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Recent studies of the synthetic selective inhibitors; With special reference to non-plasmin fibrinolytic enzyme, plasmin and plasma-kallikrein
THROMBOSIS RESEARCH, Supplement Vlllj 131-141, 1988 0049-3848/88 $3.00 + .00 Printed in the USA. Copyright (c) 1988 Pergamon Press p1c. A11 rights reserved.
REVIEW RECENT STUDIES OF THE SYNTHETIC SELECTIVE INHIBITORS; WITH SPECIAL REFERENCE TO NON-PLASMIN FIBRINOLYTIC ENZYME, PLASMIN AND PLASMA-KALLIKREIN
S.Okamoto, U.Okamoto*, A,Hijikata-Okunomiya**, K,Wanaka and Y,Okada* Kobe Research Projects on Thrombosis and Haernostasis, Saiseikai Hospital, Kobe 651 Japan, *Kobe-Gakuin University, Kobe 673 Japan and **School of Allied Medical Sciences, Kobe University, Kobe 654 Japan
I. INTRODUCTORY REMARKS In 1975, a short review was presented by Okamoto and Hijikata, in which trials to approach as possible as rationally to the highly selective inhibitors such as epsilon-amino-caproic acid (EACA) and tranexamic acid (tAMCHA) were described (1). The mode of action of these inhibitors was very unique, Fact is, years more than one decade elapsed, when it was found that they inhibit the fibrinolysis by plasmin through their binding to the lysinebinding-sites (LBS) of the heavy chain of the plasmin and plasminogen (2,3), In the following undertaking by Okamoto and his colleagues, the target enzyme was thrombin, the highly selective synthetic inhibitors of which were systematically pursued. The first step of stud&es was to examine the thrombin inhibitory activities of the homologs of N -tosyl-arginine methyl ester (TAMe), which had weak thrombin inhibitory activity, TAMe was easily hydrolyzed by thrombin; this might be understood from the fact that most of the substrates to the enzyme assumed the inhibitory activity, as it were, "substrate inhibition". It was soon found that some chemical modifications of the tosyl radical increased the stability of the compounds as well as inhibitory activity with regard to thrombin. Thus, stepwise chemical modifications were conducted, as the resu~8B, the compound called No. &05 was obtained, Ki value of which, 3,7xlO M; No. 205 was chemically N -dansyl-arginine 4ethylpiperidine amide (4).
Key words: plasmin, plasma kallikrein, elastase, thrombin, synthetic substrate
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synthetic
inhibitor,
leukocyte
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It was noticed that No. 205 showed high toxicity (i.p. LO for mice SO was 80 mg/kg) and that inhibitory activity of the compound to plasma cholinesterase was extremely strong (5). Results accumulated supported the possibility that the 4-ethylpiperidine radical of No. 205 assumed the toxicity of the compound. Therefore, instead of the ethylpiperidine, a-alanine methyl ester, the non-toxic radical, was introduced into the C-terminal of Arg. The for mice, 1000 mg/kg, Ki compound obtained _~as No. 329, of which i.p. LO SO for thrombin, 10 M order and the inhibitory act~vity to plasma cholinesterase, was very weak. Encouraged with this finding, again stepwise chemical modificat~§ns were made and finally No. BOS was obtained (6). Ki of No. 805 was 1.9xlO M, i.p. LO for mice, 700 mg/kg (Fig 1). No. BOS has been SO broadly investigated either experimentally and clinically. Its medical utility has been still under investigations in a large number of the clinical institutions in Japan (7-9). The mode of action of No. 805 (abbreviated as MO-BOS clinically) was typically competitive inhibition, This implies that the active site of thrombin takes the structure mold of No. 805. A question may remain why No. 205 inhibits both thrombin and plasma cholinesterase while No. 805 inhibits thrombin only. Hijikata-Okunomiya et al. have baen working on the problem mentioned above, and reported in 1987 that N -dansyl-arginine 4-phenylpiperidine amide was highly selective inhibitor of plasama cholinesterase. This finding would be one of the most successful cases in designing newly the highly selective synthetic inhibitors (10). It should be noteworthy that the background knowledge of the c:omparative aspects of different serine proteases has been accumulated, not only employing laboratory-experimental technique but also using the computergenerated molecular model to predict the nature of the active site as well as selective site of the enzyme concerned (11).
NH NH 2 ~I MD-805
_~
S
C
NH
CH2 CH2 CH2
SO-NHtHeo-N~CH 2 }-/ 3 eOOH
CH 3
(2R.4R)
FIG. 1 Chemical structure of
No. 805 (MD-80S)
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II. NON-PLASMIN FIBRINOLYTIC PROTEASE Although the plasmin system is known to play an important role in fibrinolysis in the blood, several studies have clearly demonstrated the presence of a plasmin-independent fibr inolysis in leukocyte by Plow et al. (1975), Bilezikian and Nossel (1977) and Kopec et al. (1978) (12-14). The protease has been purified from leukocyte extracts by using sepharose chromatography, indicating an indipendent fibrinolytic pathway from plasmin system. The authors group including Nagamatsu et al. also obtained a neutral protease capable of fibrin degradation which was extracted especially from the human spleen obta ined at the time of the autopsy of ca. 400 cases (15). The enzyme, spleen fibrinolytic protease (SFP), was found to be a neutral serine protease different from plasmin based on its mode of action against synthetic substrates and inhibitors. The physicochemical and enzymatic properties of the enzyme were found very s imilar or identical to those of leukocyte elastase (Table 1). Later an effective purific~tlon method was found using a new affinity chromatography coupled with Suc-L-Tyr-D-Leu -D-Val-pNA (16). The purified preparation contained practically no chymotrypsin-like protease act ivity. The enzyme so purified readily degraded fibrin, fibrinogen and Val-type synthetic peptide substrates, such as Suc-L-Tyr-L-Leu-L-Val-pNA and Suc-LAla-L-Tyr-L-Leu-L-Val-pNA (17 ). These substrates were not hydrolyzed by plasmin. The enzyme was inhibited by Suc-L-Tyr-D-Leu-D-Val-pNA competitively (Table 2) and by Boc-L-Ala-L-Tyr-L-Leu-L-val-CH irreversibly (Table 3) as 2Cl reported by Tsuda et al. (18). DFP and SBTI inn ibited the enzyme. Neither tAMCHA nor TLCK inhibited the enzyme. Notable was that the species difference of the non-plasmin fibrinolytic enzyme was considerably great. Pathophysiological significance of non-plasmin fibrinolysis is still obscure. However, concerning the possible role of the leukocyte and spleen in thrombolysis, we ex pect that the studies may provide some precise tools for the fibrinolysis in a broad sense. Our further studies are now continuing
