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Synthetic inhibitors of serine proteinases XVI. Influence of 3- and 4-amidinobenzyl derivatives on the formation and action of thrombin
SYNTHETIC INHIBITORS OF SERINE PROTEINASES XVI * INFLUENCE OF 3- AND 4-AMIDINOBENZYL DERIVATIVES FORMATION AND ACTION OF THROMEIN
J.
Hauptmann,
Institute Medical
(Received
9.2.1978.
F.
Markwardt
and
P.
ON THE
Walsmann
of Pharmacology and Toxicology, Academy, DDR-50 Er-fur-t, GDR
Accepted
by Editor
G. %ller-Berghaus)
ABSTRACT derivatives The effects of 3- and 4-amidinobenzyl known as competitive inhibitors of the esterolytic activity of Factor Xa and thrombin on prothrombin activation and plasma coagulation were studied. Compared to 4-amidinobenzyl derivatives, the corresponding 3-isomers are stronger inhibitors of Factor they inhibit the Factor Xa-catalyzed formation of Xa, thrombin in isolated systems and in plasma. Moreover, they are more effective in inhibiting overall plasma coagulation. The results suggest that inhibition of Factor Xa by synthetic competitive inhibitors represents effective control of blood coagulation.
INTRODUCTION The serine proteinases of the blood, thrombin and Factor Xa (F Xa) are inhibited competitively by 3- and 4-amidinobenzyl ethers and ketones (1). The inhibitor constants (Ki-values) determined with synthetic substrates revealed differences in the affinity of the inhibitors for these enzymes. Generally, 3-amidinobenzyl derivatives inhibited F Xa more strongly than thrombin, whereas 4-amidinobenzyl derivatives showed only low affinity for F Xa. Since the extrinsic and intrinsic patbways of the coagulation sequence converge at the stage of F Xa, inhibition of this enzyme and of thrombin might represent an effective interference with the coagulation process. The anticoagulant effect of some amidinobenzyl derivatives due to inhibition of thrombin has already been shown in vitro and in vivo (2, 3, 4, Therefore, the effects on blood coagulation caused by 5, 6). 735
SYNTHETIC
736
COAGLUTIOB
IIHIBITORS
Vo1.12,No.5
synthetic inhibitors with strong affinity for F Xa were studied. Moreover, the question about the mosteffective blockade of the coagulation sequence by synthetic inhibitors should be clarified. Because of the lack of specific synthetic inhibitors of F Xa and thrombin, isomeric 3- and 4-amidinobenzyl derivatives of different inhibitor strength towards F Xa and thrombin were chosen. Table 1 shows that the 3-isomers are effective inhibitors of F Xa, while the corresponding 4-isomers are less effective, the inhibitor strength towards thrombin, however, does not differ between the isomers. TABLE Inhibitor Ketones
for
Constants Inhibition
of
3of
1
and 4-Amidinobenzyl Thrombin (7, 8)
and
HN\
CH2R
H2N’ Ki-values R
No. Thrombin
-o- 01 \
Factor
Xa
Ethers Factor
C / \ -0 -
and Xa (1)
CH2R
(,uM) No. Thrombin
Factor
I
66
6.8
II
58
380
CH3
III
53
4.8
IV
36
310
OCH3
V
47
5.4
VI
26
210
Xa
-
-CO -o-/
\ -
-CO-COOH
VII
790
220
MATERIALS
VIII
6.5
9.4
AND METHODS
Amidinobenzyl derivatives: Detailed data and the provenance of the inhibitors are given elsewhere (1, 7, 8, 9, 10). Fibrinogen: Bovine fibrinogen (Behringwerke AC, Marburg/ Lahn). Human titrated plasma: Fresh blood from healthy donors was collected in 3.13 % sodium citrate (9 + 1). Plasma was obtained by centrifugation at 700 g for 5 min and pooled. Standard thrombin, lot No B-3, specific activity Thrombin: 21.7 NIH U/mg (National Institute of Health, Bethesda /Maryland) was used for standardization. Thrombin, specific activity 1055 NIH U/mg prepared from bovine blood (11) was used for clotting assays. Determination of thrombin activity determine thrombin activity, 0.2 ml mixed with 0.2 ml of 0.1 M Tris-HCl
by clottinq assay: To of fibrinogen (0.6 %) was buffer, pH 7.5, after
addition of 0.1 ml of a thrombin-containing sample the clotting A standard curve was obtained using standard time was recorded. To determine 150-values (inhibthrombin (0.01 - 6.0 NIH U/ml). itor concentration causing 50 % inhibition of thrombin activity) the fibrinogen solution was mixed with for the inhibitors, varying inhibitor concentrations and 0.1 ml of thrombin solution (6 NIH U/ml). Factor X was isolated from fresh Isolation of Factor X: bovine blood (collected in sodium citrate plus heparin) accordChromatography’ was carriedout on ing to BAJAJ and MANN (12). DEAE-cellulose instead of DEAE-Sephadex. A Factor X solution in 50 % glycerol with an activity of 920 U/ml (determined according to BACHMANW(13))was obtained. The preparation was but contained 150 NIH U of prothrombin/ml. free of thrombin, Samples of 0.2 ml of the Factor X Activation of Factor X: pH 8.0 for 45 min with solution were incubated at 37 oC and 5.8 