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Effects of acetylthrombin on protein C activation and fibrinogen clotting
THROMBOSIS RESEARCH 59; 713-722,199O 0049-3848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.
EFFECTS OF ACETYLTBRONBIN ON PROTEIN C ACTIVATION AND FIBRINOGEN CLOTTING
Kazuya Nakagomi, Katsumi Ajisaka, and Itsuro Yokota Bio-organic Chemistry Division, Meiji Institute of Health Science, Meiji Milk Products Co. Ltd., 540 Naruda, Odawara 250, Japan (Received
19.2.1990;
accepted
in revised form 12.6.1990
by Editor S. Okamoto)
ABSTRACT Thrombin was acetylated by treatment with acetic the potency for protein C activation and anhydride, and the fibrinogen clotting activity of the resultant acetylthrombin were investigated in vitro and in vivo. The acetylthrombin retained an amidolyractivityto the synthetic substrate, S-2238, and reduced both of the potency for @rotein C activation and the clotting activity. The potency for protein C activation (0.47% of that of thrombin) was retained more than the clottiny activity (0.015% of that of thrombin). The enzymatic activities of acetylthrombin were in inverse proportion to the molecular weights of the substrates, S-2238, Similarly, the inhibitory protein C, and fibrinogen. activity on acetylthrombin was dependent on the molecular weights of the inhibitors, Thromstop, I-2581, hirudin, and antithrombin-III. When acetylthrombin (5000 units/kg body weight) was infused into rabbits, the activated partial thromboplastin time was prolonged to the same extent as that following infusion of thrombin (125 units/kg), but the fibrinogen level was not decreased in contrast to the larye decrease following infusion of thrombin. It is suggested that acetylthrombin activates protein C without clotting fibrinogen -in vivo.
INTRODUCTION Thrombin is a plasma serine protease which converts ---_--__-_------~~~~~~~~~~~~~~~~~------~~~~~~~~~_~~~~~~~~~_-~---Key words: thrombin, protein C, chemical modification, acetylation 713
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fibrinogen to fibrin and activates coagulation factors (1). When thrombin conjugates thrombomodulin, the thrombin loses its clotting activity (2) and acquires a more than several thousandfold potency to activate protein C (PC) (3, 4). It is said that the thrombin changes its conformation on attachment to thrombomodulin (5). The aim of our research is to find a chemically modified thrombin which has an analoyous conformation and activity to the thrombin-thrombomodulin complex. Landaburu and Seegers (6) reported that acetylation of thrombin eliminated most of the activity for converting fibrinogen to fibrin and increased the esterase activity to ptoluenesulfonyl-L-arginine methyl ester. The potency of the acetylated thrombin for PC activation has not yet been investigated in vitro and in vivo. In the Eex study,- we prepared acetylthrombin by an improved procedure, and investigated its potency for PC activation and fibrinogen clotting activity. We infused the acetylthrombin into rabbits and examined its effects on the coagulation system.
MATERIALS AND METHODS MATERIALS The following materials were purchased from the following manufacturers: bovine thrombin from Mochida Pharmaceutical Co. Ltd., Tokyo, Japan; bovine serum albumin (BSA) from Behring Diagnostica, La Jolla, CA; human antithrombin III (AT-III) from Hoechst Tokyo, Japan Ltd., Thromstop Japan; (N'-(2naphthylsulfonylglycyl)-D,L-amidinophenylalanine-piperidide), human PC, and rabbit thrombomodulin from American Diagnostica Inc., New York, NY; hirudin (No. H7016) and bovine fibrinogen (No. F8630) from Sigma Chemical Co., St. Louis, MO; Boc-Leu-Serfrom Peptide Thr-Ary-4-methylcoumaryl-7-amide Institute Inc., (S-2238) and NaOsaka, Japan; and D-Phe-Pip-Arg-p-nitroanilide dansyl-(e-yuanidino)-phenylalanine-piperidide hydrochloride (I2581) from Daiichi Pure Chemicals Co. Ltd., Tokyo, Japan. Bovine thrombin was further purified by column on CM Sephadex C-50. The fractions containing chromatography thrombin were dialyzed against distilled water and lyophilized. Thrombin (1310 units/mg protein) was confirmed as a single band at the position of a-thrombin by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The units of thrombin and in terms of the acetylthrombin in this paper are expressed amidolytic activity. Human AT-III was further purified by column chromatography on Blue Sepharose CL-6B, and the homogeneity of the AT-III was confirmed as a single band by SDS-PAGE. Human PC further and rabbit thrombomodulin were employed without purification. Acetylation of Thrombin Ten mgof lyophilized thrombin was suspended in 5 ml of distilled water, and 5 ml of saturated sodium acetate solution was added slowly to the suspension. Acetic anhydride was added dropwise to the reaction mixture with an Eppendorf pipette (100
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ul) to yive pH 6.0, 6.5, or 7.0. Each reaction mixture immediately dialyzed against excess NaCl solution (9 mg/ml).
