Fibrinolytic activity of an isolated equimolar human plasmin-streptokinase activator complex in an experimental dog model

MICROVASCULAR

RESEARCH

Fibrinolytic

6,114-l 19 (1974)

Activity

of an Isolated

Plasmin-Streptokinase Experimental

Equimolar

Activator

Complex

Human in an

Dog Model1

KENNETH C. ROBBINS,LOUIS SUMMARIA,LILA FRIEDMAN, AND

S. FREDERICK RABINER~ Blood Center, and Division of Hemutology, Department of Medicine, Michael Reese Hospital and Medical Center, Chicago, Illiliois 60616 and Department of Medicine, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois 60637 Received Jane I,1973

Theisolatedequimolarhumanplasmin-streptokinaseactivatorcomplexwasfound to be an excellent fibrinolytic agent in an experimental dog model. It will dissolve artificially produced human clots placed in the femoral vein of the animals. There appears to be a critical dose level for fibrinolytic activity which may be approxia mately 0.1 mg per kg body weight. At the critical dose level, the activator complex appeared to be more effective than equivalent individual doses of either streptokinase or human plasmin.

Various

types of thrombolytic

enzyme preparations

and activators

of the fibrinolytic

system have been used experimentally to dissolve artificially produced clots in the dog (Backet al., 1956; Ambrus etal., 1957; Back et al., 1958 ; Frieman etal., 1960; Roschlau and Painter, 1962; Tsapogas and Flute, 1964). The preparations studied include streptokinase- and urokinase-activated human plasmin (Back et al., 1956; Ambrus et al., 1957 ; Back et al., 1958 ; Roschlau and Painter, 1962), streptokinase (Ambrus et al., 1957; Tsapogas and Flute, 1964), urokinase (Tsapogas and Flute, 1964) and mixtures of human plasmin and streptokinase (Freiman et al., 1960). Our recently completed work on the isolation of highly purified bovine plasminogen activator, an equimolar humanplasmin-streptokinase complex(Ling, Summaria, and Robbins, 1967), suggested a study of the effect of this activator complex on the dissolution of artificially produced clots in the dog. MATERIALS

AND METHODS

Equimolar human plasmin-streptokinase activator complex. The equimolar human plasmin-streptokinase activator complex3 was prepared by methods previously described (Ling, Summaria, and Robbins, 1967) from highly purified human plasminogen and crude streptokinase (Varidase, Lederle Laboratories). It was dissolved in 0.1 M l This investigation was supported in part by Grant HL-04366 from The National Heart and Lung Institute, United States Public Health Service. z Present address: Department of Medicine, Good Samaritan Hospital and Medical Center, and the University of Oregon Medical School, Portland, Oregon. 3 Abbreviations used: equimolar human plasmin-streptokina activator complex, activator; &-aminocaproic acid, s-ACA; streptokinase, SK. 114 Copyright 0 1974 by Academic Press, Inc. Allrights of reproduction in any form reserved. Printed in Great Britain