ml sodium chloride (0.154 M) containing 1 mg cephalin, ug Russell’s viper venom and 0.05 mMo1 CaC12. The solution 50 was / centrifuged and kept at 4 oC. Prothrombin was isolated and Isolation of prothrombin: purified according to the method used for Factor X (12). A prothrombin solution in 50 % glycerol with an activity of The prothrombin preparation 27,000 NIH U/ml was obtained. contained 10 NIH U of thrombin/ml and 0.3 U of Factor X/ml. Volumes of 2 ml of this solution were mixed with 18 ml of 0.02 M Tris-HCl buffer (pH 7.5) containing 0.1 M NaCl and deep frozen and stored, The activity was deter0.01 M CaC12, mined after activation by Factor Xa using the clotting assay described above. Samples of 0.4 ml of prothrombin Activation of prothrombin: solution were mixed with either 0.5 ml of Tris-HCl buffer,pH 7.5,or inhibitor solution, and activation (25 oC) was started by addition of 0.1 ml of activated Factor X. Atselected time aliquots were removed and assayed for thrombin intervals, The amount of thrombin present in the sample was calformed. culated from the clotting time referring to a curve with To compensate for disturbance of the deter, standard thrombin. mination of thrombin activity in the clotting assay by the antithrombin activity of the inhibitors a standard curve was made for each compound using that concentration which would result from transferring samples of the incubation mixture to the clotting assay. Thrombelastoqraphy: A volume of 0.2 ml of human titrated plasma was mixed with either 0.1 ml of Tris-HCl buffer, pH 7.4, or inhibitor solution containing 5 % ethanol each-and recalcified with 0.05 ml of 0.1 M CaC12. Thrombelastographic tracings were recorded with the Hellige thrombelastograph. The reaction time was measured and expressed as per cent of the control value. The thrombin generation test Thrombin qeneration in plasma: of MACFARLANE and BIGGS (14) was sliqhtly modified: 0.5 ml of human titrated plasma was mixed with-either 0.5 ml of Tris-HCl buffer, pH 7.4, or inhibitor solution containing 10 % methanol each and recalcified with 0.5 ml of 0.025 M CaCl2. The forming clot was wrapped around an applicator stick. At intervals of
738
SYXTHETIC
COAGLUTIOS
IXHIBIT'ORS
2 min, samples of 0.1 ml were withdrawn from the incubation mixture and added to 0.4 ml of fibrinogen (0.2 %). Thrombin activity was determined as described under “Activation of prothrombin”. One-staqe prothrombin time: A volume of 0.1 ml of human citrated olasma was mixed with either 0.1 ml of Tris-HCl buffer, or inhibitor solution containing 10 $Y, methanol each and pH 7.4; 0.1 ml of human brain thromboplastin (Thrombokinase AWD, VEB Arzneimittelwerk Dresden) and incubated over a period of 3 min 0.1 ml of 0.025 M CaC12 was added and the clotat 37 oC; then Results are expressed as per cent proting time was recorded. longation of the clotting time of control. RESULTS Influence on the reaction of thrombin and Factor Xa with natural substrates. The influence of amidinobenzyl derivatives on the thrombin catalyzed fibrinogen clotting was investigated in an isolated To evaluate the inhibitor activity of the derivatives system. the inhibitor concentra.. on the thrombin-fibrinogen reaction, tions causing 50 % inhibition of thrombin activity were estiThe values in Table 2 show that 4-amidinomated (150-values). benzyl derivatives inhibit fibrinogen clotting more strongly than 3..amidinobenzyl derivatives, whereas the Ki-values (cf. Table 1) are nearly the same. their
TABLE Influence of 3Thrombin-Catalyzed
and
4-Amidinobenzyl Ethers Fibrinogen Clotting 150-Values
No.
I III V VII
2
3-Derivatives 500 150 250 700
and
Ketones
on
(,uM) No. II IV VI VIII
4-Derivatives 150 30 100 8
the influence of amidinobenzyl derivatives on the F XaFurther, catalyzed prothrombin activation was studied determining the amount of thrombin formed during 30 minutes incubation (pH 7.5, 25 OC) in the presence of an inhibitor concentration of 20 uM. / The values in Table 3 are expressed as percent of thrombin formed in inhibitor-free controls. The activity of F Xa was inhibited by 3-amidinobenzyl derivatives, whereas the same concentration of the isomeric kamidinobenzyl derivatives had no influence on the time course of prothrombin activation. A representative example is shown in Fig. 1.
I-39
TABLE Influence Factor
and
of 3Xa-Catalyzed
4-Amidinobenzyl Prothrombin
Percent NO.
3 Ethers Activation
thrombin No.
42 56 32 93
II IV VI VIII
4-Oeriva
FIG. course of prothrombin
O-O
n-a
1
Influence After
60 on
the
t ives
1 Factor Xa-cataactivation.
control without inhibitor; in presence of 3-amidinobenzyl-4’ -methoxyphenyl ketone (V), 20 /uM; in presence of 4-amidinobenzyl-4’ -methoxyphenyl uM. ketone (VI), 20 /
I
.
