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The antithrombotic effect of synthetic thrombin inhibitors
THROMBOSIS Printed
RESEARCH in the United
Vol. 1, pp. 243-252,1972 Pergamon Press, Inc.
States
TH8 ANTITHROMBOTICM%I?ECTOF SY~T'IEETIC mO1E3n? lj?r~IIE;ITOP!
F. IVIarkwardt and H.-P. El&king Institute of Pharmacology and Toxicology Medical Academy Xrfurt, GOR
(Received
26.5.1972.
Accepted
by
Editor
B. Blomb&k)
ABSTRACT The pharmacokinetics,.toxicityand anticoagulantactivity of the synthetic thrombin inhibitor 4_amidinophenylpyruvic acid (APPA) which represents a new type of anticoagulant agents have been studied. In experimentalanimals lethal effects caused by thrombin infusion, the incidence of experimental thrombi and endotoxin-induced disseminated intravascularcoagulationwere prevented by the administrationof this inhibitor.
INTRODUCTION ;Niththe introduction-ofthe direct and indirect acting anticoagulants of the heparin and coumarin type, the search for new anticoagulant compounds has continued since both groups of drugs still have certain disadvantages.The development of synthetic, low molecular weight inhibitors of coagulation enzymes presents a new and exciting approach. Such substances could find potential use as oral, rapidly acting anticoagulantswhich would offer several advantages for antithrombotiotherapy. Biochemical studies of serine protease inhibitors of blood led 243
244
ANTITHROMBOTIC
EFFECT
Vol.l,No.3
us to the development of several types of synthetic thrombin inhibitors. These substances inactivate the active center of the enzyme by covalent and non-covalent bonding (l-6). Coagulation studies revealed that the competitive inhibitors of the enzyme were especially suited to affect the clotting activity (7). A study of their pharmacologicalproperties indicated that Y-amidinophenylpyruvicacid (BEPA) zas useful as an antithrombin in vivo (8). In the present study an attempt has been made to demonstrate the antithrombotic effect of this new anticoagulant in experimental animals. iUTRRIAIS AND NETHODS Thrombin: Thrombin AUD (Kombinat VZB ArzneimittelwerkDresden) 100 NM units/mg. Endotoxin: Lipopolysaccharidefrom X. coli, isolated by the method of Uestphal (9). 4-Amidinophenylpyruvicacid (APPA): Prepared by Dr.Kazmirowski (Kombinat VEB ArzneimittelwerkDresden) according to the procedure of Richter and \iJagner (10). Experimental animals: Rabbits (2.5-3.0 kg), Uistar rats (25O300 g) and white mice (Agnes Bluhm 20-25 g) of both sexes were used. Determination of 4-amidinophenylpyruvicacid (Al?PA)in bioloEica1 specimen One ml of protein-free plasma (supernatant of a mixture of 2 ml titrated plasma and 1 ml 30 % trichloroaceticacid, centrifuged) or 1 ml serum wjereincubated for 30 min at room temperature with I ml freshly filtered 2,4-dinitrophenylhydrazine(0.1 g/l00 ml N HCl). The mixture was diluted with 24 ml H20. The extinction coefficient of the brownish purple solution was measured at 390 nm, using a similarly treated control plasma or control serum as a standard. The amount of APPA was read from a standard curve which was prepared by adding increasing amounts of APPA to plasma or serum samples. Concentrations as low as IO ug AFPA/ml plasma or serum can be measured. Sx-perimentalproduction of thrombi Clotting thrombi (stasis) were produced by the method of Wessler (II) using anesthetized rabbits (hexobarbital 50 mg/kg i.v.).
