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The influence of synthetic thrombin inhibitors on the thrombin-antithrombin reaction
THROMBOSIS RESEARCH Printed in the
United
vo1.2, pp. Pergamon
States
343-348, Press,
1973 Inc.
THE INFLUENCE OF SYNTHETIC THROMBIN INHIBITORS ON THE THROMBIN-ANTITHROMBINREACTION
F. Markwardt, J. Hoffmann and E. KGrbs Institute of Pharmacology and Toxicology Medical Academy Erfurt, GDR (Received
23.1.1973; Accepted by
in revised form 26.3.1973. Editor B. Blombtick)
ABSTRACT The reaction of the antithrombin III of blood plasma with thrombin is inhibited in the presence of the reversible inhibitor, 4-amidinophenylpyruvicacid (APPA) and prevented when the active site of thrombin is chemically blocked by reaction with the irreversible inhibitor, 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF). These results support the assumption that the thrombin-antithrombinreaction proceeds like an enzyme-substratereaction.
INTRODUCTION Physiologically, the most important thrombin inhibitor in blood plasma is antithrombin III (identical to heparin-cofactor), which inactivates the enzyme in a time- and temperaturedependent reaction (1). Although the thrombin-antithrombin reaction is of decisive importance for the regulation of the blood clotting process, its mechanism has not yet been finally clarified. So far, investigations of the kinetics of this reaction indicate that it is comparable to an enzyme-substrate reaction E+AyE
kl
k2 A-E-A'
+P 4
k3
*E+P2 “-1 where E is thrombin, A is antithrombin, E A is the (adsorptive) enzyme antithrombin complex, E-A' is the product of the stoichio??
??
343
344
SYNTHETIC
THROMBIN
INHIBITORS
vo1.2,~0.4
metric reaction of the enzyme with antithrombin liberating 1 equiv of P,, and P2 is the finally released other product. According to this scheme, the formation of a thrombin-antiA) would be followed by the acylation of thrombin complex (E the enzyme, Since deacylation (k3) is very small, a very stable acyl enzyme (E-A') results and a long-term but not permanent inhibition of the enzyme is found (2, 3, 4). In connection with our work on protease inhibitors, we have developed various types of thrombin inhibitors which block its action at different steps in the above general scheme (5, 6, 7, 8). It was therefore especially interesting to investigate the influence of these inhibitors on the thrombin-antithrombinreaction in order to obtain more information about the mechanism of this reaction. For this purpose we have used the competitive inhibitor, 4-amidinophenylpyruvic acid (APPd), which forms a tight but reversible complex with thrombin, and 4-(2-aminoethyl). benzenesulfonyl fluoride (BEBSF), which irreversibly inactivates the enzyme by reacting covalently with a component of the active site. ??
MATERIALS AND METHODS Purified bovine fibrinogen was obtained from Serva, Heidelberg, and was used as an 0.6 % solution in O,g % NaCl. Thrombin was isolated from bovine plasma as described previously (9) and had 1050 NIH units/mg. Thrombin activity was determined by the following clotting assay: Diluted thrombin (0.1 ml) was added to a mixture df 0.2 ml fibrinogen and 0.2 ml of Tris-HCl buffer (0.1 M), pH Ta5, at 37’ C. The time between the addition of thrombin and the appearance of the first visible fibrin threads was measured and applied to a standard.curve to determine the thrombin concentration. dntithrombin (AT III) was prepared from bovine plasma by the method of Monkhouse (IO), purified according to Abildgaard (II), and stored at -20' C. It had 1350 antithrombin units (ATU)/mg N.
vo1.2,~0.4
SYNTHETIC
THROMBIN
INHIBITORS
345
Antithrombin activity was determined by treating 0.1 ml aliquots of thrombin solution (1000 NM units/ml) with increasing amounts of antithrombin solution, pH 7.5, and diluting the samples to 1.0 ml with Tris-HC1 buffer (0.1 M). After incubation at 37' C for 30 min, the remaining thrombin activity was determined as described above. The dependence of thrombin inactivation on the quantity of antithrombin was determined graphically. From the initial linear part of the curve the quantity of antithrombin required to inactivate 50 NIH units of thrombin was calculated and assigned an activity of 50 ATU. APPA was prepared according to Richter and Wagner (12). AEBSF was prepared by the method of Kazmirowski (13) at the Kombinat VEiB Arzneimittelwerk, Dresden. RESULTS 1. Influence of a competitive thrombin inhibitor on the thrombin-antithrombinreaction 0.1 ml of thrombin solution (500 NM units/ml) was incubated at 37' C with 0.8 ml of Tris-HCl buffer (0.1 M), pH 7e5, and 0.1 ml of antithrombin solution. At various times, aliquots of the reaction mixture were taken and diluted with Tris-HCl buffer 1 : IO to determine the remaining thrombin activity. In parallel experiments, the competitive reversible inhibitor APPA was added before the addition of antithrombin. After dilution of the aliquots the inhibitory effect of APPA on thrombin was not detectable. This was shown in control experiments in which APPA was present in the final thrombin solutions used for clotting tests in the same concentrations as above. The results in Fig. 1 show that the inactivation of thrombin by antithrombin is inhibited in the presence of the competitive inhibitor. The degree of inhibition is dependent on the concentration of APPA and is decreased by increasing concentrations of antithrombin.