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was
Measurement of Amidolytic Activity The amidolytic activity was measured by the modified ul of thrombin procedure of Swedenborg et al. (7). Fifteen solution (0 - 50 units/ml) was added to 750 ul of substrate solution (0.1 mM of S-2238 in 50 mM Tris-HCl containing 7.5 mM EDTA and 175 mM NaCl, pH 8.4). After incubation for 40 seconds at 37'C, 250 ul of glacial acetic acid was added. The absorbance at 405 nm was measured with a UV spectro$hotometer. Measurement of Clotting Activity Each solution of thrombin (0.625 - 10 units/ml) and acetylthrombin (3 - 50 my/ml) was dissolved in 20 mM Tris-HCl (pH 7.5) containing 0.1 M NaCl, 3.5 mM CaC12, and BSA (IO mg/ml). The solution was added to 200 ul of 2 mg/ml bovine fibrinogen The solution (0.1 M Tris-HCl containing 0.15 M NaCl, pH 7.5). mixture was then incubated at 37'C, and the time for fibrin clot formation was measured with a laser coagulometer LC-101 (Wako or AmelungLtd., Osaka, Pure Chemical Industries Japan) Coayulometer KC-4A (Baxter Co. Ltd., Tokyo, Japan). Measurement of Potency for PC Activation FifteenTl of throxnTl0 units/ml) or acetylthrombin (200 units/ml) was mixed with 85 ul of human PC (0.047 my/ml) in 50 mM After Tris-HCl (pH 8.0) containing 0.1 M NaCl and BSA (1 mg/ml). incubation of the mixture for 1 hour at 37'C, 150 ul of I-2581 (0.3 mM) in substrate buffer (50 mM Tris-HCl containing 0.1 M NaCl and 2 mM CaC12, pH 8.0) was added to the mixture, which was incubated for a further 15 minutes. Subsequently, 250 1-11of Boc(0.1 mM) in substrate Leu-Ser-Thr-Ary-4-methylcoumaryl-7-amide buffer was added to the mixture, which was then incubated for 10 minutes at 37OC. The reaction was stopped by adding 500 ul of 20% of liberated 7-amino-4acetic acid. The amount (v/v) rnethylcoumarine was measured with a spectrofluorophotometer (Shimadzu RF-540) with an excitation wavelength of 380 nm and emission wavelength of 460 nm. Measurement of Potency -for -PC Activation in -_ the Presence of -Thrombomodulin Five ul of thrombin (0.1 units/ml) or acetylthrombin (20 units/ml), 15 ul of rabbit thrombomodulin (30 units/ml), and 80 ul of PC (0.05 mg/ml in 50 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl, BSA (1 my/ml) and 3.5 mM CaC12) were mixed together. After incubation of the resultant mixture for 1 hour at 37"C, the same procedures as above for the measurement of the activated PC activity were carried out. In Vivo Experiments -New Zealand White (NZW) rabbits (male, 1.9 - 2.4 kg) were anesthetized with 30 mg/kg of pentobarbital sodium (Dainabbot Co. Ltd., Osaka, Japan). Additional pentobarbital sodium (15 mg/ky every 2 hours) was administered to maintain the anesthesia. Thrombin and acetylthrombin were dissolved in saline. Thrombin (125 or 62.5 units/kg), acetylthrombin (5000 units/kg), or saline
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was infused via the marginal ear vein usiny a constant infusion pump (22.5 ml per 1.5 hours). Blood samples (1 ml) were withdrawn through a catheter inserted into the femoral vein and 1 volume of trisodium citrate solution (38 mg/ml) was added to 9 volumes of the blood. Plasma was isolated by centrifugation (at 7000 rpm for 3 minutes at 4"(Z), and was placed in ice until assay. The activated partial thromboplastin time (APTT) and fibrinogen level of each plasma sample were measured immediately after isolation of the plasma, by laser coagulometry using kits (APTT-test Wako and Fibrinoyen B-test Wako). Other Methods The free amino groups in thrombin and acetylthrombin were determined with 2,4,6_trinitrobenzenesulfonic acid (8). Protein concentrations were measured by the method of Lowry -et al. (9) employing BSA as a standard. The protein concentration of AT-III was estimated by measuring the absorbance at 280 nm (A1%280 = 7.0).
RESULTS Characteristics of Acetylthrombin Three acetylthrombins were prepared by the addition of various amounts of acetic anhydride. The characteristics of these acetylthrombins are summarized in Tables 1 and 2. The potency for PC activation is shown as the normalized value of the activity of activated protein C (APC) induced by the activity of the acetylthrombin or thrombin. The remaining free amino groups of the acetylthrombins decreased according to increase in the amount of acetic anhydride. The amidolytic activity, the clotting and for PC activation of activity, the potency the acetylthrombins were reduced according to the decrease in the Although remaininy free amino groups (Tables 1 and 2). the
Table 1 -Amidolytic
and Clotting Activity of Thrombin and Acetylthrombins.
---_---_________-_---___--____--__--__----~---~~~---_~_---____-__
Specific Activity Free Amino Clotting Amidolytic Group ___-____--__---_-____--__--------_-____---_---~~~---~~~-~~~~~-~~~ (units/mg protein) (%) Acetylthrombin ** 811 (83.8) pH 6.0 33.9 ON& (0.0011) 1063 (109.8) pH 6.5 41.3 0.209 (0.015) 1405 (145.1) 71.7 pH 7.0 1310 (100) 968 (100) 100 Thrombin ____________________________-___________--__---~~----~~--~~~~-~~~ * The values relative to thrombin are indicated in parentheses. ** Not detected.
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clotting activity was decreased to a negligibly small level (less than 0.015%) with all the acetylthrombins, the potency for PC activation was retained to some extent (0.18 - 0.47%). The ratios of the remaining clotting activity to the remaining potency for PC activation of the acetylthrombin which was prepared by adding acetic anhydride to pH 7.0, were 1 : 1.8 This with thrombomodulin and 1 : 31 without thrombomodulin. Since named acetylthrombin-pH7. was acetylthrombin in terms of its acetylthrombin-pH7 was the best preparation potency for PC activation, we used acetylthrombin-pH7 in subsequent experiments.