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The clots were formed in an exposed segment of the jlugularvein . by injecting contact activated human serum l*v., followed by stasis. Per studies in non-anesthetizedanimals and for avoiding opera,ti_ve procedures the method %as modified. 1.3 ml of human serum/kg body weight was injected into the marginal ear vein of one ear of a non-anesthetizedrabbit over a period of 15 sec. Ten set later, a 2 cm segment of the other marginal ear vein was clamped off. Ten min later the isolated segment was incised and the clot removed by sucking the content into 3.8 % trisodium citrate solution. Deposition thrombi were produced by electrical current (5 mA, 60 set) in an exposed carotid artery of anesthetizedrats (lo5 g ethylurethane/kgi.p.>. The formation of the thrombotic occlusion was followed by the procedure of Hladovec (12) by measuring the drop in temperature distal to the occlusion by means of a thermistor. In control animals, the .-temperature dropped markedly II.5 + 305 min following the stimulationsmithelectrical current. Histologic examinations revealed the presence of a thrombosis. Only those animals were used which develop the drop in temperature within 30 min following the electrical stimulation.Those animals ?xhichdid not have a drop in temperaturewithin the first 30 min, did not develop a thrombosis even after 60 min or longer. Disseminated intravascularcoagulation (DIC) was'induced in anesthetized rabbits (1.2 g ethylu.rethane/kg i.p.> by infusing 75 pg endotoxin/kg/hinto a marginal ear vein. DIC was followed by fibrinogen determinationsand platelet counts (8) up to 6 hrs following the infusion of endotoxin. The animals were later sacrificed, and the number of glomeruli of both kidneys containing hyalin thrombi, were counted. RZXXJLTS 1. Toxicity In order to study the effect of AEPA on the inhibition of blood coagulation in vivo, the toxicity of the substancewas tested. The acute toxicity (LD5o) in mice was 151 (127480) mg/kg following intravenous injection, 389 (319-475) mg/kg following intraperitoneal injection, and 801 (5374380) mg/kg following oral administration.
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2. Pharmacodynamics In order to investigatewhether APPA had other pharmacodynamic effects, besides'inhibitingserine proteases, we studied its effect on a number of isolated organs. APPA had no effect on smooth muscle (guinea pig ileum) and skeletal muscle (rectus abdominis of Rana esculenta) up to a concentration of IO-3M in the incubation solution. It also had no effect on the isolated frog heart (method of Straub) and the beating guinea pig heart (method of Langendorff) up to a concentration of IO-3-iiiI. Its effect on circulation &as tested by recording blood presslureand respiratory rate in anesthetized (1.5 Q ethylurethane/kgi.p.> rabbits. Up to a concentration of IO rngAPPA/kg i.v. no effect could be measured. 3. Pharmacokinetics Since the anticoagulant effect of a synthetic thrombin inhibitor would be determined by its level in the blood related to the time following its administration,t%etested the pharmacokineticsof the compound in rabbits. AH?A in plasma and serum was determined as described above.
FIG. 1 Level of APPA in rabbit blood following the oral and intravenous administrationof 100 mg APPA/kg.
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Following the injection of 100 mg APPA/kg, the blood level dropped sharply initially, correspondingto the distribution between intra- and extravascularcompartments.The distribution equilibriumwas reached after 60 min. Thereafter, the level in plasma dropped slowly due to the elimination of APPA. The average half-life was 97.5 i 7.5 min. The extent of APPA absorption was calculated by planimetritally measuring the area under the blood level curves following intravenous and oral administrationand relating the areas to each other. Following the administrationof 50 mg APPA/kg, the rate of absorption was 33 70, following 100 mg APPA/kg, it was 17 %. According to these data, the absorption of APPA following oral administrationis incomplete and can not be improved by increasing the dose. After intravenous administration38 2 10 % of the APPA was recovered in an unaltered form in the 24 hour urine of the experimental animals. 4. Prevention of the effect of an intravenous thrombin injection The antithrombin effect of APPA in vivo was demonstrated by preventing thrombus formation follovjingthe intravenous injection of thrombin. The intravenous injection of 900 NM units of thrombin/kg over 30 set into mice (LDloo) was lethal to all animals due to massive thrombus formations in the right ventricle, atrium and pulmonary vessels. The administrationof IO mg APPA/kg i.p. prevented the lethal effect of the thrombin injection, IO min following the APPA administration,in 90 % of the experimental animals. The results are listed in Table I. 5. Inhibition of the formation of clotting and deposition thrombi The antithromboticeffect of APPA on the formation of venous clotting thrombi and arterial deposition thrombi was also tested on experimental animals models. The results are listed in Tables 2 and 3.