20
on
103 73 105 20
-3 Time lyzed
Ketones
formed
3-Derivatives
I III V VII
and
120 formation
and
action
of
thrombin
in
plasma
the effect of the inhibitors had been studied in isotheir influence on the course of coagulation of lated systems, blood plasma was investigated. Thrombelastographic recordings of the coagulation of recalcified human titrated plasma showed that 4-amidinobenzyl derivatives with low inhibitory effect on F Xa inhibit plasma coagulation to an extent corresponding to their inhibitory effect on the thrombin-fibrinogen reaction The 3-amidinobenzyl derivatives with strong in(cf. Table 2). hibitory effect on F Xa caused stronger inhibition of plasma coagulation. A comparison of the prolongation of clotting times produced by the inhibitors showed that at the same concentration the 3-isomers are more potent than the 4-isomers and that 3-isomers obviously have a steeper slope of the dose-response curve (Fig, 2). After investigation of the prothrombin activation in an isolated system, studies were performed in plasma. These experiments (thrombin generation test) showed that amidinobenzyl derivatives at concentrations of 100 /uM retard the formation of thrombin in plasma as regards the time until appearance of maximum thrombin activity. Retardation is more pronounced in case of the 3-isomers I, III, and V than in case of the cor-
740
SYXTHETIC
COAG-KLATIOE IXHIBITORS
VO1.12,SO.5
responding 4-derivatives II, IV, and VI. The curves of thrombin show that the maximum of thrombin activity activity, however, A representative example is higher in samples with inhibitors. derivatives of the influence of isomeric 3- and 4..amidinobenzyl on the course of thrombin formation in plasma is given in Fig. 3.
FIG.
2
Prolongation of plasma clotting time (reaction times from thrombelastograms of recalcified human titrated plasma) by 3.. and 4-amidinobenryl derivatives.
30 25 1
min FIG. Time
?? d M M
course
of
thrombin
generation
3 in
plasma.
control without inhibitor; in presence of 3-amidinobenzyl-4*-methoxyphenyl ketone (V), 100 /uM; in presence of 4-amidinobenzyl-4’-methoxyphenyl ketone (VI), 100 uM. /
Combination
effects
of
isomeric
3-
and
4-amidinobenzyl
derivatives In order to get further information on the overall effect of inhibition of F Xa on plasma coagulation. combinationsof inhibFrom thrombelastographic investigations it itors were studied. was obvious that using combinations of 3and &amidinobenzyl derivatives a comparatively low percentage of the corresponding stronger inhibition of 3-isomer in the combination caused plasma coagulation than was produced by the 4-isomer at maxiBY combination of two 4-amidinobenzyl derivatives, an mum. additive effect was observed only.
FIG.
4
Isobologram according to LOEWE (15); the isobole connects equieffective concentrations of combinations of the isomers I and II.
Derivative Ll[phii In further experiments, combination effects on the coagulation process were studied after adding tissue thromboplastin and Ca++ to human titrated plasma. Isoboles according to LOEWE (15) were elaborated in order to verify the supra-additive effect of inhibitor combinations. Equieffective concentrations of various combinations causing a defined effect (50 per cent prolongation of clotting time) were determined. In Fig. 4, the curve typical of potentiation is shown for the combination of isomers I and II. DISCUSSION The evaluation of the present results is rather difficult, since they were not obtained with specific synthetic inhibitors of thrombin and F Xa, but with isomeric 3and Q-amidinobenzyl derivatives of different affinities for these enzymes. In the more complex test systems used. inhibition of thrombin may have influenced the activation of prothrombin by F Xa. However, the inhibitory activity of amidinobenzyl derivatives towards thrombin and F Xa found in previous investigations with synthetic substrates of the respective enzymes was demonstrated for the reaction of the enzymes with their natural substrates in isolated systems and for the course of formation and action of thrombin in plasma. Inhibition of the activity of F Xa in plasma was also seen when the activity was
7h2
SY?;THETIC COAGFL4TIOZ; I?iHIBITORS
Vol.12,No.j
by means of a F X-test kit in normal plasma to which the inhibitors were added. It is noteworthy that3-amidinobenzyl derivatives with strong inhibitory action on F Xa cause stronger inhibition of plasma coagulation than it is caused by 4-amidinobenzyl derivatives with low inhibitory action on F Xa, while the antithrombin effect of the isomers is nearly the same. An exception represent the amidinophenylpyruvic acids (VII, VIII). Corresponding to the high affinity of 4-amidinophenylpyruvic acid for F Xa and thrombin and the low affinity of 3_amidinophenylonly the 4-isomer influences the pyruvic acid for both enzymes, formation and action of thrombin. Synthetic competitive inhibitors may influence also the nafactural processes of inactivation of activated coagulation tors in blood by interfering with their reactions with plasma was seen in the present (16). Such a phenomenon antithrombin investigation in the thrombin generation test. The irreversible F Xa and thrombin,by natural inhibitors inactivation of both, may be impaired by the synthetic competitive inhibitors. This would explain the increase in the maximum amount of thrombin formed as well as the delayed decrease in thrombin activity. The antithrombin activity of the inhibitors might also repress the proteolytic breakdown of prothrombin by thrombin itself and thus lead to increased thrombin formation (17). In studies on the inhibition of blood coagulation by diamidines effects were found that could not only be explained by the antithrombin activity (Ki-values) of the inhibitors (18). inhibition of further enzymes The authors proposed, therefore, particularly inhibition of F Xa. of the coagulation sequence, the effectiveness of the antithrombotic activity of Moreover, heparin is attributed primarily to inhibition of F Xa by some These aspects are of importance for the investigators (19). evaluation of synthetic inhibitors of blood coagulation. Hence, it would be promising to develop not only specific thrombin inhibitors but also potent inhibitors of F Xa. In light of the results presented some effects of the synthetic inhibitor 4-amidinophenylpyruvic acid already investigated in detail have to be re-evaluated. Combination of naturally occurring specific thrombin inhibitors (hirudin, reduviin) with naturally occurring inhibitors of prothrombin activation (ixodin) showed supra-additive efOn principle, potentiation is also demonstrable fects (20). with combinations of synthetic competitive inhibitors of different affinity for F Xa and thrombin. measured
REFERENCES 1.