Table --
2
Potency for Protein C Activation of Thrombrand Acetyithrombins. ____________-_____________---_________-_-----------------
Potency $or Protein C Activation (-TM 1 (+TM )
**
_________-----_______-------______----------------~~----~
Acetylthrombin pH 6.0 pH 6.5 pH 7.0
N.D.*** 0.070 (0.022) 0.087 (0.027)
0.009 (0.19) 0.019 (0.40) 0.022 (0.46)
4.75 (100) Thrombin 317.5 (100) ____----------_____----------____-----------------------* With or without thrombomodulin (TM). ** (Released APC units) / (amidolytic activity of thrombin). The values relative to thrombin are indicated in parentheses. *** Not detected.
Inhibition of Acetylthrombin-pH7 by Four Inhibitors The inzbitory effects of four thrombin specific inhibitors were compared in terms of the amidolytic activity of acetylthrombin-pH7 on the synthetic substrate, S-2238. The data in Fig. 1 show that AT-III and hirudin inhibited thrombin more strongly than they did acetylthrombin-pH7. The maximum inhibition of acetylthrombin-pH7 by AT-III was 50% and that by hirudin was 40%. Thromstop and I-2581 inhibited acetylthrombin-pH7 to almost the same extent as they did thrombin. In Vivo Effects of Acetylthrombin-pH7 -The -in vivoeffects of thrombin and acetylthrombin-pH7 on the coagulation system were examined by infusing these agents into NZW rabbits. As shown in Fig. 2, when a high dose (125 units/kg) of thrombin was infused, the APTT was prolonged and the fibrinogen level decreased with maximum effects at 2 hours after the start of infusion. The APTT returned to the pretreatment level within 5 hours. On the other hand, the fibrinogen level
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failed to recover to the pretreatment level even after 5 hours. When a low dose (62.5 units/kg) of thrombin was infused, the APTT and fibrinogen level were changed to a smaller extent than when a high dose of thrombin was infused. Acetylthrombin-pH7 also revealed a similar dose dependence for the prolongation of the APTT and fibrinogen level (data not shown). Infusion of acetylthrombin-pH7 (5000 units/kg) induced prolongation of the APTT to the same level as that with a high dose of thrombin (125 unitslky), but reduced the fibrinogen level slightly.
0 Antithrombin
IO Hirudin
III (PM)
100
IO00
(units/ml)
100
50 D P 0L-
0
I Thromstop
IO (PM)
100
0
I l-258
IO
100
I QJM>
Inhibitory Effects of Antithrombin III, Hirudin, Thromstop and I-2581 on Thrombin and Acetylthrombin Fifty ul of each thrombin (60 units/ml) was solution inhibitor mixed with 50 ul of (concentration as indicated in the figure) in 0.1 M Tris-HCl containing 0.15 M NaCl, pH 7.5, and the solution was then incubated for 30 minutes at amidolytic activities of 37OC. The residual thrombin ( 0 ) and acetylthrombin ( 0 ) were measured by usiny S-2238. Panels A, B, C, and D show the results for AT-III, hirudin, Thromstop, and I-2581, respectively.
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1I
2I
3I
4I
5I
Oo
I
I
I
I
1
2
3
4
5
Hours
Hours
Effects of Thrombin and Acetylthrombin --on APTT and Fibrinogen Level inViv0 -Four rabb;ts each were infused with saline ( 0 ), thrombin (125 units/kg, ??; 62.5 units/kg, A 1, and acetylthrombin (5000 units/kg, 0 1. Panel A shows the activated partial thromboplastin level. The units of time, and panel B shows the fibrinogen thrombin and acetylthrombin refer to the amidolytic activity. The solutions were infused for 90 minutes at a rate of 15 ml/hour. * : Significantly different from the control value at that time (Student's t-test; ~~0.05). ** : Significantly different from the control value at that time (Student's t-test; p
DISCUSSION __--_----The amidolytic activity of acetylthrombin was found to differ significantly with differences in the molecular size (molecular weight) of the substrates. When the substrate was S2238, which was small in molecular size (M.W., 6261, acetylthrombin-pH7 exhibited 140% of the enzymatic activity of thrombin. When the substrate was PC, which was large in molecular size (M.W., 62 Kd), acetylthrombin-pH7 retained 0.47% of the enzymatic activity of thrombin. When the substrate was fibrinogen, which was much larger in molecular size (M.W., 350 retained only 0.015% of the enzymatic Kd), acetylthrombin-pH7 activity of thrombin. It is suggested therefore that the enzymatic activities of acetylthrombin-pH7 are in inverse proportion to the molecular size of the substrates.