TABLE 1 Comparative Activities of Spleen Fibrinolytic Protease (SFP) and Leukocyte Elastase (LE) Substrate Suc-Tyr-Leu-Val-pNA Suc-Tyr-Leu-Ile-pNA Suc-Tyr-Leu-Ala-pNA Suc-Tyr-Leu-Leu-pNA Suc-Tyr-Leu-Phe-pNA Suc-Tyr-Leu-Gly-pNA Suc-Tyr-Leu-Met-pNA Suc-Tyr-Leu-Arg-pNA
SFP
LE
100 ,
100 ,
33 16
23
<0.2
o
o o o
13 <0.2
o
o
o o
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TABLE 2 Reversible Inhibitors of SFP Ki (mM) Suc-L-Tyr-O-Leu-O-Val-pNA LLDOOL-
0L0-
D0L-
0-
0-
L-
0.12 0.19 0.20 0.24 0.35 0.94
Substrate: Suc-L-Tyr-L-Leu-L-Val-pNA
TABLE 3 Irreversible Inhibitors of SFP
Inhibitor Boc-Tyr-Leu-Val-CH Cl Boc-Ala-Tyr-Leu-Val-CH 2Cl 2 Oan-Ala-Tyr-Leu-Val-CH Cl 2 AC-Ala-Tyr-Leu-Val-CH 2Cl
I 4) (Mxl0
Half life (sec)
0.1 0.1 0.1 0.1
31 14 23 28
K~£S~{I
(M
s
)
2240 4950 3010 2480
Substrate: Suc-Ala-Tyr-Leu-Val-pNA (0.5 mM) with the scope that non-plasmin fibrinolysis would be far more significant than ever expected, particularly in the late period of the thrombosis in vivo. An exciting task challenges us to find a new way, how to mobilize this particular enzyme in patients. III. AMlDOLYSIS AND FIBRINOLYSIS WITH PLASMIN Apart from the LBS-directed inhibitors such as EACA or t-AMCHA, the series of active-site directed inhibitors of plasmin have been studies by our group (19). Based on the facts that plasmin Spllts malnly the C-terminal of lysine in fibrinogen, our group first noticed ~-tosyl-lysine methyl ester (TLMe) was a very weak and relatively speclfic inhlbitor as well as substrate on plasmin. A number of derivatives of TLMe lncluding 5-2251 and its homologs substrates were re-examined and results obtalned were theoretically summarized according to the Free and Wilson method. Thus, the O-Ile-Phe-LyspNA was found to be the more sensltive substrate than lts homologs (Table 4) (20). Interesting was that O-Ile was signlficant, which was well coincident with the theoretical prediction on the structure of suitable substrate of plasmin, proposed by Szabo et al. (21).
about
Stimulated by the presence of the sensitive substrate mentioned above, 900 kinds of new compounds were synthesized in our laboratories and
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TABLE 4 Kinetic Parameters for the Amidolysis of Substrate by Plasmin Substrate D-Ile-Phe-Lys-pNA 3-MV-Phe-Lys-pNA Ile-Phe-Lys-pNA
kcat (s
Km (mM)
0.02 0.18
10 18
0.33
19
-1
)
3-MV: 3-methylvaleryl
Ki for plasmin
7.4 JJM Ki for thrombin
>500 pM
FIG. 2 Chemical structure of OS 153 several kinds of selective, potent and reversible inhibitors of plasmin were obtained, chemical structure of one of them called OS 153 belng shown in Fig. 2.
These inhibitors belong to lysine derivatives, characterized with the presence of NH in the chemical structure which covers the N-terminal of 2 lysine. The radical which covers the C-terminal of lysine is much complicated when compared with pNA. The structure of such synthetic inhibitors may take the structure mold of the active site of plasmin. When the activities of t-AMCHA was compared with these of OS group plasmin inhibiotrs, remarkable differences are found as shown in Table 5. Inhibiting activities of t-AMCHA (and EACA) are characterized by its strong inhibition on fibrinolysis but neither on fibrinogenolysis nor on amidolysis of S-series synthetic substrates. While OS inhibitors inhibit nearly equally not only fibr inolysis by plasmin, but also fibrinogenolysis and also _gmidolysis by plasmin, with Ki values of very low concentration such as_~O M. In particular, Ki value of OS 324 was found to be in the ord~B of 10 M. This value can be compared with that of MD-B05, Ki of which , 10 M.
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TABLE S Inhibitory Effect of t-AMCHA and OS lS3 on Amidolysis, Fibrinolysis and Fibrinogenolysis by Plasmin ' (a) Am 1' d 0 1 YS1S Ki (M) t-AMCHA OS 153
-2 4.0x10_ 6 7.4x10
i 1 YS1S ' (b) F 1' br r1no
ISO (M) -S 6.0xlO_ 6 6.1x10
Fd.b ' l ' (b) 1 r1nogeno YS1S
ISO (M) -2
1.0xlO_ S 2.0xlO
(a) Amidolysis was measured with D-Val-Leu-Lys-pNA as substrate. (b) Fibrinolysis and Fibrinogenolysis were measured using 0.04% and 0.2% fibrinogen, respectively. IV. PLASMA KALLIKREIN AND ITS HIGHLY SELECTIVE INHIBITORS It should be recalled that the action of aprotinin has been characterized by its potent inhibition toward glandular kallikrein and plasmin: And that the action of aprotinin toward plasma kallikrein (PK) is approximately 10 times weaker than that to glandular kallikrein. Highly selective synthetic inhibitors of PK has been first reported by the authors, very recently (22). In this review, the novel series of highly selective inhibitors called PKSI are introduced but briefly. Fig. 3 shows a chemical structure of one of the PK inhibitors. PKSI1007 assumed a tripod structure, the basic skeleton of which was L-arginine. This structure showed trans-4-aminomethylcyclohexanecarbonyl to be at the Nterminal of the arginine molecule and 4-ethoxycarbonylanilide to be at the Cterminal position.