346
SYNTHETIC
THROMBIN
INHIBITORS
FIG. 1 Course of thrombin (50 NIH units) inactivation after addition of 25 ATLT(O), 50 ATU (@), and 100 ATU (@) of antithrombin. Broken lines show the ffect of previous addition (10-8 M) of APX?A. Solid lines indicate the course of thrombin inactivation without APPA.
2. Influence of an irreversible inhibitor on the thrombinantithrombin reaction The addition of the irreversible inhibitor AEBSF (which forms a covalent bond with a functional group at-the active site of the enzyme in a time-dependent reaction)‘does not influence the thrombin-antithrombinreaction because it acylates the enzyme at a slower rate than the thrombin-antithrombin reaction* The acyl enzyme (aminoethylbenzenesulfonylthrombin, AEBS-thrombin) was then prepared as described earlier (7) and isolated. The irreversibly blocked enzyme was tested to determine if it could react with antithrombin. AEBS-thrombin was incubated with antithrombin under conditions which could normally lead to the complete consumption of antithrombin. The addition of unmodified thrombin to the mixture showed, however, that no reaction had occurred between AEBS-thrombin and antithrombin, since the latter had retained its full activity toward thrombin (Fig, 2).
FIG. 2 Course of inactivation of 50 Nl% units of thrombin after addition a mixture of 50 ATU of of:@, antithrombin and 50 NIH units of thrombin, preincubated for 60 min; a mixture of 50 ATU of antitR r;mbin with 50 NIH units of AEBS-thrombin, preincubated for 60 min; and () , 50 BTU of antithrombin.
SYNTHETIC
THROMBIN
INHIBITORS
347
DISCUSSION The influence of two types of thrombin inhibitors on the thrombin-antithrombinreaction supports the assumption that it proceeds like an enzyme-substratereaction. Both formation of a reversible complex with APPA and irreversible acylation by AEBSF hinder the reaction of thrombin with antithrombin. One might therefore come to the assumption that antithrombin III cannot react with thrombin if the active site of the latter is blocked in any way which interferes with its enzymatic activity. It would appear rewarding to continue investigation of the action of antithrombin on thrombin as an enzyme-substratereaction. For the therapeutic application of competitively active thrombin inhibitors in the prophylaxis of thrombosis (14), it is significant that the thrombin-antithrombinreaction in blood plasma is only slowed down but not fully stopped, since it takes place in an excess of antithrombin. (The antithrbmbin level in plasma is about four times greater than the thrombin potential.)
REFERENCES 1.
MARKWARDT, F. and LANDMANN, H. BlutgerinnungshemmendeProteine, Peptide und Aminosaurederivate. In: Handbuch der experimentellen Pharmakologie. Vol. 27. F. Markwardt (Ed.) Berlin, Heidelberg, New York, Springer 1971, p. 76.
2.
SEEGERS, W. H., YOSHINARI, M., and LANDABURU, R. H, Antithrombin as substrate for the enzyme thrombin. Thrombos. Diathes, haemorrh. (Stuttg.): 4, 293. 1960.
3.
DOMBROSE, F. A,, SEEGERS, W, H,, and SEDENSKY, J, A. Antithrombin. Inhibition of thrombin and autoprothrombinC (F-Xa) as a mutual depletion system. Thrombos. Diathes. haemorrh. (Stuttg.): 26, 103. 1971.
4.
MARKWARDT, F. and WALSMANN, P, Untersuchungen iiberden Mechanismus der Antithrombinwirkung des Heparins. HoppeSeyler's Z. physiol. Chem.: 317, 64, 1959.
5.