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Similar findings were obtained for the inhibitory effects of four inhibitors of different molecular sizes. As shown in Fig. IA and IB, AT-III and hirudin inhibited thrombin more strongly than they did acetylthrombin. In contrast, Thromstop and I-2581 inhibited acetylthrombin and thrombin to almost the same extent (Fig. IC and ID). From the viewpoint of molecular size, AT-III and hirudin are much larger than S-2238, while Thromstop and I2581 are of almost the same size as S-2238. It is suggested that inhibitors of high molecular size have more difficulty in inhibiting the amidolytic activity of acetylthrombin-pH7 than do inhibitors of low molecular size. In addition to the difference in molecular sizes of PC and fibrinogen, the following possibility should also be considered. PC and fibrinogen may require different steric or ionic circumstances around the binding site of thrombin. The acetylated amino residues might change the circumstances of the binding site so as to become less favorable for fibrinogen than for PC. Decreases of enzyme activity with increase in the molecular size of substrates have often been observed in the case of modification by polymers such as polyethylene glycol (IO) or dextran (11). It should be noted that similar changes of enzyme activity were observed even in the modification by modifiers of small molecular size. Infusion of acetylthrombin-pH7 into rabbits prolonged the APTT, but did not reduce the fibrinogen level so significantly (Fig. 2). These observations are similar to the results reported by Colucci et al. (12) and Burdick and Schaub (13), who injected -7 APC into squirrel monkeys and cats, respectively. These treatments also prolonged the APTT without affecting the thrombin time. The present and previous data suggest that acetylthrombinpH7 converted PC to APC, which caused a prolongation of the APTT. Comp and Esmon (14) reported that, when activated PC was infused into dogs, plasminogen activator activity was generated in the animals. In order to confirm our assumption that acetylthrombin activated PC in vivo as well as in vitro, we examined the amounts of plasmin?geGtivator activity before and after infusion of acetylthrombin in rabbits. However, the changes in plastninogen activator activity level were found to remain within the limits of experimental error (data not shown). The results thus failed to provide direct evidence for or ayainst the activation of PC by acetylthrombin in vivo, because the amounts by acetylthrombin, could of activated PC, if produced in vi= have been too small to generateplaxogen activator activity. The prolongation of APTT by acetylthrombin-pH7 was much greater than had been expected from the in vitro results. In the a potency for in vitro experiments, acetylthrombin-pH7yesd PC mation which amounted to only 0.027% of that of thrombin. To induce the same effect of APTT prolongation in rabbits as that the required obtained with thrombin, it was thought approximately 3700-fold that of acetylthrombin-pH7 dose would be needed was thrombin. However, the amount of acetylthrombin-pH7 found to be only 40-fold that of thrombin in our in vivo The in vitro observation that AT-III inmbited experiments. thrombin more stronTy than acetylthrombin-pH7 might explain this in vivo effect of acetylthrombin-pH7. --
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The present results differed from the reports of Landaburu et al. (15) and Pechet -et al. (16), who found that acetylthrombin -for this reduced the fibrinogen level -in vivo. The reason discrepancy between our data and theirscould be related to the differences in acetylation procedure and dose of infusion. They acetylated thrombin by adding acetic anhydride to pH 5.6 or 6.0 and precipitated the resultant acetylthrombin with ammonium sulfate and acetone. Although their acetylthrombin led to rapid plasma clot formation, our acetylthrombin did so only very slowly thus exhibited in vitro (data not shown). Our acetylthrombin different -in vivo effects from their acetylthrombin. In conclusion, our acetylthrombin, which was prepared by an improved procedure of Landaburu and Seegers, retained a potency for PC activation but had an extremely reduced clotting activity. Although we could not obtain direct confirmation in the present study, the acetylthrombin appeared to activate PC in vivo as well fibrinogen level. without reducing the vitro as in ----Acetalhrombin-pH7 is a very useful reagent for investigating the in vivo effects of endogenous APC, like thrombin-thrombomodulin complex (12). The possibility of preparing a chemically modified thrombin which could be a useful as an antithrombotic drug, has been demonstrated.
REFERENCES 1
FENTON II, J. W., Thrombin 370, 468-495, 1981. Sci.,
2
K. W., Complex ESMON, C. T., ESMON, N. L., and HARRIS, formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J. Biol. Chem., 257, 7944-7947, 1982. --
3
of an ESMON, C. T. and OWEN, W. G., Identification endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc. Natl. -Acad. Sci. U.S.A., 78, 2249-2252, 1981.
4
of natural ESMON, C. T., The regulation pathway. Science, 235, 1348-1352, 1987.
5
JOHNSON, A. J., ESMON, N. L., LAUE, T. M., and ESMON, C. T., Structural than es required for activation of protein C are induced by Ca2? binding to a high affinity site that dose not contain y-carboxyglutamic acid. -J. Biol. Chem., 258, 5554-5560, 1983.
6
LANDABURU, R. H. and SEEGERS, W. H., The acetylation of thrombin. -Can. J. Biochem. Physiol., 37, 1361-1366, 1959.
7
SWEDENBORG, J., DRYJSKI, M., FREBELIUS, S., and OLSSON, P., Uptake and inactivation of thrombin on rabbit aortic endothelium studied with two different substrates. Thromb.
specificity.
Ann. --
-N.Y.
-Acad.
anticoagulant
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Hemostas., 54, 828-832,
Vol. 59, No. 4
1985.
8
FIELD, R., The rapid determination of amino Enzymol., 25, 464-468, 1972. TNBS. Methods
9
LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and R. J., Protein measurement with the Folin-phenol Chem _J. -Biol. AI -193, 265-275, 1951.
groups
with
RANDALL, reagent.
10
BEGER, Jr, H. and PIZZO, S. V., Preparation of polyethylene plasminogen glycol-tissue activator adducts that retain functional activity: characteristics and behavior in three animal species. Blood, 7l_, 1641-1647, 1988.
11
MARSHALL, J. J. and RABINOWITZ, M. characterization of a dextran-trypsin Chem.,
251,
1081-1087,
1976.
L., Preparation and conjugate. J. Biol. -
12
of COLUCCI, M., STASSEN, J. M., and COLLEN, D., Influence C activation on blood coagulation and fibrinolysis protein in squirrel monkeys. -~ J. Clin. Invest., 74, 200-204, 1984.
13
M. D. and SCHAUB, R. G., Human protein C produces BURDICK, and increased fibrinolytic activity in the anticoagulation cat. Thromb. Res., 45, 413-94, 1987.
14
of fibrinolytic COMP, P. C. and ESMON, C. T., Generation activity by infusion of activated protein C into dogs. J. Clin. Invest., 68, 1221-1228, 1981.
15
LANDABURU, R. H., GIAVEDONI, E., and SANTILLAN, R., Thrombin and acetylated thrombin in the activation of fibrinolysis. Can. J. Physiol. Pharmacol., 46, 809-813, 1968. --
16
B., PECHET, L., ENGEL, A. M., GOLDSTEIN, C., and GLEASER, thrombin and its acetylated effects of infusing The and fibrinolysis. derivative. I. Studies of coagulation Thrombos. Diathes. Haemorrh., 20, 190-201, 1968.