STURZEBECHER, J,, MARKWARDT, F.. inhibitors of serine proteinases. Xa by derivatives of benzamidine. 1976.
and WALSMANN, P. Synthetic XIV. Inhibition of factor Thrombos. Res. 9, 637,
2.
MARKWAROT, F.. KLUCKING, H.-P.. and NOWAK, G. Antithrombinund Antiplasminwirkung von 4-Amidinophenylbrenztraubensaure Thrombos. Diathes. haemorrh. (Stuttq.1 24, 240, in vivo. 1970.
i-01.12
,X0. j
SYXTHETIC
COXGVIATIOS
Gerinnungsphysiologische , F. 3. MARKWARDT synthetischer Thtombininhibitoren. haemorrh. (Stuttq.) 27, 99, 1972.
Analyse det Thrombos. Oiathes.
4.
MARKWAROT, F., and KLUCKING, H.-P. fect of synthetic thrombin inhibitors. 243, 1972.
5.
HAUPTMANN, J., von aromatischen brinolysevorgange.
HOFFMANN, 3., Bisamidinen Acta biol.
,i7
I?r?IIBITORS
The
antithrombotic Thrombos. Res.
ef1,
Zur Wirkung and MARKWARDT, F. auf Blutgerinnungsund Fi35. 653, 1976. med. qerm.
The NOWAK, G., and HOFFMANN, J. 6. MARKWARDT, F., of drugs on disseminated intravascular coagulation II. Effects of naturally occurring and synthetic inhibitors. Thrombos. Res. 11, 275, 1977. 7. WALSMANN,
Wirkung
influence (DIG). thrombin
P.,
HORN, H., LANDMANN, H., MARKWARDT, F., Synthetische Inhibitoren J. , and WAGNER, G. der Serinproteinasen. 5. Mitteilung: Uber die Hemmung van Plasmin und Thrombin durch araliphatische AmidinoTrypsin, verbindungen mit Atherstruktur sowie Ester der 3- und 4Amidinophenoxyessigsgure. Pharmazie 30, 378, 1975.
STURZEBECHER
,
MARKWARDT, F. ?? STURZEBECHER, J. ?? VOIGT, B., 8. WALSMANN, P., and WAGNER, G. Synthetische Inhibitoren der Serinproteinasen Uber die Hemmung von Trypsin, Plasmin und 9. Mitteilung: Thrombin durch Amidinophenyl-, Amidinobenzylund R(Amidinophenyl)-sthylarylketone. Pharmazie 31, 733, 1976. and HORN, H. 9. WAGNER, G., bindungen mit Atherstruktur.
Synthese aromatischer Pharmazie 30, 353,
Amidinover1975.
10.
WAGNER, G., and VOIGT, 8. Synthese von Amidinobenzylaryl-, und Amidinobenzylalkoxymethylketonen. AmidinobenzylalkylPharmazie 31, 432, 1976.
11.
WALSMANN. Pharmazie
12.
BAJAJ, S. P., and bovine prothrombin by trypsin-activated 1973.
13.
BACHMANN, F., DUCKERT, F. v and KOLLER, F. The StuartPrower factor assay and its clinical significance. Thrombos. Diathes heamorrh. (Stuttq.) 2, 24, 1958.
14.
MACFARLANE, R. test. J. clin.
G., and BIGGS, R. Path. 6, 3, 1953.
15.
The LOEWE, S. combined drugs.
problem of synergism Arzneimittel-Forsch.
16.
MARKWARDT, F., HOFFMANN, J., and KURBS, E. The influence of synthetic thrombin inhibitors on the thrombin-antithrombin reaction. Thrombos. Res. 2, 343, 1974.
17.