MARKWARDT, F,, LANDMANN, H,, and WALSMANN, P, Comparative studies on the inhibition of trypsin, plasmin, and thrombin by derivatives of benzylamine and benzamidine. Eur. J. Biochem.: 6, 502. 1968,
348
SYNTHETIC
THROMBIN
INHIBITORS
Vo1.2,~0.4
6, MARKWARDT, F., WAGNER, G., WAISMANN, P., HORN, H., and STffRZEBECHER,J, Inhibition of serine proteinases by amidinophenyl esters of aromatic carbonic acids. Acta biol, med. germ.: 28, K 19. 1972. 7. WAISMANN, P,, RICHTER, M., and MARKWARDT, F.
Inaktivierung sin und Thrombin durch 4-Amidinobenzolsulfofluorid von T und 4rp2-Aminoathyl)-benzolsulfofluorid. Acta biol. med. germ.: 28, 577. 1972.
8. MARKWARDT, F., RICHTER, Me, WALSMANN, P., and LANDMANN, H, The inhibition of trypsin, plasmin and thrombin by benzyl 4-guanidinobenzoate and 4'-nitrobenzyl 4-guanidinobenzoate. FEBS Letters: 8, 1700 1970. 9. WAISMANN, P,
tfberdie Reinigung von Thrombinpraparaten. Pharmazie: 23, 401. 1968.
10. MONKHOUSE, F(‘. C, Preparation and assay of plasma antithrombin. In: Methods in Enzymolo Vol. 19. S. P. Colowick and N 0 Kaplan ( 1) New York and London, Academic Press, 1970; p: 915e 11. ABILDGAARD, U. Purification of two progressive antithrombins of human plasma. Stand. J. clin, Lab. Invest.: 19, 190. 1967. 12. RICHTER, Pp,and WAGNER, G. Verfahren zur Herstellung von 4-Amidinophenylbrenztraubensaure. Wirtschaftspatent C 25. 2, 19719 07~/153316, 13. KAZMIROWSKI, H.-G.
Synthese antiproteolytischwirksamer Derivate des Benzolsulfofluorids. Pharmazie: 26, 390. 19710
14. MARKWARDT, F. and KIX)CKING,H.-P. The antithrombotic effect of synthetic thrombin inhibitors. Thrombos. Res.: 1, 243. 1972.
United
vo1.2, pp. Pergamon
States
343-348, Press,
1973 Inc.
THE INFLUENCE OF SYNTHETIC THROMBIN INHIBITORS ON THE THROMBIN-ANTITHROMBINREACTION
F. Markwardt, J. Hoffmann and E. KGrbs Institute of Pharmacology and Toxicology Medical Academy Erfurt, GDR (Received
23.1.1973; Accepted by
in revised form 26.3.1973. Editor B. Blombtick)
ABSTRACT The reaction of the antithrombin III of blood plasma with thrombin is inhibited in the presence of the reversible inhibitor, 4-amidinophenylpyruvicacid (APPA) and prevented when the active site of thrombin is chemically blocked by reaction with the irreversible inhibitor, 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF). These results support the assumption that the thrombin-antithrombinreaction proceeds like an enzyme-substratereaction.
INTRODUCTION Physiologically, the most important thrombin inhibitor in blood plasma is antithrombin III (identical to heparin-cofactor), which inactivates the enzyme in a time- and temperaturedependent reaction (1). Although the thrombin-antithrombin reaction is of decisive importance for the regulation of the blood clotting process, its mechanism has not yet been finally clarified. So far, investigations of the kinetics of this reaction indicate that it is comparable to an enzyme-substrate reaction E+AyE
kl
k2 A-E-A'
+P 4
k3
*E+P2 “-1 where E is thrombin, A is antithrombin, E A is the (adsorptive) enzyme antithrombin complex, E-A' is the product of the stoichio??
??
343
344
SYNTHETIC
THROMBIN
INHIBITORS
vo1.2,~0.4
metric reaction of the enzyme with antithrombin liberating 1 equiv of P,, and P2 is the finally released other product. According to this scheme, the formation of a thrombin-antiA) would be followed by the acylation of thrombin complex (E the enzyme, Since deacylation (k3) is very small, a very stable acyl enzyme (E-A') results and a long-term but not permanent inhibition of the enzyme is found (2, 3, 4). In connection with our work on protease inhibitors, we have developed various types of thrombin inhibitors which block its action at different steps in the above general scheme (5, 6, 7, 8). It was therefore especially interesting to investigate the influence of these inhibitors on the thrombin-antithrombinreaction in order to obtain more information about the mechanism of this reaction. For this purpose we have used the competitive inhibitor, 4-amidinophenylpyruvic acid (APPd), which forms a tight but reversible complex with thrombin, and 4-(2-aminoethyl). benzenesulfonyl fluoride (BEBSF), which irreversibly inactivates the enzyme by reacting covalently with a component of the active site. ??