EFFECTS OF ACETYLTBRONBIN ON PROTEIN C ACTIVATION AND FIBRINOGEN CLOTTING
Kazuya Nakagomi, Katsumi Ajisaka, and Itsuro Yokota Bio-organic Chemistry Division, Meiji Institute of Health Science, Meiji Milk Products Co. Ltd., 540 Naruda, Odawara 250, Japan (Received
19.2.1990;
accepted
in revised form 12.6.1990
by Editor S. Okamoto)
ABSTRACT Thrombin was acetylated by treatment with acetic the potency for protein C activation and anhydride, and the fibrinogen clotting activity of the resultant acetylthrombin were investigated in vitro and in vivo. The acetylthrombin retained an amidolyractivityto the synthetic substrate, S-2238, and reduced both of the potency for @rotein C activation and the clotting activity. The potency for protein C activation (0.47% of that of thrombin) was retained more than the clottiny activity (0.015% of that of thrombin). The enzymatic activities of acetylthrombin were in inverse proportion to the molecular weights of the substrates, S-2238, Similarly, the inhibitory protein C, and fibrinogen. activity on acetylthrombin was dependent on the molecular weights of the inhibitors, Thromstop, I-2581, hirudin, and antithrombin-III. When acetylthrombin (5000 units/kg body weight) was infused into rabbits, the activated partial thromboplastin time was prolonged to the same extent as that following infusion of thrombin (125 units/kg), but the fibrinogen level was not decreased in contrast to the larye decrease following infusion of thrombin. It is suggested that acetylthrombin activates protein C without clotting fibrinogen -in vivo.
INTRODUCTION Thrombin is a plasma serine protease which converts ---_--__-_------~~~~~~~~~~~~~~~~~------~~~~~~~~~_~~~~~~~~~_-~---Key words: thrombin, protein C, chemical modification, acetylation 713
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fibrinogen to fibrin and activates coagulation factors (1). When thrombin conjugates thrombomodulin, the thrombin loses its clotting activity (2) and acquires a more than several thousandfold potency to activate protein C (PC) (3, 4). It is said that the thrombin changes its conformation on attachment to thrombomodulin (5). The aim of our research is to find a chemically modified thrombin which has an analoyous conformation and activity to the thrombin-thrombomodulin complex. Landaburu and Seegers (6) reported that acetylation of thrombin eliminated most of the activity for converting fibrinogen to fibrin and increased the esterase activity to ptoluenesulfonyl-L-arginine methyl ester. The potency of the acetylated thrombin for PC activation has not yet been investigated in vitro and in vivo. In the Eex study,- we prepared acetylthrombin by an improved procedure, and investigated its potency for PC activation and fibrinogen clotting activity. We infused the acetylthrombin into rabbits and examined its effects on the coagulation system.
MATERIALS AND METHODS MATERIALS The following materials were purchased from the following manufacturers: bovine thrombin from Mochida Pharmaceutical Co. Ltd., Tokyo, Japan; bovine serum albumin (BSA) from Behring Diagnostica, La Jolla, CA; human antithrombin III (AT-III) from Hoechst Tokyo, Japan Ltd., Thromstop Japan; (N'-(2naphthylsulfonylglycyl)-D,L-amidinophenylalanine-piperidide), human PC, and rabbit thrombomodulin from American Diagnostica Inc., New York, NY; hirudin (No. H7016) and bovine fibrinogen (No. F8630) from Sigma Chemical Co., St. Louis, MO; Boc-Leu-Serfrom Peptide Thr-Ary-4-methylcoumaryl-7-amide Institute Inc., (S-2238) and NaOsaka, Japan; and D-Phe-Pip-Arg-p-nitroanilide dansyl-(e-yuanidino)-phenylalanine-piperidide hydrochloride (I2581) from Daiichi Pure Chemicals Co. Ltd., Tokyo, Japan. Bovine thrombin was further purified by column on CM Sephadex C-50. The fractions containing chromatography thrombin were dialyzed against distilled water and lyophilized. Thrombin (1310 units/mg protein) was confirmed as a single band at the position of a-thrombin by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The units of thrombin and in terms of the acetylthrombin in this paper are expressed amidolytic activity. Human AT-III was further purified by column chromatography on Blue Sepharose CL-6B, and the homogeneity of the AT-III was confirmed as a single band by SDS-PAGE. Human PC further and rabbit thrombomodulin were employed without purification. Acetylation of Thrombin Ten mgof lyophilized thrombin was suspended in 5 ml of distilled water, and 5 ml of saturated sodium acetate solution was added slowly to the suspension. Acetic anhydride was added dropwise to the reaction mixture with an Eppendorf pipette (100
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EFFECTS OF ACETYLTHROMBIN
ul) to yive pH 6.0, 6.5, or 7.0. Each reaction mixture immediately dialyzed against excess NaCl solution (9 mg/ml).