EQUIMOLARPLASMIN-SKACTIVATORCOMPLEX

115

s-aminocaproic acid3-0. 1 A4NaCl, pH 7.1, at a concentration of 4-8 mg per ml and was stored in the frozen state. Each milligram of activator contains approximately 0.61 mg of human plasmin and 0.39 mg of streptokinase3 calculated from the molecular weights of the two components of the activator, human plasmin and SK, which are 75,400(Barlow, Summaria, and Robbins, 1969) and 47,600 (DeRenzo et al., 1967), respectively. The specific proteolytic activity of the activator varied between 4.2 and 7.9 caseinunits per mg of protein and the specific bovine plasminogen activator activity of the activator varied between 393 and 542 activator units per mg of protein (Ling, Summaria, and Robbins, 1967). Human plasmin. Human plasmin was prepared by methods previously described (Robbins et al., 1965).The specific proteolytic activity varied between 18 and 24 casein units per mg of protein. The enyzme was diluted from concentrated solutions, 20-30 mg of protein per ml, to a final concentration of 0.4 mg per ml in the s-ACA-NaCI, pH 7.1, buffer before use. We are assuming the specific activity of pure plasmin to be approximately 30 casein units per mg of protein (highest specific activity obtained in our laboratory). Streptokinase. The SK used was a commercial preparation, Varidase; the specific activity was approximately 5000 units per mg. It was dissolved in the e-ACA-NaCl, pH 7.1, buffer before use. Since pure SK is assumedto have a specific activity of approximately 100,000units per mg of protein (DeRenzo et al., 1967),the Varidase preparation contained 5 % SK. 13’1-Labeled human$brinogen. Human fibrinogen (Kabi, and Fraction I, American National Red Cross), containingplasminogen, was labeled with 1311according to methods previously described (Ambrus et al., 1956) and stored in the frozen state. Bovine thrombin. Bovine thrombin was prepared by methods previously described (Rabiner and Robbins, 1962)from bovine topical thrombin (Parke, Davis & Co.). Heparin. The heparin used was a commercial preparation (Organon). In vivo clot Zysis. The method used in this study had been previously described (Ambrus et al., 1956). Mongrel dogs, 15-20 kg, were anesthetized, the femoral vein exposed, and a 1311-labeledclot (1.O ml fibrinogen and 0.1 ml thrombin mixed in a syringe and injected) placed in situ with semiconstricting ligatures. The animals were then given 2-3 mg per kg of heparin in a single intravenous injection. The activator (or other agents) was injected into the other femoral vein. The initial dose was usually given within 1 hr after the clot was placed and stabilized (the animal was discarded if there was a continuous fall in radioactivity). The second dose was usually given 45 min to 1hr later. Additional dosesweregiven in a similar manner. The fall in radioactivity at the clot site was continuously monitored with a special columnated probe attached to a Packard Auto-Gamma SpectrometerNo. 410A. The counts varied between 12,000 and 162,000cpm with a mean value of 66,000 cpm.The final measurement of radioactivity of the clot was determined 1 hr after the last dose of activator was given. Control experiments were carried out under the sameconditions, asfollows : (a) without activator and (b) with equivalent amounts of human plasmin and SK given separately, within l-2 min, plasmin being given first. The percentage lysis was calculated from the counts obtained at 1 min before injection of the activator complex, or plasmin and streptokinase components, and from the final counts obtained 1 hr after the last dose (2nd or 3rd) was given.

116

ROBBINSET AL.

RESULTS Activator and individualplasmin and streptokinase components at doses of 0.10 mgper kg body weight. A group of 21 animals were used in these experiments. Nine dogs were injected with three doses, and two dogs were injected with two doses, of 0.10 mg activator per kg of body weight. Eight dogs were injected with three doses of the equivalent amount of human plasmin (0.061 mg per kg body weight, calculated as pure plasmin) and SK (0.039 mg per kg body weight) as individual components. Two control animals were prepared in which the clots were monitored for a total period of 4 hr. The results of these experiments are recorded in Table 1, Expt A. The percentage of lysis TABLE 1 THE F~BRINOLYTIC ACTNITYOFAN EQUIMOLAR HUMANPLASMIN-STREPTOKINASE ACTRJATOR COMPLEX IN THEDoG

Experiment no.

A

B

C

D

Preparation 1 Control 2 Activator 3 Human plasmin streptokinase 1 Activator 2 Human plasmin streptokinase 3 Activator 4 Human plasmin streptokinase 5 Activator 1 Activator 2 Human plasmin streptokinase 3 Streptokinase 4 Human plasmin 1 Human plasmin

aOne dog-two

mg/kg

+

+

+

+

Animals (no.)

Doses (no.)

% Lysis O-20 20-40 40-60 60-80

80-100

-

2

0

0

0

0.100 0.061 0.039 0 050 0.030 0.020 0.025 0.015 0.010 0.013 0.300 0.183 0.117 0.117 0.183 0.20-4.0

11 8

3 3

1 2

3” 3

2' 2

1

4

0

1

3 3

2 2

0 0

1 1

2 2

0 0

0 0

2 2

1 1

1 1

1 1

0 0

0 0

0 0

2 2

12

-20

0

0

0

0

1 1

0 0

1 0

1 0

0 0

0 1

1 1

0 1

0 0

0

0

1

20

3

9

6

0 3

0 2

2 0 0

1

2

doses.

obtained was divided into five categories (O-20 %, 20-40 %, 40-60 %, 60-80 %, 80-100 %) as shown in the Table. These experiments indicate that the activator appears to be a more effective fibrinolytic agent than the individual components at the concentrations tested. It is important to note that the fibrinolytic effect of a single dose of the activator at this concentration diminishes rapidly and disappears within 1 hr. In most of the dogs the fibrinolytic effect was greatest with the first and second doses. The effectiveness of the third dose varied; This may have been due to changes in the clot itself making it less susceptible to lysis (Rabiner and Robbins, 1962). Activator and individual plasmin and streptokinase components at doses Less than 0.10 mg per kg body weight. A group of 12 animals were used in these experiments.