STENN, K. S., and BLOUT, E. R. Mechanism of bovine prothrombin activation by an insoluble preparation of bovine factor Xa (Thrombokinase). Biochemistry 11, 4502, 1972.
P. 23,
Uber 401,
die Reinigung 1968.
von
Thrombinprgparaten.
MANN, K. G. Simultaneous purification and factor X. Activation of prothrombin factor X. J. biol. Chem. 248, 7729,
A thrombin and 3,
generation
antagonism 285, 1953.
of
of
744
SYXTHETIC
COXGCZATIO?i
EiiIBITORS
CHENG, M. C.-F., and TIDWELL, compounds with an aromatic thrombin, pancreatic kallikrein, J. Med. Chem. 19, 643, 1976.
Vo1.12,Xo.j
18.
GERATZ, J.-D., bis(benzamidino) Inhibitors of and complement.
R. R. Novel central link. trypsin,
19.
Effect of heparin on the neutralization YIN, E. T. factor X and thrombin by the plasma alpha-2-globulin Thrombos. Giathes. haemorrh. (Stuttq.), 33, itor.
20.
MARKWARDT, F., SCHULZ, E., and EWERT, 0. Uber die Wirkung von Kombinationen gerinnungshemmender Stoffe. Thrombos. Diathes. haemorrh. (Stuttg.) 7, 304, 1962.
of inhib43, 1975.
J.
Hauptmann,
Institute Medical
(Received
9.2.1978.
F.
Markwardt
and
P.
ON THE
Walsmann
of Pharmacology and Toxicology, Academy, DDR-50 Er-fur-t, GDR
Accepted
by Editor
G. %ller-Berghaus)
ABSTRACT derivatives The effects of 3- and 4-amidinobenzyl known as competitive inhibitors of the esterolytic activity of Factor Xa and thrombin on prothrombin activation and plasma coagulation were studied. Compared to 4-amidinobenzyl derivatives, the corresponding 3-isomers are stronger inhibitors of Factor they inhibit the Factor Xa-catalyzed formation of Xa, thrombin in isolated systems and in plasma. Moreover, they are more effective in inhibiting overall plasma coagulation. The results suggest that inhibition of Factor Xa by synthetic competitive inhibitors represents effective control of blood coagulation.
INTRODUCTION The serine proteinases of the blood, thrombin and Factor Xa (F Xa) are inhibited competitively by 3- and 4-amidinobenzyl ethers and ketones (1). The inhibitor constants (Ki-values) determined with synthetic substrates revealed differences in the affinity of the inhibitors for these enzymes. Generally, 3-amidinobenzyl derivatives inhibited F Xa more strongly than thrombin, whereas 4-amidinobenzyl derivatives showed only low affinity for F Xa. Since the extrinsic and intrinsic patbways of the coagulation sequence converge at the stage of F Xa, inhibition of this enzyme and of thrombin might represent an effective interference with the coagulation process. The anticoagulant effect of some amidinobenzyl derivatives due to inhibition of thrombin has already been shown in vitro and in vivo (2, 3, 4, Therefore, the effects on blood coagulation caused by 5, 6). 735
SYNTHETIC
736
COAGLUTIOB
IIHIBITORS
Vo1.12,No.5
synthetic inhibitors with strong affinity for F Xa were studied. Moreover, the question about the mosteffective blockade of the coagulation sequence by synthetic inhibitors should be clarified. Because of the lack of specific synthetic inhibitors of F Xa and thrombin, isomeric 3- and 4-amidinobenzyl derivatives of different inhibitor strength towards F Xa and thrombin were chosen. Table 1 shows that the 3-isomers are effective inhibitors of F Xa, while the corresponding 4-isomers are less effective, the inhibitor strength towards thrombin, however, does not differ between the isomers. TABLE Inhibitor Ketones
for
Constants Inhibition
of
3of
1
and 4-Amidinobenzyl Thrombin (7, 8)
and
HN\
CH2R
H2N’ Ki-values R
No. Thrombin
-o- 01 \
Factor
Xa
Ethers Factor
C / \ -0 -
and Xa (1)
CH2R
(,uM) No. Thrombin
Factor
I
66
6.8
II
58
380
CH3
III
53
4.8
IV
36
310
OCH3
V
47
5.4
VI
26
210
Xa
-
-CO -o-/
\ -
-CO-COOH
VII
790
220
MATERIALS
VIII
6.5
9.4
AND METHODS
Amidinobenzyl derivatives: Detailed data and the provenance of the inhibitors are given elsewhere (1, 7, 8, 9, 10). Fibrinogen: Bovine fibrinogen (Behringwerke AC, Marburg/ Lahn). Human titrated plasma: Fresh blood from healthy donors was collected in 3.13 % sodium citrate (9 + 1). Plasma was obtained by centrifugation at 700 g for 5 min and pooled. Standard thrombin, lot No B-3, specific activity Thrombin: 21.7 NIH U/mg (National Institute of Health, Bethesda /Maryland) was used for standardization. Thrombin, specific activity 1055 NIH U/mg prepared from bovine blood (11) was used for clotting assays. Determination of thrombin activity determine thrombin activity, 0.2 ml mixed with 0.2 ml of 0.1 M Tris-HCl
by clottinq assay: To of fibrinogen (0.6 %) was buffer, pH 7.5, after