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EFFECT
TABI8 1 The Effect of APPA on Lethal Thrombin Injections in White Nice Humber of animals n
i.p. administration Xumber of dead animals IO min before thrombin after thrombin injection .p+ injection (900 NJ3 units/kg) n %
44
0.1 ml 0.9 % NaCl solution
46
0.1 ml APPA solution (IO
44
100
4
9
m&g)
< 0.001
+ Significance calculated by the X2-test
TABI 2 The Bffect of APPA on the Formation of Clotting Thrombi in the Jugular Vein (I) and Marginal Ear Vein (II) of Rabbits Number of APPA dose in mgjkg Animals with thrombi p+' i.v.+ anti_als n % 21 IO
0
0
< 0.001
11
5
7
63
n. s.
29
87
-
0
0
11
++
90
12
33
+
19
IO
4 min prior to the injection of serum Significance calculated by the X2-test
<
0.001
I
II
ANTITHROMBOTIC
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EFFECT
TABLE 3 The Xffect of APPA on the E'ormation of Deposition Thrombi in the Carotid Artery of Rats. The Thrombi Were Induced by 'Vessel Yall Damage Using Electrical Current Rumber of animals
BPPA dose in+mg/kg i.v.
n
18
10
4
94 22
12
5
7
58
IO
205
9
90
50
+
Animals with thrombotic vessel occlusions within 30 min follov;ing electrical current n 510
‘i?
P
f-l-
d 0,001 < 0.001
n. s.
5 min prior to the vessel wall lesion
++ Significance
calculated by the X2-test
According to these data, a single intravenous injection of IO mg/kg APPA prevented the formation of a clotting thrombus, produced by the injection of serum plus stasis in.an isolated vessel segment. The same dose was sufficient to prevent the formation of a deposition thrombus in an arterial segment of rats, produced by vessel wall lesion by means of electrical current. 6, The effect on disseminated intravascularcoagulation
Of special interest mas the effect of the synthetic inhibitors on disseminated intravascularcoagulation or consumption coagulopathy, as it is observed in the generalizedSanarelli-Shwartzman phenomenon. The phenomenon was produced by the intravenous injection of endotoxin in rabbits and followed by changes in the fibrinogen levels and number of circulatingplatelets. After terminati_on of the experiments,histologic examinationsmere performed to assess the degree of microthrombosis.The results listed in Table 4 indicate that the drop in fibrinogen levels and platelet counts, as seen in the control animals, could be prevented-by the simultaneou-u administration of AlEPA.This pro-
tective effect of APPA was also seen in a reduction of the number of microthrombi
in the organs investigated.
250
ANTITHROMBOTIC
EFFECT
Vol.l,No.3
'PABIdS4
The 3ffect of APPA on the tindotoxinInduced Coagulation Changes in Rabbits Xu$er
Infusion
animals n 7
7
Crop in Number of Drop in platelet count fibrinogen levels glomeruli containing in %+ in %+ microthrombi in %
endotoxin
75 w&dh
67 + 17
36 2 12
71 2 25
2.52 12
17 2 12
22 +,11
(p< O.ool)++
(p( O.l)++
(p( o.ool)++
5 w-sA=Uk@;
i.v. initially, 2 mg APPA/kg/h continuously endotoxin 75 IJ.@;/kdh
+ 6 hours after the start of the infusion ++ Significance calculated by the t-test EISCUSSION The present studies have demonstrated that intravascular thrombus formation could be prevented by the infusion of a synthetic thrombin inhibitor. These findings are of fundamental importance since so far no anticoagulantwith a similar mode of action in vivo has been studied. The differences to the known anticoagulantsare apparent since the protease inhibitors are synthetic, low molecular weight compounds aith a direct action which can be administered orally. Already at this time, the inhibitor tested can serve as a useful pharmacologicalmodel for compounds of this nature and allows an estimate of the potential therapeutic effect of this class of 1 dru?!~~vie~ring these results, it also must be kept in mind that the tested inhibitor not only has an effect on thrombin, but alSO on try-psin(13) and other serum proteases of the blood with similar specificity as trypsin, such as plasmin and kallikrein (7, 14). The effects could play a major role in the treatment of consumption coagulopathy in which the fibrinolytic system and kinin forming system are also activated.