715
was
Measurement of Amidolytic Activity The amidolytic activity was measured by the modified ul of thrombin procedure of Swedenborg et al. (7). Fifteen solution (0 - 50 units/ml) was added to 750 ul of substrate solution (0.1 mM of S-2238 in 50 mM Tris-HCl containing 7.5 mM EDTA and 175 mM NaCl, pH 8.4). After incubation for 40 seconds at 37'C, 250 ul of glacial acetic acid was added. The absorbance at 405 nm was measured with a UV spectro$hotometer. Measurement of Clotting Activity Each solution of thrombin (0.625 - 10 units/ml) and acetylthrombin (3 - 50 my/ml) was dissolved in 20 mM Tris-HCl (pH 7.5) containing 0.1 M NaCl, 3.5 mM CaC12, and BSA (IO mg/ml). The solution was added to 200 ul of 2 mg/ml bovine fibrinogen The solution (0.1 M Tris-HCl containing 0.15 M NaCl, pH 7.5). mixture was then incubated at 37'C, and the time for fibrin clot formation was measured with a laser coagulometer LC-101 (Wako or AmelungLtd., Osaka, Pure Chemical Industries Japan) Coayulometer KC-4A (Baxter Co. Ltd., Tokyo, Japan). Measurement of Potency for PC Activation FifteenTl of throxnTl0 units/ml) or acetylthrombin (200 units/ml) was mixed with 85 ul of human PC (0.047 my/ml) in 50 mM After Tris-HCl (pH 8.0) containing 0.1 M NaCl and BSA (1 mg/ml). incubation of the mixture for 1 hour at 37'C, 150 ul of I-2581 (0.3 mM) in substrate buffer (50 mM Tris-HCl containing 0.1 M NaCl and 2 mM CaC12, pH 8.0) was added to the mixture, which was incubated for a further 15 minutes. Subsequently, 250 1-11of Boc(0.1 mM) in substrate Leu-Ser-Thr-Ary-4-methylcoumaryl-7-amide buffer was added to the mixture, which was then incubated for 10 minutes at 37OC. The reaction was stopped by adding 500 ul of 20% of liberated 7-amino-4acetic acid. The amount (v/v) rnethylcoumarine was measured with a spectrofluorophotometer (Shimadzu RF-540) with an excitation wavelength of 380 nm and emission wavelength of 460 nm. Measurement of Potency -for -PC Activation in -_ the Presence of -Thrombomodulin Five ul of thrombin (0.1 units/ml) or acetylthrombin (20 units/ml), 15 ul of rabbit thrombomodulin (30 units/ml), and 80 ul of PC (0.05 mg/ml in 50 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl, BSA (1 my/ml) and 3.5 mM CaC12) were mixed together. After incubation of the resultant mixture for 1 hour at 37"C, the same procedures as above for the measurement of the activated PC activity were carried out. In Vivo Experiments -New Zealand White (NZW) rabbits (male, 1.9 - 2.4 kg) were anesthetized with 30 mg/kg of pentobarbital sodium (Dainabbot Co. Ltd., Osaka, Japan). Additional pentobarbital sodium (15 mg/ky every 2 hours) was administered to maintain the anesthesia. Thrombin and acetylthrombin were dissolved in saline. Thrombin (125 or 62.5 units/kg), acetylthrombin (5000 units/kg), or saline
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was infused via the marginal ear vein usiny a constant infusion pump (22.5 ml per 1.5 hours). Blood samples (1 ml) were withdrawn through a catheter inserted into the femoral vein and 1 volume of trisodium citrate solution (38 mg/ml) was added to 9 volumes of the blood. Plasma was isolated by centrifugation (at 7000 rpm for 3 minutes at 4"(Z), and was placed in ice until assay. The activated partial thromboplastin time (APTT) and fibrinogen level of each plasma sample were measured immediately after isolation of the plasma, by laser coagulometry using kits (APTT-test Wako and Fibrinoyen B-test Wako). Other Methods The free amino groups in thrombin and acetylthrombin were determined with 2,4,6_trinitrobenzenesulfonic acid (8). Protein concentrations were measured by the method of Lowry -et al. (9) employing BSA as a standard. The protein concentration of AT-III was estimated by measuring the absorbance at 280 nm (A1%280 = 7.0).
RESULTS Characteristics of Acetylthrombin Three acetylthrombins were prepared by the addition of various amounts of acetic anhydride. The characteristics of these acetylthrombins are summarized in Tables 1 and 2. The potency for PC activation is shown as the normalized value of the activity of activated protein C (APC) induced by the activity of the acetylthrombin or thrombin. The remaining free amino groups of the acetylthrombins decreased according to increase in the amount of acetic anhydride. The amidolytic activity, the clotting and for PC activation of activity, the potency the acetylthrombins were reduced according to the decrease in the Although remaininy free amino groups (Tables 1 and 2). the
Table 1 -Amidolytic
and Clotting Activity of Thrombin and Acetylthrombins.
---_---_________-_---___--____--__--__----~---~~~---_~_---____-__
Specific Activity Free Amino Clotting Amidolytic Group ___-____--__---_-____--__--------_-____---_---~~~---~~~-~~~~~-~~~ (units/mg protein) (%) Acetylthrombin ** 811 (83.8) pH 6.0 33.9 ON& (0.0011) 1063 (109.8) pH 6.5 41.3 0.209 (0.015) 1405 (145.1) 71.7 pH 7.0 1310 (100) 968 (100) 100 Thrombin ____________________________-___________--__---~~----~~--~~~~-~~~ * The values relative to thrombin are indicated in parentheses. ** Not detected.
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clotting activity was decreased to a negligibly small level (less than 0.015%) with all the acetylthrombins, the potency for PC activation was retained to some extent (0.18 - 0.47%). The ratios of the remaining clotting activity to the remaining potency for PC activation of the acetylthrombin which was prepared by adding acetic anhydride to pH 7.0, were 1 : 1.8 This with thrombomodulin and 1 : 31 without thrombomodulin. Since named acetylthrombin-pH7. was acetylthrombin in terms of its acetylthrombin-pH7 was the best preparation potency for PC activation, we used acetylthrombin-pH7 in subsequent experiments.
Table --
2
Potency for Protein C Activation of Thrombrand Acetyithrombins. ____________-_____________---_________-_-----------------
Potency $or Protein C Activation (-TM 1 (+TM )
**
_________-----_______-------______----------------~~----~
Acetylthrombin pH 6.0 pH 6.5 pH 7.0
N.D.*** 0.070 (0.022) 0.087 (0.027)
0.009 (0.19) 0.019 (0.40) 0.022 (0.46)
4.75 (100) Thrombin 317.5 (100) ____----------_____----------____-----------------------* With or without thrombomodulin (TM). ** (Released APC units) / (amidolytic activity of thrombin). The values relative to thrombin are indicated in parentheses. *** Not detected.