FIG. 3 Chemical structure of PKSI-1007
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TABLE 6 Enzyme Inhibition of PKSI-Inhibitors P.Kallikrein Ki (pM) PKSI-I007 (Index) PKSI-OlBO (Index) PKSI-0527 (Index)
B.O (1) 0.47 (1) O.Bl (1)
G.Kallikrein Ki (pM) 120
Plasmin Ki (pM) 270
(15) 53 (113) >500 (>617)
(34) 20 (43) 390 (4Bl)
Thrombin Ki (pM)
FXa Ki (pM)
>500 >500 (>63) (>63) >500 330 (>1064) (702) >500 >500 (>617) (>617)
Substrate: S-2302 for P.kallikrein, S-2266 for G.kallikrein, S-2251 for plasmin, S-223B for thrombin and S-2222 for factor Xa. The mode of the inhibition of PKSI inhibitors was competitive toward PK as well as the different enzyme examined. Inhibitory effects of PKSI inhibitors on the enzymes were measured using the synthetic chromogenic substrates, as shown in Table 6. (a) PKSI-1007 showed Ki value of B.O pM for PK. By contrast, of 120 pM for glandular kallikrein, 270 pM for plasmin and more than 500 pM for thrombin and factor Xa were obtained. (b) PKSI-OlBO was more potent inhibitor than PKSI-1007, Ki value for PK being 0.47 pM. (c) PKSI0527 showed that Ki value for PK was O.Bl pM, while Ki values for plasmin, glandular kallikrein, thrombin and factor Xa were 390 pM, >500flM, >500pM and >500 pM, respectively. High selectivity toward PK was thus remarkably demonstrated, so far as the enzymes examined concerned. In particular, Ki values of PKSI-0527 for glandular ka llikrein, plasmin, thrombin and factor Xa were hundreds times larger than that for PK, as indicated by the figures in the parenthesis in Table 6. In addition, it can be noted that i.v. L0 was over 50 100 mg/kg in our preliminary toxicity test, which encourages us to extend the studies toward the in vivo applications.
Ki
As the preliminary test toward in vivo studies, the intact human plasma was employed and the inhibitory effect of PKSI-OlBO on the bradykinin (BK) formation by kaolin as well as effect on PTT were examined. As shown in Fig. 4, the amount of BK formed (left ordinate) in the kaolin-activated human plasma decreased by increasing the concentration of PKSI-01BO (abscissa) gradually. Results concluded the inhibition of BK formation by PKSI-0180 in such a contact activated system. Fig 4 also showed that PKSI-0180 was able to prolong PTT. The right ordinate indicated the ratio of PTT with different concentration of PKSI-01BO to PTT without inhibitor. The results concluded that PK inhibitor was able to prolong PTT presumably by inhbiting PK of the contact system. In the following studies, the ascites of tumor bearing mice was employed. The Sarcoma 1BO was inoculated into the abdominal cavity of mice and the ascites on the 8th day was obtained. The ascites collected underwent the acid-treatment to get rid of the kininase activity. The samples were incu0C bated at 37 and the aliquots taken out were measured of the amount of BK formed. In the control aliquots, the amount of BK showed remarkable increase with the time course of incubation. The presence of PKSI-0527 of the final concentration of 10 pM and 100 pM in the ascites showed the significant
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FIG. 4 fnhibitory effect of PKSI-01BO on bradykinin formation by kaolin and PT1 BK formation: The intact human plasma containing o-phenanthroline 0C was incubated with PKSI-01BO and kaolin (0.2 mg/ml) at 37 for 2 min. BK levels in the plasma were measured by EIA method using a MARKIT-A-Bradykinin kit (Dainippon Pharmaceutical Co.). PTT was measured with PTT reagent purchased from Behring institute.
600
'::' 400
C---
..•..
,
.....
.- -.. ;'~
",
~ 800
"
'ii
..•.
:;
",'
,'10JlM
... 200
;'
,,' ;'100pM
" ,
III
.. -..r:r .:: .. --V
100
o o
10
"
,A'
.-"
-",- ,.-
20
TI.. 8(.. IIl)
FIG. 5 Inhibitory effect of PKSI-0527 On BK formation in acid-treated ascites of sarcoma 180 mice
80
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decrease of the amount of BK, indicating clearly the dose-dependency of the concentrat ion of PKSI-0527. The PKSI-Ol80 showed s i milar effect in suppress ing the BK formation in the ascites (Fig. 5). PKSI-l007, 0180 and 0527 were obtained as the highly selective inhibitors. However, possible role of PK concerning with the contact system should involve the compl icated problems in health and disease. The present studies may provide some tools for the studies in this field, the correlation between coagulation, BK formation, f ibrinolysis and possibly platelets aggreagat ion in the contact system would be e x t r eme l y complicated, even so we expect that results obtained by using PKSI-inhibitors cast light on a promising perspect ive to us. Needless to say, the further stud ies are urgently required and they are now under investigation in our laboratori es.
V. SIGNIFICANCE OF PROTEOLYTIC REACTION --- The Present and the Future --Recent studies of the different proteases revealed the inherent ability of the enzyme to cl eave their substrates with the unexpectedly high selectiv ity, which could be e no ug h compared with that of the antigen-antibody reaction. In fact, such a high selectivity provided the systematlc and stepwise activation of the protea ses as seen 1n the coagulation cascade. The present paper dealt mainly with the results of our investigations conduc ted to provide the h ighly selective synthet ic inhibitors which may be the excellent tools for revealing significance of the target proteases, even so fina l goal should be to find the clinical ut ilities of synthetic compounds. These studies long continued more than 40 years have been very much encouraged by know ing the presence of natural i nh i b i t o r s with h igh select ivity jus t as U PI . 2 Studies of ours have been developing fortunately and selective synthet ic i nhibitors of trypsin are now under investigation. At the same time, I have learned from Homma that some kinds of virus including AIDS virus may require the activation by trypsin-like protease i n each multiplication step (23). This reminded us the activation of the zymogen to the active enzyme by som e certain proteases. The future of the investigation of proteases seems to be far more promising than our expectation, which has been cherishing in our mind.
ACKNOWLEDGEMENT The authors express the cordial thanks to their close co-workers in d ifferent institutions who contributed immensely. Dr. T. Naito and his co workers, the institute of Showa Denko have been working closely together for the r ecent several years, their contributions be ing appreciated. Sp ec ial thanks to Dr. R. Kikumoto and his co-workers, central laboratories, Mitsubishi Chemical Industri es for the studies of MD-805 and its homologs. To those other close friends who supported us, we ask their pardon for missing even names due to the space a l l o t t e d .
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REFFERENCES 1.
OKAMOTO,S. and HIJIKATA,A. Rational Approach to Proteinase Inhbitors, In: Drug Design VI. E.J.Ariens (Ed.) San Francisco: Academic Press, 1975, pp. 143-169.
2.
IWAMOTO, M. Plasminogen-plasmin system. IX. Specific binding of tranexamic acid to plasmin. Thrombos.Diathes.Haemorrh.(Stuttg.) 33, 573-585, 1975.
3.
MARKUS,G., PRIORE,R.L. and WISSLER,F.C. The binding of tranexamic acid to native (G1u) and modified (Lys) human plasminogen and its effect on conformation. J.Bio1.Chem. 254, 1211-1216, 1979.