ANTITHROMBOTIC
Vol.l,No.3
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2.
3.
4.
5.
6.
7.
8.
9. 70.
11.
12. 13.
14.
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F L!A&QmwiN, 8. , and ‘uLmA1NN, P. Comparative studies on the inhibition of tryp;in, plasmin and thrombin by derivatives of benzylamine and benzamidine. Xu_r. J. biochem.: 6, 502. 1qc-Js. P., and LAiEXANN, H. [email protected]$ARDT, P., RICHT_lQ?, Id., YiAIi3WiTN, The inhibition of try-psin, plasmin and thrombin by benzyl 4-guanidinobenzoate and 4' -nitrobenzyl 4-guanidinobenzoate. FtiBS Letters: 8, 170. 1970. 1{&RKiiARDT,Pa, 'iiALSM.A,NlN, Pep RICHTLR, Id.9KLOCKETG, Ha-P. DlWLRT, J., and LAXDMiNN, H. Aminoalkylbenzolsulfofluoride Pharmazie: 26, 401. 1971. als Fermentinhibitoren. P., RICHThR, hl.,and EWS&$DT, P. Inaktivierung $ALSM,tiTfl, von Tr sin und Thrombin durch 4-Amidinobenzolsulfofluorid und 4-$ 2-Aminoathyl)-benzolsulfofluorid. Acta biol. med. merman..* 28; 573. 1972. A~~UjXDT, %'.,~;~ALSWl!7N, P., and KAZEIIRO'EKI, H.-G. Untersuchungen iiber den Linflul3 von Ring-Substitutionen auf die thrombinhemmende %irkung von Benzylamin- und Benzanidinderivaten. Pharmazie: 24, 400. 1969. lWES;;ARDT, F., WALSFANN, I?., and LANDMANN, H. Hemmung der Thrombin-, Plasmin- und Trypsinwirkung durch Alkyl- und Alkoxybenzamidine. Pbarmazie: 25, 551. 1970. MARK;&RDT, P. Gerinnungsphysiologische Analyse der liirkung synthetischer Thrombininhibitoren. Thrombos. Diathes. haemorrh. (Stuttg.): 27, 99. 1972. WW;;ARDT, 2’. , KLCiCKING,H.-P., and NO::.AK,G. Antithrombinund Antiplasminviirkung von 4-Amidinophenylbrenztraubensaure (AEFA) in vivo. Thrombos. Diathes. haernorrh. (stuttg.): 24, 240. 1970. s,3TElAL, O., LtiYXRITZ, O., and EEmR, E'. Uber die i;xtraktion von Bakterien mit Phenol/*iasser. Z. Baturforach. B : 7, 148. 1952. RICXTER, I?. and SiiAGN%R,G. Verfahren zur Herstellung .von 4-Amidinophenylbrenztraubensglure. Virtschaftspatent C 07c/153316, 25. 2. 1971. ;&SSmR, Se tixperimental production of thrombosis by administration of serum. In: Blood coagulation, hemorrha#Te and thrombosis. L. Iii.Tocantlns and L A K lend ) TJew York and London, Grune and Stratto:, iq6grap. t4:: HMCO'aC, J. Experimental arterial thrombosis in rats with continuous registration. Thrombos. Diathes. haemorrh. (Stuttg.): 26, 407. 1971. GERkTZ, J. D. p-Amidinophenylpyruvic acid: a new highly effective inhibitor of enterokinase and trypsin. Arch. Biochem.: 118, 91. 1967. IMWARDT, F., KIXjCKIIVG, H.-P*, and NCXAK, G. Hemmung der Xininbildung im Elut durch p-Amidinophenylbrenztraubensgure. 3xoerientia (Basell: 27, 812. 1971. f;~;QpJ$~Ep,
e
,
RESEARCH in the United
Vol. 1, pp. 243-252,1972 Pergamon Press, Inc.
States
TH8 ANTITHROMBOTICM%I?ECTOF SY~T'IEETIC mO1E3n? lj?r~IIE;ITOP!