addition of 0.1 ml of a thrombin-containing sample the clotting A standard curve was obtained using standard time was recorded. To determine 150-values (inhibthrombin (0.01 - 6.0 NIH U/ml). itor concentration causing 50 % inhibition of thrombin activity) the fibrinogen solution was mixed with for the inhibitors, varying inhibitor concentrations and 0.1 ml of thrombin solution (6 NIH U/ml). Factor X was isolated from fresh Isolation of Factor X: bovine blood (collected in sodium citrate plus heparin) accordChromatography’ was carriedout on ing to BAJAJ and MANN (12). DEAE-cellulose instead of DEAE-Sephadex. A Factor X solution in 50 % glycerol with an activity of 920 U/ml (determined according to BACHMANW(13))was obtained. The preparation was but contained 150 NIH U of prothrombin/ml. free of thrombin, Samples of 0.2 ml of the Factor X Activation of Factor X: pH 8.0 for 45 min with solution were incubated at 37 oC and 5.8 ml sodium chloride (0.154 M) containing 1 mg cephalin, ug Russell’s viper venom and 0.05 mMo1 CaC12. The solution 50 was / centrifuged and kept at 4 oC. Prothrombin was isolated and Isolation of prothrombin: purified according to the method used for Factor X (12). A prothrombin solution in 50 % glycerol with an activity of The prothrombin preparation 27,000 NIH U/ml was obtained. contained 10 NIH U of thrombin/ml and 0.3 U of Factor X/ml. Volumes of 2 ml of this solution were mixed with 18 ml of 0.02 M Tris-HCl buffer (pH 7.5) containing 0.1 M NaCl and deep frozen and stored, The activity was deter0.01 M CaC12, mined after activation by Factor Xa using the clotting assay described above. Samples of 0.4 ml of prothrombin Activation of prothrombin: solution were mixed with either 0.5 ml of Tris-HCl buffer,pH 7.5,or inhibitor solution, and activation (25 oC) was started by addition of 0.1 ml of activated Factor X. Atselected time aliquots were removed and assayed for thrombin intervals, The amount of thrombin present in the sample was calformed. culated from the clotting time referring to a curve with To compensate for disturbance of the deter, standard thrombin. mination of thrombin activity in the clotting assay by the antithrombin activity of the inhibitors a standard curve was made for each compound using that concentration which would result from transferring samples of the incubation mixture to the clotting assay. Thrombelastoqraphy: A volume of 0.2 ml of human titrated plasma was mixed with either 0.1 ml of Tris-HCl buffer, pH 7.4, or inhibitor solution containing 5 % ethanol each-and recalcified with 0.05 ml of 0.1 M CaC12. Thrombelastographic tracings were recorded with the Hellige thrombelastograph. The reaction time was measured and expressed as per cent of the control value. The thrombin generation test Thrombin qeneration in plasma: of MACFARLANE and BIGGS (14) was sliqhtly modified: 0.5 ml of human titrated plasma was mixed with-either 0.5 ml of Tris-HCl buffer, pH 7.4, or inhibitor solution containing 10 % methanol each and recalcified with 0.5 ml of 0.025 M CaCl2. The forming clot was wrapped around an applicator stick. At intervals of
738
SYXTHETIC
COAGLUTIOS
IXHIBIT'ORS
2 min, samples of 0.1 ml were withdrawn from the incubation mixture and added to 0.4 ml of fibrinogen (0.2 %). Thrombin activity was determined as described under “Activation of prothrombin”. One-staqe prothrombin time: A volume of 0.1 ml of human citrated olasma was mixed with either 0.1 ml of Tris-HCl buffer, or inhibitor solution containing 10 $Y, methanol each and pH 7.4; 0.1 ml of human brain thromboplastin (Thrombokinase AWD, VEB Arzneimittelwerk Dresden) and incubated over a period of 3 min 0.1 ml of 0.025 M CaC12 was added and the clotat 37 oC; then Results are expressed as per cent proting time was recorded. longation of the clotting time of control. RESULTS Influence on the reaction of thrombin and Factor Xa with natural substrates. The influence of amidinobenzyl derivatives on the thrombin catalyzed fibrinogen clotting was investigated in an isolated To evaluate the inhibitor activity of the derivatives system. the inhibitor concentra.. on the thrombin-fibrinogen reaction, tions causing 50 % inhibition of thrombin activity were estiThe values in Table 2 show that 4-amidinomated (150-values). benzyl derivatives inhibit fibrinogen clotting more strongly than 3..amidinobenzyl derivatives, whereas the Ki-values (cf. Table 1) are nearly the same. their
TABLE Influence of 3Thrombin-Catalyzed
and
4-Amidinobenzyl Ethers Fibrinogen Clotting 150-Values
No.