Inhibition of Acetylthrombin-pH7 by Four Inhibitors The inzbitory effects of four thrombin specific inhibitors were compared in terms of the amidolytic activity of acetylthrombin-pH7 on the synthetic substrate, S-2238. The data in Fig. 1 show that AT-III and hirudin inhibited thrombin more strongly than they did acetylthrombin-pH7. The maximum inhibition of acetylthrombin-pH7 by AT-III was 50% and that by hirudin was 40%. Thromstop and I-2581 inhibited acetylthrombin-pH7 to almost the same extent as they did thrombin. In Vivo Effects of Acetylthrombin-pH7 -The -in vivoeffects of thrombin and acetylthrombin-pH7 on the coagulation system were examined by infusing these agents into NZW rabbits. As shown in Fig. 2, when a high dose (125 units/kg) of thrombin was infused, the APTT was prolonged and the fibrinogen level decreased with maximum effects at 2 hours after the start of infusion. The APTT returned to the pretreatment level within 5 hours. On the other hand, the fibrinogen level
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failed to recover to the pretreatment level even after 5 hours. When a low dose (62.5 units/kg) of thrombin was infused, the APTT and fibrinogen level were changed to a smaller extent than when a high dose of thrombin was infused. Acetylthrombin-pH7 also revealed a similar dose dependence for the prolongation of the APTT and fibrinogen level (data not shown). Infusion of acetylthrombin-pH7 (5000 units/kg) induced prolongation of the APTT to the same level as that with a high dose of thrombin (125 unitslky), but reduced the fibrinogen level slightly.
0 Antithrombin
IO Hirudin
III (PM)
100
IO00
(units/ml)
100
50 D P 0L-
0
I Thromstop
IO (PM)
100
0
I l-258
IO
100
I QJM>
Inhibitory Effects of Antithrombin III, Hirudin, Thromstop and I-2581 on Thrombin and Acetylthrombin Fifty ul of each thrombin (60 units/ml) was solution inhibitor mixed with 50 ul of (concentration as indicated in the figure) in 0.1 M Tris-HCl containing 0.15 M NaCl, pH 7.5, and the solution was then incubated for 30 minutes at amidolytic activities of 37OC. The residual thrombin ( 0 ) and acetylthrombin ( 0 ) were measured by usiny S-2238. Panels A, B, C, and D show the results for AT-III, hirudin, Thromstop, and I-2581, respectively.
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00t
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1I
2I
3I
4I
5I
Oo
I
I
I
I
1
2
3
4
5
Hours
Hours
Effects of Thrombin and Acetylthrombin --on APTT and Fibrinogen Level inViv0 -Four rabb;ts each were infused with saline ( 0 ), thrombin (125 units/kg, ??; 62.5 units/kg, A 1, and acetylthrombin (5000 units/kg, 0 1. Panel A shows the activated partial thromboplastin level. The units of time, and panel B shows the fibrinogen thrombin and acetylthrombin refer to the amidolytic activity. The solutions were infused for 90 minutes at a rate of 15 ml/hour. * : Significantly different from the control value at that time (Student's t-test; ~~0.05). ** : Significantly different from the control value at that time (Student's t-test; p
DISCUSSION __--_----The amidolytic activity of acetylthrombin was found to differ significantly with differences in the molecular size (molecular weight) of the substrates. When the substrate was S2238, which was small in molecular size (M.W., 6261, acetylthrombin-pH7 exhibited 140% of the enzymatic activity of thrombin. When the substrate was PC, which was large in molecular size (M.W., 62 Kd), acetylthrombin-pH7 retained 0.47% of the enzymatic activity of thrombin. When the substrate was fibrinogen, which was much larger in molecular size (M.W., 350 retained only 0.015% of the enzymatic Kd), acetylthrombin-pH7 activity of thrombin. It is suggested therefore that the enzymatic activities of acetylthrombin-pH7 are in inverse proportion to the molecular size of the substrates.