F. IVIarkwardt and H.-P. El&king Institute of Pharmacology and Toxicology Medical Academy Xrfurt, GOR
(Received
26.5.1972.
Accepted
by
Editor
B. Blomb&k)
ABSTRACT The pharmacokinetics,.toxicityand anticoagulantactivity of the synthetic thrombin inhibitor 4_amidinophenylpyruvic acid (APPA) which represents a new type of anticoagulant agents have been studied. In experimentalanimals lethal effects caused by thrombin infusion, the incidence of experimental thrombi and endotoxin-induced disseminated intravascularcoagulationwere prevented by the administrationof this inhibitor.
INTRODUCTION ;Niththe introduction-ofthe direct and indirect acting anticoagulants of the heparin and coumarin type, the search for new anticoagulant compounds has continued since both groups of drugs still have certain disadvantages.The development of synthetic, low molecular weight inhibitors of coagulation enzymes presents a new and exciting approach. Such substances could find potential use as oral, rapidly acting anticoagulantswhich would offer several advantages for antithrombotiotherapy. Biochemical studies of serine protease inhibitors of blood led 243
244
ANTITHROMBOTIC
EFFECT
Vol.l,No.3
us to the development of several types of synthetic thrombin inhibitors. These substances inactivate the active center of the enzyme by covalent and non-covalent bonding (l-6). Coagulation studies revealed that the competitive inhibitors of the enzyme were especially suited to affect the clotting activity (7). A study of their pharmacologicalproperties indicated that Y-amidinophenylpyruvicacid (BEPA) zas useful as an antithrombin in vivo (8). In the present study an attempt has been made to demonstrate the antithrombotic effect of this new anticoagulant in experimental animals. iUTRRIAIS AND NETHODS Thrombin: Thrombin AUD (Kombinat VZB ArzneimittelwerkDresden) 100 NM units/mg. Endotoxin: Lipopolysaccharidefrom X. coli, isolated by the method of Uestphal (9). 4-Amidinophenylpyruvicacid (APPA): Prepared by Dr.Kazmirowski (Kombinat VEB ArzneimittelwerkDresden) according to the procedure of Richter and \iJagner (10). Experimental animals: Rabbits (2.5-3.0 kg), Uistar rats (25O300 g) and white mice (Agnes Bluhm 20-25 g) of both sexes were used. Determination of 4-amidinophenylpyruvicacid (Al?PA)in bioloEica1 specimen One ml of protein-free plasma (supernatant of a mixture of 2 ml titrated plasma and 1 ml 30 % trichloroaceticacid, centrifuged) or 1 ml serum wjereincubated for 30 min at room temperature with I ml freshly filtered 2,4-dinitrophenylhydrazine(0.1 g/l00 ml N HCl). The mixture was diluted with 24 ml H20. The extinction coefficient of the brownish purple solution was measured at 390 nm, using a similarly treated control plasma or control serum as a standard. The amount of APPA was read from a standard curve which was prepared by adding increasing amounts of APPA to plasma or serum samples. Concentrations as low as IO ug AFPA/ml plasma or serum can be measured. Sx-perimentalproduction of thrombi Clotting thrombi (stasis) were produced by the method of Wessler (II) using anesthetized rabbits (hexobarbital 50 mg/kg i.v.).