I III V VII
2
3-Derivatives 500 150 250 700
and
Ketones
on
(,uM) No. II IV VI VIII
4-Derivatives 150 30 100 8
the influence of amidinobenzyl derivatives on the F XaFurther, catalyzed prothrombin activation was studied determining the amount of thrombin formed during 30 minutes incubation (pH 7.5, 25 OC) in the presence of an inhibitor concentration of 20 uM. / The values in Table 3 are expressed as percent of thrombin formed in inhibitor-free controls. The activity of F Xa was inhibited by 3-amidinobenzyl derivatives, whereas the same concentration of the isomeric kamidinobenzyl derivatives had no influence on the time course of prothrombin activation. A representative example is shown in Fig. 1.
I-39
TABLE Influence Factor
and
of 3Xa-Catalyzed
4-Amidinobenzyl Prothrombin
Percent NO.
3 Ethers Activation
thrombin No.
42 56 32 93
II IV VI VIII
4-Oeriva
FIG. course of prothrombin
O-O
n-a
1
Influence After
60 on
the
t ives
1 Factor Xa-cataactivation.
control without inhibitor; in presence of 3-amidinobenzyl-4’ -methoxyphenyl ketone (V), 20 /uM; in presence of 4-amidinobenzyl-4’ -methoxyphenyl uM. ketone (VI), 20 /
I
.
20
on
103 73 105 20
-3 Time lyzed
Ketones
formed
3-Derivatives
I III V VII
and
120 formation
and
action
of
thrombin
in
plasma
the effect of the inhibitors had been studied in isotheir influence on the course of coagulation of lated systems, blood plasma was investigated. Thrombelastographic recordings of the coagulation of recalcified human titrated plasma showed that 4-amidinobenzyl derivatives with low inhibitory effect on F Xa inhibit plasma coagulation to an extent corresponding to their inhibitory effect on the thrombin-fibrinogen reaction The 3-amidinobenzyl derivatives with strong in(cf. Table 2). hibitory effect on F Xa caused stronger inhibition of plasma coagulation. A comparison of the prolongation of clotting times produced by the inhibitors showed that at the same concentration the 3-isomers are more potent than the 4-isomers and that 3-isomers obviously have a steeper slope of the dose-response curve (Fig, 2). After investigation of the prothrombin activation in an isolated system, studies were performed in plasma. These experiments (thrombin generation test) showed that amidinobenzyl derivatives at concentrations of 100 /uM retard the formation of thrombin in plasma as regards the time until appearance of maximum thrombin activity. Retardation is more pronounced in case of the 3-isomers I, III, and V than in case of the cor-
740
SYXTHETIC
COAG-KLATIOE IXHIBITORS
VO1.12,SO.5
responding 4-derivatives II, IV, and VI. The curves of thrombin show that the maximum of thrombin activity activity, however, A representative example is higher in samples with inhibitors. derivatives of the influence of isomeric 3- and 4..amidinobenzyl on the course of thrombin formation in plasma is given in Fig. 3.
FIG.
2
Prolongation of plasma clotting time (reaction times from thrombelastograms of recalcified human titrated plasma) by 3.. and 4-amidinobenryl derivatives.
30 25 1
min FIG. Time
?? d M M
course
of
thrombin
generation
3 in
plasma.
control without inhibitor; in presence of 3-amidinobenzyl-4*-methoxyphenyl ketone (V), 100 /uM; in presence of 4-amidinobenzyl-4’-methoxyphenyl ketone (VI), 100 uM. /
Combination
effects
of
isomeric
3-
and
4-amidinobenzyl
derivatives In order to get further information on the overall effect of inhibition of F Xa on plasma coagulation. combinationsof inhibFrom thrombelastographic investigations it itors were studied. was obvious that using combinations of 3and &amidinobenzyl derivatives a comparatively low percentage of the corresponding stronger inhibition of 3-isomer in the combination caused plasma coagulation than was produced by the 4-isomer at maxiBY combination of two 4-amidinobenzyl derivatives, an mum. additive effect was observed only.
FIG.
4
Isobologram according to LOEWE (15); the isobole connects equieffective concentrations of combinations of the isomers I and II.