MATERIALS AND METHODS Purified bovine fibrinogen was obtained from Serva, Heidelberg, and was used as an 0.6 % solution in O,g % NaCl. Thrombin was isolated from bovine plasma as described previously (9) and had 1050 NIH units/mg. Thrombin activity was determined by the following clotting assay: Diluted thrombin (0.1 ml) was added to a mixture df 0.2 ml fibrinogen and 0.2 ml of Tris-HCl buffer (0.1 M), pH Ta5, at 37’ C. The time between the addition of thrombin and the appearance of the first visible fibrin threads was measured and applied to a standard.curve to determine the thrombin concentration. dntithrombin (AT III) was prepared from bovine plasma by the method of Monkhouse (IO), purified according to Abildgaard (II), and stored at -20' C. It had 1350 antithrombin units (ATU)/mg N.
vo1.2,~0.4
SYNTHETIC
THROMBIN
INHIBITORS
345
Antithrombin activity was determined by treating 0.1 ml aliquots of thrombin solution (1000 NM units/ml) with increasing amounts of antithrombin solution, pH 7.5, and diluting the samples to 1.0 ml with Tris-HC1 buffer (0.1 M). After incubation at 37' C for 30 min, the remaining thrombin activity was determined as described above. The dependence of thrombin inactivation on the quantity of antithrombin was determined graphically. From the initial linear part of the curve the quantity of antithrombin required to inactivate 50 NIH units of thrombin was calculated and assigned an activity of 50 ATU. APPA was prepared according to Richter and Wagner (12). AEBSF was prepared by the method of Kazmirowski (13) at the Kombinat VEiB Arzneimittelwerk, Dresden. RESULTS 1. Influence of a competitive thrombin inhibitor on the thrombin-antithrombinreaction 0.1 ml of thrombin solution (500 NM units/ml) was incubated at 37' C with 0.8 ml of Tris-HCl buffer (0.1 M), pH 7e5, and 0.1 ml of antithrombin solution. At various times, aliquots of the reaction mixture were taken and diluted with Tris-HCl buffer 1 : IO to determine the remaining thrombin activity. In parallel experiments, the competitive reversible inhibitor APPA was added before the addition of antithrombin. After dilution of the aliquots the inhibitory effect of APPA on thrombin was not detectable. This was shown in control experiments in which APPA was present in the final thrombin solutions used for clotting tests in the same concentrations as above. The results in Fig. 1 show that the inactivation of thrombin by antithrombin is inhibited in the presence of the competitive inhibitor. The degree of inhibition is dependent on the concentration of APPA and is decreased by increasing concentrations of antithrombin.
346
SYNTHETIC
THROMBIN
INHIBITORS
FIG. 1 Course of thrombin (50 NIH units) inactivation after addition of 25 ATLT(O), 50 ATU (@), and 100 ATU (@) of antithrombin. Broken lines show the ffect of previous addition (10-8 M) of APX?A. Solid lines indicate the course of thrombin inactivation without APPA.
2. Influence of an irreversible inhibitor on the thrombinantithrombin reaction The addition of the irreversible inhibitor AEBSF (which forms a covalent bond with a functional group at-the active site of the enzyme in a time-dependent reaction)‘does not influence the thrombin-antithrombinreaction because it acylates the enzyme at a slower rate than the thrombin-antithrombin reaction* The acyl enzyme (aminoethylbenzenesulfonylthrombin, AEBS-thrombin) was then prepared as described earlier (7) and isolated. The irreversibly blocked enzyme was tested to determine if it could react with antithrombin. AEBS-thrombin was incubated with antithrombin under conditions which could normally lead to the complete consumption of antithrombin. The addition of unmodified thrombin to the mixture showed, however, that no reaction had occurred between AEBS-thrombin and antithrombin, since the latter had retained its full activity toward thrombin (Fig, 2).
FIG. 2 Course of inactivation of 50 Nl% units of thrombin after addition a mixture of 50 ATU of of:@, antithrombin and 50 NIH units of thrombin, preincubated for 60 min; a mixture of 50 ATU of antitR r;mbin with 50 NIH units of AEBS-thrombin, preincubated for 60 min; and () , 50 BTU of antithrombin.