EFFECTS OF ACETYLTHROMBIN
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Similar findings were obtained for the inhibitory effects of four inhibitors of different molecular sizes. As shown in Fig. IA and IB, AT-III and hirudin inhibited thrombin more strongly than they did acetylthrombin. In contrast, Thromstop and I-2581 inhibited acetylthrombin and thrombin to almost the same extent (Fig. IC and ID). From the viewpoint of molecular size, AT-III and hirudin are much larger than S-2238, while Thromstop and I2581 are of almost the same size as S-2238. It is suggested that inhibitors of high molecular size have more difficulty in inhibiting the amidolytic activity of acetylthrombin-pH7 than do inhibitors of low molecular size. In addition to the difference in molecular sizes of PC and fibrinogen, the following possibility should also be considered. PC and fibrinogen may require different steric or ionic circumstances around the binding site of thrombin. The acetylated amino residues might change the circumstances of the binding site so as to become less favorable for fibrinogen than for PC. Decreases of enzyme activity with increase in the molecular size of substrates have often been observed in the case of modification by polymers such as polyethylene glycol (IO) or dextran (11). It should be noted that similar changes of enzyme activity were observed even in the modification by modifiers of small molecular size. Infusion of acetylthrombin-pH7 into rabbits prolonged the APTT, but did not reduce the fibrinogen level so significantly (Fig. 2). These observations are similar to the results reported by Colucci et al. (12) and Burdick and Schaub (13), who injected -7 APC into squirrel monkeys and cats, respectively. These treatments also prolonged the APTT without affecting the thrombin time. The present and previous data suggest that acetylthrombinpH7 converted PC to APC, which caused a prolongation of the APTT. Comp and Esmon (14) reported that, when activated PC was infused into dogs, plasminogen activator activity was generated in the animals. In order to confirm our assumption that acetylthrombin activated PC in vivo as well as in vitro, we examined the amounts of plasmin?geGtivator activity before and after infusion of acetylthrombin in rabbits. However, the changes in plastninogen activator activity level were found to remain within the limits of experimental error (data not shown). The results thus failed to provide direct evidence for or ayainst the activation of PC by acetylthrombin in vivo, because the amounts by acetylthrombin, could of activated PC, if produced in vi= have been too small to generateplaxogen activator activity. The prolongation of APTT by acetylthrombin-pH7 was much greater than had been expected from the in vitro results. In the a potency for in vitro experiments, acetylthrombin-pH7yesd PC mation which amounted to only 0.027% of that of thrombin. To induce the same effect of APTT prolongation in rabbits as that the required obtained with thrombin, it was thought approximately 3700-fold that of acetylthrombin-pH7 dose would be needed was thrombin. However, the amount of acetylthrombin-pH7 found to be only 40-fold that of thrombin in our in vivo The in vitro observation that AT-III inmbited experiments. thrombin more stronTy than acetylthrombin-pH7 might explain this in vivo effect of acetylthrombin-pH7. --
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721
The present results differed from the reports of Landaburu et al. (15) and Pechet -et al. (16), who found that acetylthrombin -for this reduced the fibrinogen level -in vivo. The reason discrepancy between our data and theirscould be related to the differences in acetylation procedure and dose of infusion. They acetylated thrombin by adding acetic anhydride to pH 5.6 or 6.0 and precipitated the resultant acetylthrombin with ammonium sulfate and acetone. Although their acetylthrombin led to rapid plasma clot formation, our acetylthrombin did so only very slowly thus exhibited in vitro (data not shown). Our acetylthrombin different -in vivo effects from their acetylthrombin. In conclusion, our acetylthrombin, which was prepared by an improved procedure of Landaburu and Seegers, retained a potency for PC activation but had an extremely reduced clotting activity. Although we could not obtain direct confirmation in the present study, the acetylthrombin appeared to activate PC in vivo as well fibrinogen level. without reducing the vitro as in ----Acetalhrombin-pH7 is a very useful reagent for investigating the in vivo effects of endogenous APC, like thrombin-thrombomodulin complex (12). The possibility of preparing a chemically modified thrombin which could be a useful as an antithrombotic drug, has been demonstrated.
REFERENCES 1
FENTON II, J. W., Thrombin 370, 468-495, 1981. Sci.,
2
K. W., Complex ESMON, C. T., ESMON, N. L., and HARRIS, formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J. Biol. Chem., 257, 7944-7947, 1982. --
3
of an ESMON, C. T. and OWEN, W. G., Identification endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc. Natl. -Acad. Sci. U.S.A., 78, 2249-2252, 1981.
4
of natural ESMON, C. T., The regulation pathway. Science, 235, 1348-1352, 1987.
5
JOHNSON, A. J., ESMON, N. L., LAUE, T. M., and ESMON, C. T., Structural than es required for activation of protein C are induced by Ca2? binding to a high affinity site that dose not contain y-carboxyglutamic acid. -J. Biol. Chem., 258, 5554-5560, 1983.
6
LANDABURU, R. H. and SEEGERS, W. H., The acetylation of thrombin. -Can. J. Biochem. Physiol., 37, 1361-1366, 1959.
7
SWEDENBORG, J., DRYJSKI, M., FREBELIUS, S., and OLSSON, P., Uptake and inactivation of thrombin on rabbit aortic endothelium studied with two different substrates. Thromb.
specificity.
Ann. --
-N.Y.
-Acad.
anticoagulant
722
EFFECTS OF ACETYLTHROMBIN
Hemostas., 54, 828-832,
Vol. 59, No. 4
1985.
8
FIELD, R., The rapid determination of amino Enzymol., 25, 464-468, 1972. TNBS. Methods
9
LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and R. J., Protein measurement with the Folin-phenol Chem _J. -Biol. AI -193, 265-275, 1951.
groups
with
RANDALL, reagent.
10
BEGER, Jr, H. and PIZZO, S. V., Preparation of polyethylene plasminogen glycol-tissue activator adducts that retain functional activity: characteristics and behavior in three animal species. Blood, 7l_, 1641-1647, 1988.
11
MARSHALL, J. J. and RABINOWITZ, M. characterization of a dextran-trypsin Chem.,
251,
1081-1087,
1976.
L., Preparation and conjugate. J. Biol. -
12
of COLUCCI, M., STASSEN, J. M., and COLLEN, D., Influence C activation on blood coagulation and fibrinolysis protein in squirrel monkeys. -~ J. Clin. Invest., 74, 200-204, 1984.
13
M. D. and SCHAUB, R. G., Human protein C produces BURDICK, and increased fibrinolytic activity in the anticoagulation cat. Thromb. Res., 45, 413-94, 1987.
14
of fibrinolytic COMP, P. C. and ESMON, C. T., Generation activity by infusion of activated protein C into dogs. J. Clin. Invest., 68, 1221-1228, 1981.
15
LANDABURU, R. H., GIAVEDONI, E., and SANTILLAN, R., Thrombin and acetylated thrombin in the activation of fibrinolysis. Can. J. Physiol. Pharmacol., 46, 809-813, 1968. --
16
B., PECHET, L., ENGEL, A. M., GOLDSTEIN, C., and GLEASER, thrombin and its acetylated effects of infusing The and fibrinolysis. derivative. I. Studies of coagulation Thrombos. Diathes. Haemorrh., 20, 190-201, 1968.