Vol.l,No.3
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245
The clots were formed in an exposed segment of the jlugularvein . by injecting contact activated human serum l*v., followed by stasis. Per studies in non-anesthetizedanimals and for avoiding opera,ti_ve procedures the method %as modified. 1.3 ml of human serum/kg body weight was injected into the marginal ear vein of one ear of a non-anesthetizedrabbit over a period of 15 sec. Ten set later, a 2 cm segment of the other marginal ear vein was clamped off. Ten min later the isolated segment was incised and the clot removed by sucking the content into 3.8 % trisodium citrate solution. Deposition thrombi were produced by electrical current (5 mA, 60 set) in an exposed carotid artery of anesthetizedrats (lo5 g ethylurethane/kgi.p.>. The formation of the thrombotic occlusion was followed by the procedure of Hladovec (12) by measuring the drop in temperature distal to the occlusion by means of a thermistor. In control animals, the .-temperature dropped markedly II.5 + 305 min following the stimulationsmithelectrical current. Histologic examinations revealed the presence of a thrombosis. Only those animals were used which develop the drop in temperature within 30 min following the electrical stimulation.Those animals ?xhichdid not have a drop in temperaturewithin the first 30 min, did not develop a thrombosis even after 60 min or longer. Disseminated intravascularcoagulation (DIC) was'induced in anesthetized rabbits (1.2 g ethylu.rethane/kg i.p.> by infusing 75 pg endotoxin/kg/hinto a marginal ear vein. DIC was followed by fibrinogen determinationsand platelet counts (8) up to 6 hrs following the infusion of endotoxin. The animals were later sacrificed, and the number of glomeruli of both kidneys containing hyalin thrombi, were counted. RZXXJLTS 1. Toxicity In order to study the effect of AEPA on the inhibition of blood coagulation in vivo, the toxicity of the substancewas tested. The acute toxicity (LD5o) in mice was 151 (127480) mg/kg following intravenous injection, 389 (319-475) mg/kg following intraperitoneal injection, and 801 (5374380) mg/kg following oral administration.
246
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2. Pharmacodynamics In order to investigatewhether APPA had other pharmacodynamic effects, besides'inhibitingserine proteases, we studied its effect on a number of isolated organs. APPA had no effect on smooth muscle (guinea pig ileum) and skeletal muscle (rectus abdominis of Rana esculenta) up to a concentration of IO-3M in the incubation solution. It also had no effect on the isolated frog heart (method of Straub) and the beating guinea pig heart (method of Langendorff) up to a concentration of IO-3-iiiI. Its effect on circulation &as tested by recording blood presslureand respiratory rate in anesthetized (1.5 Q ethylurethane/kgi.p.> rabbits. Up to a concentration of IO rngAPPA/kg i.v. no effect could be measured. 3. Pharmacokinetics Since the anticoagulant effect of a synthetic thrombin inhibitor would be determined by its level in the blood related to the time following its administration,t%etested the pharmacokineticsof the compound in rabbits. AH?A in plasma and serum was determined as described above.
FIG. 1 Level of APPA in rabbit blood following the oral and intravenous administrationof 100 mg APPA/kg.
Vol.l,No.3
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247
Following the injection of 100 mg APPA/kg, the blood level dropped sharply initially, correspondingto the distribution between intra- and extravascularcompartments.The distribution equilibriumwas reached after 60 min. Thereafter, the level in plasma dropped slowly due to the elimination of APPA. The average half-life was 97.5 i 7.5 min. The extent of APPA absorption was calculated by planimetritally measuring the area under the blood level curves following intravenous and oral administrationand relating the areas to each other. Following the administrationof 50 mg APPA/kg, the rate of absorption was 33 70, following 100 mg APPA/kg, it was 17 %. According to these data, the absorption of APPA following oral administrationis incomplete and can not be improved by increasing the dose. After intravenous administration38 2 10 % of the APPA was recovered in an unaltered form in the 24 hour urine of the experimental animals. 4. Prevention of the effect of an intravenous thrombin injection The antithrombin effect of APPA in vivo was demonstrated by preventing thrombus formation follovjingthe intravenous injection of thrombin. The intravenous injection of 900 NM units of thrombin/kg over 30 set into mice (LDloo) was lethal to all animals due to massive thrombus formations in the right ventricle, atrium and pulmonary vessels. The administrationof IO mg APPA/kg i.p. prevented the lethal effect of the thrombin injection, IO min following the APPA administration,in 90 % of the experimental animals. The results are listed in Table I. 5. Inhibition of the formation of clotting and deposition thrombi The antithromboticeffect of APPA on the formation of venous clotting thrombi and arterial deposition thrombi was also tested on experimental animals models. The results are listed in Tables 2 and 3.