4.
HIJIKATA,A., OKAMOTO,S., KIKUMOTO,R. and TAMAO,Y. Kinetic studies on the selectivity of a synthetic thrombin-inhibitor using synthetic peptide substrates. Thrombos.Haemostas.(Stuttg.) 1, 1039-1045, 1979.
5.
NAGANO,S., OKAMOTO,S., IKEZAWA,K., MIMURA,K., MATSUOKA,A., HIJIKATA,A. and TAMAO,Y. Fluorescence studies on the mode of action of two synthetic thrombin inhibitors, No.205 and No.80S. Thrombos.Haemostas. 46, 4S, 1981
6.
OKAMOTO,S., HIJIKATA,A., KIKUMOTO,R., TONOMURA,S., HARA,H., NINOMIYA,K., MARUYAMA,A., SUGANO,M. and TAMAO,Y. Potent inhibition of thrombin by the newly synthesized arginine derivative no.805. The importance of stereostructure of its hydrophobic carboxamide portion. Biochem.Biophys.Res. £Q!.!!!!ll!n • .1Ql, 440-446, 1981.
7.
OTA,K., KAWAGUCHI, H., NAKAGAWA,S., KOSHIKAWA,S., MAEDA,K. MATSUI,N., HIRASAWA,Y. and SASAOKA,T. Clinical evaluation of a new thrombin inhibitor available for haemodialysis. Proc.Eur.Dialysis Transp1ant.Assoc. 20, 144-149, 1983.
8.
KUMON,K., TANAKA,K., NAKAJIMA,N., NAITO,Y. and FUJITA,T. Anticoagulation with a synthetic thrombin inhibitor after cardiovascular surgery and for treatment of disseminated intravascular coagulation. Crit.Care Med. 11, 1039-1043, 1984.
9.
YONEKAWA,Y., HANDA,H., OKAMOTO,S., KAMIJO,Y., ODA,y., ISHIKAWA,J., TSUDA,H., SHIMIZU,Y., SATOH,M., YAMAGAMI,T., YANO,I., HORIKAWA,Y. and TSUDA,E. Treatment of cerebral infarction in the acute stage with synthetic antithrombin MD-805: Clinical study among multiple institutions. Arch.Jpn.Chir. 55, 711-722, 1986.
10. HIJIKATA-OKUNOMIYA,A., OKAMOTO,S., KIKUMOTO,R. and TAMAO,Y. Stereogeometry of the active sites of serine enzymes gathered from synthetic thrombin-inhibitors. Thrombos.Haemostas. 58, 489, 1987. 11. HIJIKATA-OKUNOMIYA,A., OKAMOTO,S., KIKUMOTO,R., TAMAO,Y., OHKUBO.K., TEZUKA,T., TONOMURA,S. and MATSUMOTO,O. Similarity and dissimilarity in the stereogeometry of the active sites of thrombin, trypsin, plasmin and glandular kallikrein. Thromb.Res. 45, 451-462, 1987. 12. P1ow,E.F. and EDGINGTON,T.S. An alternative pathway for fibrinolysis. I. The cleavage of fibrinogen by leukocyte protease at physiologic pH. J.Clin.Invest. 56, 30-38, 1975.
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13. BILEZIKIAN,S.B. and NOSSEL,H.L. Unique pattern of fibrinogen cleavage by human leukocyte proteases. Blood 50, 21-28, 1977. 14. KOPEC,M., WEGRZYNOWICZ,Z., ARDELT,W., TEISSEYRE,E. and LATALLO,Z.S. Effect of e1astolytic enzymes from human leukocyte granules and from pig pancreas on plasminogen, fibrinogen, and fibrin. In: Progress in Chemical Fibrinolysis and Thrombolysis. J.F.Davidson, R.M.Rowan, M.M.Samama and P.C.Desnoyers (Eds.) New York: Raven Press, 3, 1978, pp. 417-425. 15. NAGAMATSU,Y., OKAMOTO,U., OKADA,N., AMEMIYA,T., HIRASE,F. and KUBOTA,A. Statistical characteristics of malignant tumor cases found in spleen fibrinolytic protease (SFP) activity distribution of 308 autopsy cases. IGAKU NO AYUMI (in Japanese)-l£!, 273-275, 1983. 16. NAGAMATSU,Y., OKAMOTO,U., TSUDA,Y. and OKADA,Y. Human leukocyte elastaselike proteinase purified by affinity chromatography with Suc-L-Tyr-D-LeuD-Val-pNA, and its identification with human spleen fibrinolytic proteinase. Thromos.Haemostas.(Stuttg.) 51, 243-247, 1984. 17. OKAMOTO,U., NAGAMATSU,Y., TSUDA,Y. and OKADA,Y. A new polypeptide substrate, Suc-Tyr-Leu-Val-pNA, specific for spleen fibrinolytic proteinase (SFP). Biochem.Biophys.Res.Comrnun. 97, 28-32, 1980. 18. Tsuda,Y., OKADA,Y., NAGAMATSU,Y. and OKAMOTO,U. Suynthesis of peptide chloromethyl ketones and examination of their inhibitory effects on human spleen fibrinolytic proteinase (SFP) and human leukocyte elastase (LE). Chem.Pharm.Bull. 35, 3576-3584, 1987. 19. OKAMOTO,S., OKAMOTO,U., OKADA,Y., HIJIKATA-OKUNOMIYA,A., WANAKA,K., HORIE,N. and NAITO,T. Coding, decoding and noise of proteinase: a rational approach to plasmin inhibitors. In: Fundamental and clinical fibrinolysis, F.J.Caste11ino, P.J.Gaffney, M.M.Samama and A.Takada (Eds.), Amsterdam-Oxford-New York: Excerpta Medica,-1987, pp.67-82. 20. OKADA,Y. TSUDA,Y., TENO,N., WANAKA,K., SASAKI,K., HIJIKATA,A., NAlTO,T. and OKAMOTO,S. Synthesis of plasmin substrates and relationship between their structure and plasmin activity. Int.J.Peptide Protein Res. ~, 7985, 1986. 21. Cs.-SZABO,G., POZSGAY,M. and ELODI,P. Investigatioo of the substratebinding site of human plasmin using tripeptidyl-p-nitroanilide substrates. Thromb.Res. £Q, 199-206, 1980. 22. OKAMOTO,S., OKAMOTO,U., WANAKA,K., HIJIKATA-OKUNOMIYA,A., BOHGAKI,M., NAITO,T., HORIE,N. and OKADA,Y. Highly selective synthetic inhibitors with regard to plasma-kallikrein activities. KININ V. (in press). 23. TASHIRO,M. and HOMMA,M. Evidence of proteolytic activation of virus in mouse lung. Archives of Virology 22, 127-137, 1983.
