Utako Okamoto

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.

A new polypeptide substrate, Suc-Tyr-Leu-Val-pNA, specific for spleen fibrinolytic proteinase (SFP)

Summary In a search for an adequate synthetic substrate for human spleen fibrinolytic proteinase (SFP), Suc-Tyr-Leu-Val-pNA was newly synthesized. Its degradation by SFP was compared with that of other synthetic polypeptide substrates. Suc-Tyr-Leu-Val-pNA was degraded by SFP with a high K cat /K m , but it was not degraded by the following serine proteinases, plasmin, trypsin, thrombin, chymotrypsin, urokinase and milk plasminogen-activator, or by pancreatic elastase to any practical extent.

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.

A partially degraded form of human plasminogen in circulating blood

A method for separate measurement of the amounts of the native form and partially degraded form of plasminogen and plasmin in human circulating blood was devised based on their different mobilities on disc polyacrylamide gel electrophoresis. The relative amounts in the circulating blood as estimated by the method were as follows: 78.5% native form, 6.1% partially degraded form and 15.5% plasmin, in the resting state of healthy adults. The partially degraded form increased to about 50% after strenuous exercise load. The present study showed that relatively large amounts of the partially degraded form can be produced in the circulating blood, even though abundant plasmin inhibitors exist there. These data suggest that the partially degraded form may play an important role in thrombolysis in vivo.

New modified activated partial thromboplastin time and prothrombin time methods using a synthetic chromogenic substrate in combination with diazotization

A chromogenic substrate, H-D-Phe-Pip-Arg-pNA (S-2238) is a highly specific substrate to thrombin and releases p-nitroaniline (pNA) by the action of thrombin. We describe new modified APTT and PT methods using S-2238 in combination with the diazotization of pNA. In the modified APTT method, 100 microliter citrated plasma (diluted to 10-fold), 90 microliter 1 mM S-2238, 100 microliter 20 mM CaCl2 and 100 microliter Actin were mixed in an ice-bath, then incubated for 8 min at 37 degrees C. The reaction was stopped, and the generated pNA was diazotized by adding the following solutions sequentially: 975 microliter 0.04% sodium nitrite, 975 microliter 0.3% ammonium sulfamate and 975 microliter 0.07% N-(l-naphthyl)-ethylenediamine dihydrochloride. Diazotization changed pNA from yellow to pink. Then, absorbance at 545 nm was read, and values were expressed as thrombin units/ml plasma. In the modified PT method, 100 microliter citrated plasma (diluted to 20-fold), 90 microliter 1 mM S-2238 and 200 microliter tissue thromboplastin-C solution were mixed and processed as above. Correlations of the present modified APTT and APTT methods, and of modified PT and PT methods were significant (r = 0.426, p less than 0.01 and r = 0.561, p less than 0.01, respectively).

Increase of Nonplasmin Fibrinolytic Activity in the Lung and Spleen of Streptozotocin-Induced Diabetic Rats

In streptozotocin-induced diabetes in rats, nonplasmin fibrinolytic activity was found to be significantly increased in the lung and spleen, but not in the liver and kidney. The plasminogen-activator activities, either soluble or insoluble, were almost simultaneously increased in the lung, suggesting the possible release of plasminogen-activator from the lung to the blood. It seems likely that plasminogen-activator of the usual type and nonplasmin protease are also involved in thrombotic disorders in the experimental diabetes.

Fibrinolytic and Amidolytic Activities of Elastase- and Chymotrypsin-Like Proteases in Spleen and Leukocytes of Some Mammals

Nonplasmin-mediated fibrinolytic activities were extracted with 2 M NaCIO4 from the spleen and leukocytes of nine species of mammals. The amidolytic and fibrinolytic activities of extracts were measured using Suc-Ala-Tyr-Leu-Val-pNA for elastase-like protease (ELP) and Suc-Tyr-Leu-Phe-pNA (or Suc-Ile-Pro-Phe-pNA) for chymotrypsin-like protease (CLP). ELP and the fibrinolytic activity in the human spleen and leukocytes exhibited the highest values among the nine species. The relative order of amidolysis by CLP was as follows: hamster greater than rat greater than mouse greater than dog in the spleen and hamster greater than rat = ox greater than dog in leukocytes. Neither fibrinolytic nor amidolytic activity was measurable in spleens and leukocytes of pigs and rabbits. The ratios of ELP to CLP activity in the spleen or leukocytes of each of the species varied significantly from those observed in humans. The amidolytic activity of the spleen in control mice was low; however, that of mice which had been intraperitoneally inoculated with sarcoma 180 cells was enhanced and further accompanied by an increase in the number of granulocytes and in fibrinolytic activity. These results suggest that the enhancement of spleen amidolytic and fibrinolytic activities is induced by an increase of granulocytes in the circulation.

The level of granulocyte elastase (ELP) in children suffering from inflammatory diseases.

In order to study on the elastolysis and fibrinolysis in inflammatory process, the level of granulocyte elastase (ELP) was assayed in inflammatory diseases. The assay was carried out in children suffering from inflammatory diseases such as Kawasaki disease (MCLS), pneumonia, allergic purpura and others.ELP was extracted from granulocytes by 2mol/l KSCN and measured by using the artificial substrate, Suc-Ala-Tyr-Leu-Val-pNA. The biological activity was expressed as amidolytic activity per cell. The ELP activity of normal newborn babies was 9.0±3.1 nU/cell and it increased by age, attaining to the level of 16.1±5.8 nU/cell of normal adults. In MCLS, pneumonia and aseptic meningitis, the level of ELP decreased during the 2nd and 3rd weeks of illness, and it returned to the normal range up to the 4th or 5th week. On the other hand, the level of ELP was not reduced in upper respiratory diseases in which the inflammation was moderate. In allergic purpura, the level of ELP increased and returned to the normal range after the 5th week. Although the causes of the decreased level of ELP in inflammatory diseases are not clear, it can be suspected that ELP is released from granulocytes and consumed during inflammatory process.