248
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EFFECT
TABI8 1 The Effect of APPA on Lethal Thrombin Injections in White Nice Humber of animals n
i.p. administration Xumber of dead animals IO min before thrombin after thrombin injection .p+ injection (900 NJ3 units/kg) n %
44
0.1 ml 0.9 % NaCl solution
46
0.1 ml APPA solution (IO
44
100
4
9
m&g)
< 0.001
+ Significance calculated by the X2-test
TABI 2 The Bffect of APPA on the Formation of Clotting Thrombi in the Jugular Vein (I) and Marginal Ear Vein (II) of Rabbits Number of APPA dose in mgjkg Animals with thrombi p+' i.v.+ anti_als n % 21 IO
0
0
< 0.001
11
5
7
63
n. s.
29
87
-
0
0
11
++
90
12
33
+
19
IO
4 min prior to the injection of serum Significance calculated by the X2-test
<
0.001
I
II
ANTITHROMBOTIC
Vol.l,No.3
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EFFECT
TABLE 3 The Xffect of APPA on the E'ormation of Deposition Thrombi in the Carotid Artery of Rats. The Thrombi Were Induced by 'Vessel Yall Damage Using Electrical Current Rumber of animals
BPPA dose in+mg/kg i.v.
n
18
10
4
94 22
12
5
7
58
IO
205
9
90
50
+
Animals with thrombotic vessel occlusions within 30 min follov;ing electrical current n 510
‘i?
P
f-l-
d 0,001 < 0.001
n. s.
5 min prior to the vessel wall lesion
++ Significance
calculated by the X2-test
According to these data, a single intravenous injection of IO mg/kg APPA prevented the formation of a clotting thrombus, produced by the injection of serum plus stasis in.an isolated vessel segment. The same dose was sufficient to prevent the formation of a deposition thrombus in an arterial segment of rats, produced by vessel wall lesion by means of electrical current. 6, The effect on disseminated intravascularcoagulation
Of special interest mas the effect of the synthetic inhibitors on disseminated intravascularcoagulation or consumption coagulopathy, as it is observed in the generalizedSanarelli-Shwartzman phenomenon. The phenomenon was produced by the intravenous injection of endotoxin in rabbits and followed by changes in the fibrinogen levels and number of circulatingplatelets. After terminati_on of the experiments,histologic examinationsmere performed to assess the degree of microthrombosis.The results listed in Table 4 indicate that the drop in fibrinogen levels and platelet counts, as seen in the control animals, could be prevented-by the simultaneou-u administration of AlEPA.This pro-
tective effect of APPA was also seen in a reduction of the number of microthrombi
in the organs investigated.
250
ANTITHROMBOTIC
EFFECT
Vol.l,No.3
'PABIdS4
The 3ffect of APPA on the tindotoxinInduced Coagulation Changes in Rabbits Xu$er
Infusion
animals n 7
7
Crop in Number of Drop in platelet count fibrinogen levels glomeruli containing in %+ in %+ microthrombi in %
endotoxin
75 w&dh
67 + 17
36 2 12
71 2 25
2.52 12
17 2 12
22 +,11
(p< O.ool)++
(p( O.l)++
(p( o.ool)++
5 w-sA=Uk@;
i.v. initially, 2 mg APPA/kg/h continuously endotoxin 75 IJ.@;/kdh
+ 6 hours after the start of the infusion ++ Significance calculated by the t-test EISCUSSION The present studies have demonstrated that intravascular thrombus formation could be prevented by the infusion of a synthetic thrombin inhibitor. These findings are of fundamental importance since so far no anticoagulantwith a similar mode of action in vivo has been studied. The differences to the known anticoagulantsare apparent since the protease inhibitors are synthetic, low molecular weight compounds aith a direct action which can be administered orally. Already at this time, the inhibitor tested can serve as a useful pharmacologicalmodel for compounds of this nature and allows an estimate of the potential therapeutic effect of this class of 1 dru?!~~vie~ring these results, it also must be kept in mind that the tested inhibitor not only has an effect on thrombin, but alSO on try-psin(13) and other serum proteases of the blood with similar specificity as trypsin, such as plasmin and kallikrein (7, 14). The effects could play a major role in the treatment of consumption coagulopathy in which the fibrinolytic system and kinin forming system are also activated.
ANTITHROMBOTIC
Vol.l,No.3
1.
2.
3.
4.
5.
6.
7.
8.
9. 70.
11.
12. 13.
14.
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