Sendai
REVIEW RECENT STUDIES OF THE SYNTHETIC SELECTIVE INHIBITORS; WITH SPECIAL REFERENCE TO NON-PLASMIN FIBRINOLYTIC ENZYME, PLASMIN AND PLASMA-KALLIKREIN
S.Okamoto, U.Okamoto*, A,Hijikata-Okunomiya**, K,Wanaka and Y,Okada* Kobe Research Projects on Thrombosis and Haernostasis, Saiseikai Hospital, Kobe 651 Japan, *Kobe-Gakuin University, Kobe 673 Japan and **School of Allied Medical Sciences, Kobe University, Kobe 654 Japan
I. INTRODUCTORY REMARKS In 1975, a short review was presented by Okamoto and Hijikata, in which trials to approach as possible as rationally to the highly selective inhibitors such as epsilon-amino-caproic acid (EACA) and tranexamic acid (tAMCHA) were described (1). The mode of action of these inhibitors was very unique, Fact is, years more than one decade elapsed, when it was found that they inhibit the fibrinolysis by plasmin through their binding to the lysinebinding-sites (LBS) of the heavy chain of the plasmin and plasminogen (2,3), In the following undertaking by Okamoto and his colleagues, the target enzyme was thrombin, the highly selective synthetic inhibitors of which were systematically pursued. The first step of stud&es was to examine the thrombin inhibitory activities of the homologs of N -tosyl-arginine methyl ester (TAMe), which had weak thrombin inhibitory activity, TAMe was easily hydrolyzed by thrombin; this might be understood from the fact that most of the substrates to the enzyme assumed the inhibitory activity, as it were, "substrate inhibition". It was soon found that some chemical modifications of the tosyl radical increased the stability of the compounds as well as inhibitory activity with regard to thrombin. Thus, stepwise chemical modifications were conducted, as the resu~8B, the compound called No. &05 was obtained, Ki value of which, 3,7xlO M; No. 205 was chemically N -dansyl-arginine 4ethylpiperidine amide (4).
Key words: plasmin, plasma kallikrein, elastase, thrombin, synthetic substrate
131
synthetic
inhibitor,
leukocyte
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It was noticed that No. 205 showed high toxicity (i.p. LO for mice SO was 80 mg/kg) and that inhibitory activity of the compound to plasma cholinesterase was extremely strong (5). Results accumulated supported the possibility that the 4-ethylpiperidine radical of No. 205 assumed the toxicity of the compound. Therefore, instead of the ethylpiperidine, a-alanine methyl ester, the non-toxic radical, was introduced into the C-terminal of Arg. The for mice, 1000 mg/kg, Ki compound obtained _~as No. 329, of which i.p. LO SO for thrombin, 10 M order and the inhibitory act~vity to plasma cholinesterase, was very weak. Encouraged with this finding, again stepwise chemical modificat~§ns were made and finally No. BOS was obtained (6). Ki of No. 805 was 1.9xlO M, i.p. LO for mice, 700 mg/kg (Fig 1). No. BOS has been SO broadly investigated either experimentally and clinically. Its medical utility has been still under investigations in a large number of the clinical institutions in Japan (7-9). The mode of action of No. 805 (abbreviated as MO-BOS clinically) was typically competitive inhibition, This implies that the active site of thrombin takes the structure mold of No. 805. A question may remain why No. 205 inhibits both thrombin and plasma cholinesterase while No. 805 inhibits thrombin only. Hijikata-Okunomiya et al. have baen working on the problem mentioned above, and reported in 1987 that N -dansyl-arginine 4-phenylpiperidine amide was highly selective inhibitor of plasama cholinesterase. This finding would be one of the most successful cases in designing newly the highly selective synthetic inhibitors (10). It should be noteworthy that the background knowledge of the c:omparative aspects of different serine proteases has been accumulated, not only employing laboratory-experimental technique but also using the computergenerated molecular model to predict the nature of the active site as well as selective site of the enzyme concerned (11).
NH NH 2 ~I MD-805
_~
S
C
NH
CH2 CH2 CH2
SO-NHtHeo-N~CH 2 }-/ 3 eOOH
CH 3
(2R.4R)
FIG. 1 Chemical structure of
No. 805 (MD-80S)
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II. NON-PLASMIN FIBRINOLYTIC PROTEASE Although the plasmin system is known to play an important role in fibrinolysis in the blood, several studies have clearly demonstrated the presence of a plasmin-independent fibr inolysis in leukocyte by Plow et al. (1975), Bilezikian and Nossel (1977) and Kopec et al. (1978) (12-14). The protease has been purified from leukocyte extracts by using sepharose chromatography, indicating an indipendent fibrinolytic pathway from plasmin system. The authors group including Nagamatsu et al. also obtained a neutral protease capable of fibrin degradation which was extracted especially from the human spleen obta ined at the time of the autopsy of ca. 400 cases (15). The enzyme, spleen fibrinolytic protease (SFP), was found to be a neutral serine protease different from plasmin based on its mode of action against synthetic substrates and inhibitors. The physicochemical and enzymatic properties of the enzyme were found very s imilar or identical to those of leukocyte elastase (Table 1). Later an effective purific~tlon method was found using a new affinity chromatography coupled with Suc-L-Tyr-D-Leu -D-Val-pNA (16). The purified preparation contained practically no chymotrypsin-like protease act ivity. The enzyme so purified readily degraded fibrin, fibrinogen and Val-type synthetic peptide substrates, such as Suc-L-Tyr-L-Leu-L-Val-pNA and Suc-LAla-L-Tyr-L-Leu-L-Val-pNA (17 ). These substrates were not hydrolyzed by plasmin. The enzyme was inhibited by Suc-L-Tyr-D-Leu-D-Val-pNA competitively (Table 2) and by Boc-L-Ala-L-Tyr-L-Leu-L-val-CH irreversibly (Table 3) as 2Cl reported by Tsuda et al. (18). DFP and SBTI inn ibited the enzyme. Neither tAMCHA nor TLCK inhibited the enzyme. Notable was that the species difference of the non-plasmin fibrinolytic enzyme was considerably great. Pathophysiological significance of non-plasmin fibrinolysis is still obscure. However, concerning the possible role of the leukocyte and spleen in thrombolysis, we ex pect that the studies may provide some precise tools for the fibrinolysis in a broad sense. Our further studies are now continuing