SYNTHETIC
THROMBIN
INHIBITORS
347
DISCUSSION The influence of two types of thrombin inhibitors on the thrombin-antithrombinreaction supports the assumption that it proceeds like an enzyme-substratereaction. Both formation of a reversible complex with APPA and irreversible acylation by AEBSF hinder the reaction of thrombin with antithrombin. One might therefore come to the assumption that antithrombin III cannot react with thrombin if the active site of the latter is blocked in any way which interferes with its enzymatic activity. It would appear rewarding to continue investigation of the action of antithrombin on thrombin as an enzyme-substratereaction. For the therapeutic application of competitively active thrombin inhibitors in the prophylaxis of thrombosis (14), it is significant that the thrombin-antithrombinreaction in blood plasma is only slowed down but not fully stopped, since it takes place in an excess of antithrombin. (The antithrbmbin level in plasma is about four times greater than the thrombin potential.)
REFERENCES 1.
MARKWARDT, F. and LANDMANN, H. BlutgerinnungshemmendeProteine, Peptide und Aminosaurederivate. In: Handbuch der experimentellen Pharmakologie. Vol. 27. F. Markwardt (Ed.) Berlin, Heidelberg, New York, Springer 1971, p. 76.
2.
SEEGERS, W. H., YOSHINARI, M., and LANDABURU, R. H, Antithrombin as substrate for the enzyme thrombin. Thrombos. Diathes, haemorrh. (Stuttg.): 4, 293. 1960.
3.
DOMBROSE, F. A,, SEEGERS, W, H,, and SEDENSKY, J, A. Antithrombin. Inhibition of thrombin and autoprothrombinC (F-Xa) as a mutual depletion system. Thrombos. Diathes. haemorrh. (Stuttg.): 26, 103. 1971.
4.
MARKWARDT, F. and WALSMANN, P, Untersuchungen iiberden Mechanismus der Antithrombinwirkung des Heparins. HoppeSeyler's Z. physiol. Chem.: 317, 64, 1959.
5.
MARKWARDT, F,, LANDMANN, H,, and WALSMANN, P, Comparative studies on the inhibition of trypsin, plasmin, and thrombin by derivatives of benzylamine and benzamidine. Eur. J. Biochem.: 6, 502. 1968,
348
SYNTHETIC
THROMBIN
INHIBITORS
Vo1.2,~0.4
6, MARKWARDT, F., WAGNER, G., WAISMANN, P., HORN, H., and STffRZEBECHER,J, Inhibition of serine proteinases by amidinophenyl esters of aromatic carbonic acids. Acta biol, med. germ.: 28, K 19. 1972. 7. WAISMANN, P,, RICHTER, M., and MARKWARDT, F.
Inaktivierung sin und Thrombin durch 4-Amidinobenzolsulfofluorid von T und 4rp2-Aminoathyl)-benzolsulfofluorid. Acta biol. med. germ.: 28, 577. 1972.
8. MARKWARDT, F., RICHTER, Me, WALSMANN, P., and LANDMANN, H, The inhibition of trypsin, plasmin and thrombin by benzyl 4-guanidinobenzoate and 4'-nitrobenzyl 4-guanidinobenzoate. FEBS Letters: 8, 1700 1970. 9. WAISMANN, P,
tfberdie Reinigung von Thrombinpraparaten. Pharmazie: 23, 401. 1968.
10. MONKHOUSE, F(‘. C, Preparation and assay of plasma antithrombin. In: Methods in Enzymolo Vol. 19. S. P. Colowick and N 0 Kaplan ( 1) New York and London, Academic Press, 1970; p: 915e 11. ABILDGAARD, U. Purification of two progressive antithrombins of human plasma. Stand. J. clin, Lab. Invest.: 19, 190. 1967. 12. RICHTER, Pp,and WAGNER, G. Verfahren zur Herstellung von 4-Amidinophenylbrenztraubensaure. Wirtschaftspatent C 25. 2, 19719 07~/153316, 13. KAZMIROWSKI, H.-G.
Synthese antiproteolytischwirksamer Derivate des Benzolsulfofluorids. Pharmazie: 26, 390. 19710
14. MARKWARDT, F. and KIX)CKING,H.-P. The antithrombotic effect of synthetic thrombin inhibitors. Thrombos. Res.: 1, 243. 1972.