-directed plasmin (PL) inhibitor, N-(4-aminomethylbenzoyl)-4-(3-picolyloxy)-L-phenylalanine n-hexylamide dihydrochloride (PASI-535) was found. We characterized PASI-535 and analyzed the actions of PL, comparing with those of PASI-535 and tranexamic acid (t-AMCHA). (1) PASI-535 strongly inhibited not only fibrinolysis (IC50: 2.9 x 10(-6) M) but also amidolysis (Ki value: 2.9 x 10(-6) M) and fibrinogenolysis (IC50: 4.5 x 10(-6) M) induced by PL. While t-AMCHA which strongly inhibited fibrinolysis (IC50: 6.0 x 10(-5) M), rarely inhibited amidolysis (Ki value: 4.0 x 10(-2) M) and fibrinogenolysis (IC50: 1.0 x 10(-2) M). (2) PL is able to liberate kinins by degrading kininogen. This kinin-generation by PL was inhibited by 2 x 10(-5) M PASI-535. However, it was little inhibited even by 1 x 10(-3) M t-AMCHA. (3) The inhibitory effect of PASI-535 and t-AMCHA on tumor growth was studied. In sarcoma-180 bearing mice, ascites retention and the increase of tumor cells were markedly suppressed by subcutaneous injection of PASI-535, either 30 mg/kg/day or 50 mg/kg/day, for 5 days, and the inhibitory effect was dose-dependent. Although t-AMCHA also reduced both ascites retention and the increase of tumor cells, it needed approximately 40 times (2 g/kg/day) the amount of PASI-535 to exert these effects. PASI-535 may be a useful tool in analyzing the multiplicity of PL actions. Moreover, PASI-535 can be used as an antifibrinolytic drug which has a mechanism of function different from that of t-AMCHA.

Effects of Argatroban on Thrombin-Induced Events in Cultured Vascular Smooth Muscle Cells

BRIEF COMMUNICATION Effects of Argatroban on Thrombin-Induced Events in Cultured Vascular Smooth Muscle Cells A. Hijikata-Okunomiya1, Y. Nakaya2, N. Inoue3, A. Takahashi3, T. Taniguchi3, K. Wanaka2, Y. Tsuda4 Y. Okada4 and Y. Ishikawa1 1Faculty of Health Science, Kobe University School of Medicine; 2Kobe Research Projects on Thrombosis and Haemostasis; and 3First Department of Internal Medicine, Kobe University School of Medicine, Kobe; 4Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan.

IN VITRO AND IN VIVO PROPERTIES OF SYNTHETIC INHIBITORS OF THROMBIN: RECENT ADVANCES

The purpose of our study of synthetic inhibitors of thrombin was to find out novel compounds which inhibit thrombin greatly and selectively and to establish their structural features. In the process of the research, we recognized that synthetic inhibitors of thrombin should have additional properties, such that the acute and subacute toxicities are low, and that the Vd (volume of distribution) is small We considered that our thrombin inhibitor should meet these requirements, in perspective of using it in animal experiments and further even in clinical trials.