TABLE 1 Comparative Activities of Spleen Fibrinolytic Protease (SFP) and Leukocyte Elastase (LE) Substrate Suc-Tyr-Leu-Val-pNA Suc-Tyr-Leu-Ile-pNA Suc-Tyr-Leu-Ala-pNA Suc-Tyr-Leu-Leu-pNA Suc-Tyr-Leu-Phe-pNA Suc-Tyr-Leu-Gly-pNA Suc-Tyr-Leu-Met-pNA Suc-Tyr-Leu-Arg-pNA
SFP
LE
100 ,
100 ,
33 16
23
<0.2
o
o o o
13 <0.2
o
o
o o
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TABLE 2 Reversible Inhibitors of SFP Ki (mM) Suc-L-Tyr-O-Leu-O-Val-pNA LLDOOL-
0L0-
D0L-
0-
0-
L-
0.12 0.19 0.20 0.24 0.35 0.94
Substrate: Suc-L-Tyr-L-Leu-L-Val-pNA
TABLE 3 Irreversible Inhibitors of SFP
Inhibitor Boc-Tyr-Leu-Val-CH Cl Boc-Ala-Tyr-Leu-Val-CH 2Cl 2 Oan-Ala-Tyr-Leu-Val-CH Cl 2 AC-Ala-Tyr-Leu-Val-CH 2Cl
I 4) (Mxl0
Half life (sec)
0.1 0.1 0.1 0.1
31 14 23 28
K~£S~{I
(M
s
)
2240 4950 3010 2480
Substrate: Suc-Ala-Tyr-Leu-Val-pNA (0.5 mM) with the scope that non-plasmin fibrinolysis would be far more significant than ever expected, particularly in the late period of the thrombosis in vivo. An exciting task challenges us to find a new way, how to mobilize this particular enzyme in patients. III. AMlDOLYSIS AND FIBRINOLYSIS WITH PLASMIN Apart from the LBS-directed inhibitors such as EACA or t-AMCHA, the series of active-site directed inhibitors of plasmin have been studies by our group (19). Based on the facts that plasmin Spllts malnly the C-terminal of lysine in fibrinogen, our group first noticed ~-tosyl-lysine methyl ester (TLMe) was a very weak and relatively speclfic inhlbitor as well as substrate on plasmin. A number of derivatives of TLMe lncluding 5-2251 and its homologs substrates were re-examined and results obtalned were theoretically summarized according to the Free and Wilson method. Thus, the O-Ile-Phe-LyspNA was found to be the more sensltive substrate than lts homologs (Table 4) (20). Interesting was that O-Ile was signlficant, which was well coincident with the theoretical prediction on the structure of suitable substrate of plasmin, proposed by Szabo et al. (21).
about
Stimulated by the presence of the sensitive substrate mentioned above, 900 kinds of new compounds were synthesized in our laboratories and
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TABLE 4 Kinetic Parameters for the Amidolysis of Substrate by Plasmin Substrate D-Ile-Phe-Lys-pNA 3-MV-Phe-Lys-pNA Ile-Phe-Lys-pNA
kcat (s
Km (mM)
0.02 0.18
10 18
0.33
19
-1
)
3-MV: 3-methylvaleryl
Ki for plasmin
7.4 JJM Ki for thrombin
>500 pM
FIG. 2 Chemical structure of OS 153 several kinds of selective, potent and reversible inhibitors of plasmin were obtained, chemical structure of one of them called OS 153 belng shown in Fig. 2.
These inhibitors belong to lysine derivatives, characterized with the presence of NH in the chemical structure which covers the N-terminal of 2 lysine. The radical which covers the C-terminal of lysine is much complicated when compared with pNA. The structure of such synthetic inhibitors may take the structure mold of the active site of plasmin. When the activities of t-AMCHA was compared with these of OS group plasmin inhibiotrs, remarkable differences are found as shown in Table 5. Inhibiting activities of t-AMCHA (and EACA) are characterized by its strong inhibition on fibrinolysis but neither on fibrinogenolysis nor on amidolysis of S-series synthetic substrates. While OS inhibitors inhibit nearly equally not only fibr inolysis by plasmin, but also fibrinogenolysis and also _gmidolysis by plasmin, with Ki values of very low concentration such as_~O M. In particular, Ki value of OS 324 was found to be in the ord~B of 10 M. This value can be compared with that of MD-B05, Ki of which , 10 M.
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TABLE S Inhibitory Effect of t-AMCHA and OS lS3 on Amidolysis, Fibrinolysis and Fibrinogenolysis by Plasmin ' (a) Am 1' d 0 1 YS1S Ki (M) t-AMCHA OS 153
-2 4.0x10_ 6 7.4x10
i 1 YS1S ' (b) F 1' br r1no
ISO (M) -S 6.0xlO_ 6 6.1x10
Fd.b ' l ' (b) 1 r1nogeno YS1S
ISO (M) -2
1.0xlO_ S 2.0xlO
(a) Amidolysis was measured with D-Val-Leu-Lys-pNA as substrate. (b) Fibrinolysis and Fibrinogenolysis were measured using 0.04% and 0.2% fibrinogen, respectively. IV. PLASMA KALLIKREIN AND ITS HIGHLY SELECTIVE INHIBITORS It should be recalled that the action of aprotinin has been characterized by its potent inhibition toward glandular kallikrein and plasmin: And that the action of aprotinin toward plasma kallikrein (PK) is approximately 10 times weaker than that to glandular kallikrein. Highly selective synthetic inhibitors of PK has been first reported by the authors, very recently (22). In this review, the novel series of highly selective inhibitors called PKSI are introduced but briefly. Fig. 3 shows a chemical structure of one of the PK inhibitors. PKSI1007 assumed a tripod structure, the basic skeleton of which was L-arginine. This structure showed trans-4-aminomethylcyclohexanecarbonyl to be at the Nterminal of the arginine molecule and 4-ethoxycarbonylanilide to be at the Cterminal position.