Derivative Ll[phii In further experiments, combination effects on the coagulation process were studied after adding tissue thromboplastin and Ca++ to human titrated plasma. Isoboles according to LOEWE (15) were elaborated in order to verify the supra-additive effect of inhibitor combinations. Equieffective concentrations of various combinations causing a defined effect (50 per cent prolongation of clotting time) were determined. In Fig. 4, the curve typical of potentiation is shown for the combination of isomers I and II. DISCUSSION The evaluation of the present results is rather difficult, since they were not obtained with specific synthetic inhibitors of thrombin and F Xa, but with isomeric 3and Q-amidinobenzyl derivatives of different affinities for these enzymes. In the more complex test systems used. inhibition of thrombin may have influenced the activation of prothrombin by F Xa. However, the inhibitory activity of amidinobenzyl derivatives towards thrombin and F Xa found in previous investigations with synthetic substrates of the respective enzymes was demonstrated for the reaction of the enzymes with their natural substrates in isolated systems and for the course of formation and action of thrombin in plasma. Inhibition of the activity of F Xa in plasma was also seen when the activity was
7h2
SY?;THETIC COAGFL4TIOZ; I?iHIBITORS
Vol.12,No.j
by means of a F X-test kit in normal plasma to which the inhibitors were added. It is noteworthy that3-amidinobenzyl derivatives with strong inhibitory action on F Xa cause stronger inhibition of plasma coagulation than it is caused by 4-amidinobenzyl derivatives with low inhibitory action on F Xa, while the antithrombin effect of the isomers is nearly the same. An exception represent the amidinophenylpyruvic acids (VII, VIII). Corresponding to the high affinity of 4-amidinophenylpyruvic acid for F Xa and thrombin and the low affinity of 3_amidinophenylonly the 4-isomer influences the pyruvic acid for both enzymes, formation and action of thrombin. Synthetic competitive inhibitors may influence also the nafactural processes of inactivation of activated coagulation tors in blood by interfering with their reactions with plasma was seen in the present (16). Such a phenomenon antithrombin investigation in the thrombin generation test. The irreversible F Xa and thrombin,by natural inhibitors inactivation of both, may be impaired by the synthetic competitive inhibitors. This would explain the increase in the maximum amount of thrombin formed as well as the delayed decrease in thrombin activity. The antithrombin activity of the inhibitors might also repress the proteolytic breakdown of prothrombin by thrombin itself and thus lead to increased thrombin formation (17). In studies on the inhibition of blood coagulation by diamidines effects were found that could not only be explained by the antithrombin activity (Ki-values) of the inhibitors (18). inhibition of further enzymes The authors proposed, therefore, particularly inhibition of F Xa. of the coagulation sequence, the effectiveness of the antithrombotic activity of Moreover, heparin is attributed primarily to inhibition of F Xa by some These aspects are of importance for the investigators (19). evaluation of synthetic inhibitors of blood coagulation. Hence, it would be promising to develop not only specific thrombin inhibitors but also potent inhibitors of F Xa. In light of the results presented some effects of the synthetic inhibitor 4-amidinophenylpyruvic acid already investigated in detail have to be re-evaluated. Combination of naturally occurring specific thrombin inhibitors (hirudin, reduviin) with naturally occurring inhibitors of prothrombin activation (ixodin) showed supra-additive efOn principle, potentiation is also demonstrable fects (20). with combinations of synthetic competitive inhibitors of different affinity for F Xa and thrombin. measured
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MARKWAROT, F.. KLUCKING, H.-P.. and NOWAK, G. Antithrombinund Antiplasminwirkung von 4-Amidinophenylbrenztraubensaure Thrombos. Diathes. haemorrh. (Stuttq.1 24, 240, in vivo. 1970.
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The NOWAK, G., and HOFFMANN, J. 6. MARKWARDT, F., of drugs on disseminated intravascular coagulation II. Effects of naturally occurring and synthetic inhibitors. Thrombos. Res. 11, 275, 1977. 7. WALSMANN,
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HORN, H., LANDMANN, H., MARKWARDT, F., Synthetische Inhibitoren J. , and WAGNER, G. der Serinproteinasen. 5. Mitteilung: Uber die Hemmung van Plasmin und Thrombin durch araliphatische AmidinoTrypsin, verbindungen mit Atherstruktur sowie Ester der 3- und 4Amidinophenoxyessigsgure. Pharmazie 30, 378, 1975.
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MARKWARDT, F. ?? STURZEBECHER, J. ?? VOIGT, B., 8. WALSMANN, P., and WAGNER, G. Synthetische Inhibitoren der Serinproteinasen Uber die Hemmung von Trypsin, Plasmin und 9. Mitteilung: Thrombin durch Amidinophenyl-, Amidinobenzylund R(Amidinophenyl)-sthylarylketone. Pharmazie 31, 733, 1976. and HORN, H. 9. WAGNER, G., bindungen mit Atherstruktur.
Synthese aromatischer Pharmazie 30, 353,
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WAGNER, G., and VOIGT, 8. Synthese von Amidinobenzylaryl-, und Amidinobenzylalkoxymethylketonen. AmidinobenzylalkylPharmazie 31, 432, 1976.
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BACHMANN, F., DUCKERT, F. v and KOLLER, F. The StuartPrower factor assay and its clinical significance. Thrombos. Diathes heamorrh. (Stuttq.) 2, 24, 1958.
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MARKWARDT, F., HOFFMANN, J., and KURBS, E. The influence of synthetic thrombin inhibitors on the thrombin-antithrombin reaction. Thrombos. Res. 2, 343, 1974.
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STENN, K. S., and BLOUT, E. R. Mechanism of bovine prothrombin activation by an insoluble preparation of bovine factor Xa (Thrombokinase). Biochemistry 11, 4502, 1972.
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MANN, K. G. Simultaneous purification and factor X. Activation of prothrombin factor X. J. biol. Chem. 248, 7729,
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CHENG, M. C.-F., and TIDWELL, compounds with an aromatic thrombin, pancreatic kallikrein, J. Med. Chem. 19, 643, 1976.
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GERATZ, J.-D., bis(benzamidino) Inhibitors of and complement.
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