FIG. 3 Chemical structure of PKSI-1007
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TABLE 6 Enzyme Inhibition of PKSI-Inhibitors P.Kallikrein Ki (pM) PKSI-I007 (Index) PKSI-OlBO (Index) PKSI-0527 (Index)
B.O (1) 0.47 (1) O.Bl (1)
G.Kallikrein Ki (pM) 120
Plasmin Ki (pM) 270
(15) 53 (113) >500 (>617)
(34) 20 (43) 390 (4Bl)
Thrombin Ki (pM)
FXa Ki (pM)
>500 >500 (>63) (>63) >500 330 (>1064) (702) >500 >500 (>617) (>617)
Substrate: S-2302 for P.kallikrein, S-2266 for G.kallikrein, S-2251 for plasmin, S-223B for thrombin and S-2222 for factor Xa. The mode of the inhibition of PKSI inhibitors was competitive toward PK as well as the different enzyme examined. Inhibitory effects of PKSI inhibitors on the enzymes were measured using the synthetic chromogenic substrates, as shown in Table 6. (a) PKSI-1007 showed Ki value of B.O pM for PK. By contrast, of 120 pM for glandular kallikrein, 270 pM for plasmin and more than 500 pM for thrombin and factor Xa were obtained. (b) PKSI-OlBO was more potent inhibitor than PKSI-1007, Ki value for PK being 0.47 pM. (c) PKSI0527 showed that Ki value for PK was O.Bl pM, while Ki values for plasmin, glandular kallikrein, thrombin and factor Xa were 390 pM, >500flM, >500pM and >500 pM, respectively. High selectivity toward PK was thus remarkably demonstrated, so far as the enzymes examined concerned. In particular, Ki values of PKSI-0527 for glandular ka llikrein, plasmin, thrombin and factor Xa were hundreds times larger than that for PK, as indicated by the figures in the parenthesis in Table 6. In addition, it can be noted that i.v. L0 was over 50 100 mg/kg in our preliminary toxicity test, which encourages us to extend the studies toward the in vivo applications.
Ki
As the preliminary test toward in vivo studies, the intact human plasma was employed and the inhibitory effect of PKSI-OlBO on the bradykinin (BK) formation by kaolin as well as effect on PTT were examined. As shown in Fig. 4, the amount of BK formed (left ordinate) in the kaolin-activated human plasma decreased by increasing the concentration of PKSI-01BO (abscissa) gradually. Results concluded the inhibition of BK formation by PKSI-0180 in such a contact activated system. Fig 4 also showed that PKSI-0180 was able to prolong PTT. The right ordinate indicated the ratio of PTT with different concentration of PKSI-01BO to PTT without inhibitor. The results concluded that PK inhibitor was able to prolong PTT presumably by inhbiting PK of the contact system. In the following studies, the ascites of tumor bearing mice was employed. The Sarcoma 1BO was inoculated into the abdominal cavity of mice and the ascites on the 8th day was obtained. The ascites collected underwent the acid-treatment to get rid of the kininase activity. The samples were incu0C bated at 37 and the aliquots taken out were measured of the amount of BK formed. In the control aliquots, the amount of BK showed remarkable increase with the time course of incubation. The presence of PKSI-0527 of the final concentration of 10 pM and 100 pM in the ascites showed the significant
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FIG. 4 fnhibitory effect of PKSI-01BO on bradykinin formation by kaolin and PT1 BK formation: The intact human plasma containing o-phenanthroline 0C was incubated with PKSI-01BO and kaolin (0.2 mg/ml) at 37 for 2 min. BK levels in the plasma were measured by EIA method using a MARKIT-A-Bradykinin kit (Dainippon Pharmaceutical Co.). PTT was measured with PTT reagent purchased from Behring institute.
600
'::' 400
C---
..•..
,
.....
.- -.. ;'~
",
~ 800
"
'ii
..•.
:;
",'
,'10JlM
... 200
;'
,,' ;'100pM
" ,
III
.. -..r:r .:: .. --V
100
o o
10
"
,A'
.-"
-",- ,.-
20
TI.. 8(.. IIl)
FIG. 5 Inhibitory effect of PKSI-0527 On BK formation in acid-treated ascites of sarcoma 180 mice
80
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decrease of the amount of BK, indicating clearly the dose-dependency of the concentrat ion of PKSI-0527. The PKSI-Ol80 showed s i milar effect in suppress ing the BK formation in the ascites (Fig. 5). PKSI-l007, 0180 and 0527 were obtained as the highly selective inhibitors. However, possible role of PK concerning with the contact system should involve the compl icated problems in health and disease. The present studies may provide some tools for the studies in this field, the correlation between coagulation, BK formation, f ibrinolysis and possibly platelets aggreagat ion in the contact system would be e x t r eme l y complicated, even so we expect that results obtained by using PKSI-inhibitors cast light on a promising perspect ive to us. Needless to say, the further stud ies are urgently required and they are now under investigation in our laboratori es.
V. SIGNIFICANCE OF PROTEOLYTIC REACTION --- The Present and the Future --Recent studies of the different proteases revealed the inherent ability of the enzyme to cl eave their substrates with the unexpectedly high selectiv ity, which could be e no ug h compared with that of the antigen-antibody reaction. In fact, such a high selectivity provided the systematlc and stepwise activation of the protea ses as seen 1n the coagulation cascade. The present paper dealt mainly with the results of our investigations conduc ted to provide the h ighly selective synthet ic inhibitors which may be the excellent tools for revealing significance of the target proteases, even so fina l goal should be to find the clinical ut ilities of synthetic compounds. These studies long continued more than 40 years have been very much encouraged by know ing the presence of natural i nh i b i t o r s with h igh select ivity jus t as U PI . 2 Studies of ours have been developing fortunately and selective synthet ic i nhibitors of trypsin are now under investigation. At the same time, I have learned from Homma that some kinds of virus including AIDS virus may require the activation by trypsin-like protease i n each multiplication step (23). This reminded us the activation of the zymogen to the active enzyme by som e certain proteases. The future of the investigation of proteases seems to be far more promising than our expectation, which has been cherishing in our mind.
ACKNOWLEDGEMENT The authors express the cordial thanks to their close co-workers in d ifferent institutions who contributed immensely. Dr. T. Naito and his co workers, the institute of Showa Denko have been working closely together for the r ecent several years, their contributions be ing appreciated. Sp ec ial thanks to Dr. R. Kikumoto and his co-workers, central laboratories, Mitsubishi Chemical Industri es for the studies of MD-805 and its homologs. To those other close friends who supported us, we ask their pardon for missing even names due to the space a l l o t t e d .
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REFFERENCES 1.
OKAMOTO,S. and HIJIKATA,A. Rational Approach to Proteinase Inhbitors, In: Drug Design VI. E.J.Ariens (Ed.) San Francisco: Academic Press, 1975, pp. 143-169.
2.
IWAMOTO, M. Plasminogen-plasmin system. IX. Specific binding of tranexamic acid to plasmin. Thrombos.Diathes.Haemorrh.(Stuttg.) 33, 573-585, 1975.
3.
MARKUS,G., PRIORE,R.L. and WISSLER,F.C. The binding of tranexamic acid to native (G1u) and modified (Lys) human plasminogen and its effect on conformation. J.Bio1.Chem. 254, 1211-1216, 1979.
4.
HIJIKATA,A., OKAMOTO,S., KIKUMOTO,R. and TAMAO,Y. Kinetic studies on the selectivity of a synthetic thrombin-inhibitor using synthetic peptide substrates. Thrombos.Haemostas.(Stuttg.) 1, 1039-1045, 1979.
5.
NAGANO,S., OKAMOTO,S., IKEZAWA,K., MIMURA,K., MATSUOKA,A., HIJIKATA,A. and TAMAO,Y. Fluorescence studies on the mode of action of two synthetic thrombin inhibitors, No.205 and No.80S. Thrombos.Haemostas. 46, 4S, 1981
6.
OKAMOTO,S., HIJIKATA,A., KIKUMOTO,R., TONOMURA,S., HARA,H., NINOMIYA,K., MARUYAMA,A., SUGANO,M. and TAMAO,Y. Potent inhibition of thrombin by the newly synthesized arginine derivative no.805. The importance of stereostructure of its hydrophobic carboxamide portion. Biochem.Biophys.Res. £Q!.!!!!ll!n • .1Ql, 440-446, 1981.
7.
OTA,K., KAWAGUCHI, H., NAKAGAWA,S., KOSHIKAWA,S., MAEDA,K. MATSUI,N., HIRASAWA,Y. and SASAOKA,T. Clinical evaluation of a new thrombin inhibitor available for haemodialysis. Proc.Eur.Dialysis Transp1ant.Assoc. 20, 144-149, 1983.
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KUMON,K., TANAKA,K., NAKAJIMA,N., NAITO,Y. and FUJITA,T. Anticoagulation with a synthetic thrombin inhibitor after cardiovascular surgery and for treatment of disseminated intravascular coagulation. Crit.Care Med. 11, 1039-1043, 1984.
9.
YONEKAWA,Y., HANDA,H., OKAMOTO,S., KAMIJO,Y., ODA,y., ISHIKAWA,J., TSUDA,H., SHIMIZU,Y., SATOH,M., YAMAGAMI,T., YANO,I., HORIKAWA,Y. and TSUDA,E. Treatment of cerebral infarction in the acute stage with synthetic antithrombin MD-805: Clinical study among multiple institutions. Arch.Jpn.Chir. 55, 711-722, 1986.
10. HIJIKATA-OKUNOMIYA,A., OKAMOTO,S., KIKUMOTO,R. and TAMAO,Y. Stereogeometry of the active sites of serine enzymes gathered from synthetic thrombin-inhibitors. Thrombos.Haemostas. 58, 489, 1987. 11. HIJIKATA-OKUNOMIYA,A., OKAMOTO,S., KIKUMOTO,R., TAMAO,Y., OHKUBO.K., TEZUKA,T., TONOMURA,S. and MATSUMOTO,O. Similarity and dissimilarity in the stereogeometry of the active sites of thrombin, trypsin, plasmin and glandular kallikrein. Thromb.Res. 45, 451-462, 1987. 12. P1ow,E.F. and EDGINGTON,T.S. An alternative pathway for fibrinolysis. I. The cleavage of fibrinogen by leukocyte protease at physiologic pH. J.Clin.Invest. 56, 30-38, 1975.
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13. BILEZIKIAN,S.B. and NOSSEL,H.L. Unique pattern of fibrinogen cleavage by human leukocyte proteases. Blood 50, 21-28, 1977. 14. KOPEC,M., WEGRZYNOWICZ,Z., ARDELT,W., TEISSEYRE,E. and LATALLO,Z.S. Effect of e1astolytic enzymes from human leukocyte granules and from pig pancreas on plasminogen, fibrinogen, and fibrin. In: Progress in Chemical Fibrinolysis and Thrombolysis. J.F.Davidson, R.M.Rowan, M.M.Samama and P.C.Desnoyers (Eds.) New York: Raven Press, 3, 1978, pp. 417-425. 15. NAGAMATSU,Y., OKAMOTO,U., OKADA,N., AMEMIYA,T., HIRASE,F. and KUBOTA,A. Statistical characteristics of malignant tumor cases found in spleen fibrinolytic protease (SFP) activity distribution of 308 autopsy cases. IGAKU NO AYUMI (in Japanese)-l£!, 273-275, 1983. 16. NAGAMATSU,Y., OKAMOTO,U., TSUDA,Y. and OKADA,Y. Human leukocyte elastaselike proteinase purified by affinity chromatography with Suc-L-Tyr-D-LeuD-Val-pNA, and its identification with human spleen fibrinolytic proteinase. Thromos.Haemostas.(Stuttg.) 51, 243-247, 1984. 17. OKAMOTO,U., NAGAMATSU,Y., TSUDA,Y. and OKADA,Y. A new polypeptide substrate, Suc-Tyr-Leu-Val-pNA, specific for spleen fibrinolytic proteinase (SFP). Biochem.Biophys.Res.Comrnun. 97, 28-32, 1980. 18. Tsuda,Y., OKADA,Y., NAGAMATSU,Y. and OKAMOTO,U. Suynthesis of peptide chloromethyl ketones and examination of their inhibitory effects on human spleen fibrinolytic proteinase (SFP) and human leukocyte elastase (LE). Chem.Pharm.Bull. 35, 3576-3584, 1987. 19. OKAMOTO,S., OKAMOTO,U., OKADA,Y., HIJIKATA-OKUNOMIYA,A., WANAKA,K., HORIE,N. and NAITO,T. Coding, decoding and noise of proteinase: a rational approach to plasmin inhibitors. In: Fundamental and clinical fibrinolysis, F.J.Caste11ino, P.J.Gaffney, M.M.Samama and A.Takada (Eds.), Amsterdam-Oxford-New York: Excerpta Medica,-1987, pp.67-82. 20. OKADA,Y. TSUDA,Y., TENO,N., WANAKA,K., SASAKI,K., HIJIKATA,A., NAlTO,T. and OKAMOTO,S. Synthesis of plasmin substrates and relationship between their structure and plasmin activity. Int.J.Peptide Protein Res. ~, 7985, 1986. 21. Cs.-SZABO,G., POZSGAY,M. and ELODI,P. Investigatioo of the substratebinding site of human plasmin using tripeptidyl-p-nitroanilide substrates. Thromb.Res. £Q, 199-206, 1980. 22. OKAMOTO,S., OKAMOTO,U., WANAKA,K., HIJIKATA-OKUNOMIYA,A., BOHGAKI,M., NAITO,T., HORIE,N. and OKADA,Y. Highly selective synthetic inhibitors with regard to plasma-kallikrein activities. KININ V. (in press). 23. TASHIRO,M. and HOMMA,M. Evidence of proteolytic activation of virus in mouse lung. Archives of Virology 22, 127-137, 1983.
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