EQUIMOLAR

PLASMIN-SK

ACTIVATOR

COMPLEX

117

Three dogs were injected with two doses of 0.05 mg activator per kg of body weight and three dogs were injected with two dosesof the equivalent amounts of human plasmin and SK. Two dogs were injected with a single dose of 0.025 mg activator per kg of body weight and two dogs were injected with a single dose of the equivalent amounts of human plasmin and SK. Two dogs were injected with a single dose of 0.0125mg activator per kg body weight. The results of these experiments are recorded in Table 1, Expt B. These experiments show that there was little fibrinolytic activity and very little difference between the fibrinolytic effect of the activator and the individual components given at doseslower than 0.10 mg per kg body weight. We believe that the fibrinolytic effect seenwith the individual components is obtained with SK and not with plasmin. Activator and individualplasmin and streptokinase components at doses Greater than 0.10 mg per kg body weight. A group of six animals were used in these experiments.

Two dogs were injected with a single dose of 0.30 mg of activator per kg body weight and one dog wasinjected with a single doseof the equivalent amounts of human plasmin (0.181 mg) and SK (0.117 mg). Two dogs were injected with a single dose of 0.117 mg SK per kg body weight and one dog was injected with a single dose of 0. I8 1 mg of human plasmin per kg of body weight. The results of these experiments are recorded in Table I, Expt C. It is rather obvious that a single dose of SK at three times the dose given in Expt 1A is an excellent fibrinolytic agent whether alone, or with human plasmin, whereasthe plasmin alone has little fibrinolytic effect. The high SK dosemay be necessary to activate sufficient plasminogen, circulating or clot, to produce the fibrinolytic effect. The high dose of activator was not fibrinolytically effective in the two animals studied. There appears to be a critical dose for the fibrinolytic effect of the activator. In these experiments (Table 1, Expts A, B, and C), the critical effective dose was approximately 0.10 mg per kg body weight. Human plasmin. Human plasmin was injected into a group of 20 animals (nonheparinized) at concentrations of 0.2-4.0 mg per kg of body weight, in three doses.The results of theseexperiments are recorded in Table 1, Expt D. Since the human plasmin dose was not found to be related to fibrinolytic effect, the entire group was tabulated as shown in the table. It appearsthat human plasmin, in massive doses,is not as effective a fibrinolytic agent as the activator given in small doses. DISCUSSION The isolated equimolar human plasmin-streptokinase activator complex will dissolve artificially produced human clots in the experimental dog model (Table 1). There appears to be a critical dosefor fibrinolytic activity. The fibrinolytic effect of the activator, particularly at a critical doselevel of 0.10 mg per kg body weight, is apparently due to the activator itself sinceequivalent amounts of human plasmin and streptokinase given separately did not produce a comparable fibrinolytic effect. It had beenpreviously reported that, in vitro, dog plasminogen is not readily activated by low concentrations of SK, but is readily activated by high concentrations of SK (Wulf and Mertz, 1969) which may in part be due to inhibitors and antibodies to SK usually found in dogs (Cliffton and Mootse, 1967). But, dog plasminogen is apparently readily activated, in vitro, by human plasmin-SK mixtures containing the sameamount of SK that will not activate the plasminogen directly (Wulf and Mertz, 1969).The excellent fibrinolytic

118

ROBBINS ET AL..

effect of SK alone in our animal experiments is probably due to the excessively high concentrations of this activator used. In Expt IA, a significant difference in clot lysis was found between activator (0.1 mg per kg of body weight) and the individual components, plasmin and SK, when the percentage of lysis categories were combined into two groups, O-60% and 60-100% In Expt lB, using the sametwo groups of categories,it is obvious that there is no differencebetween lower dosesof activator and the individual components. But, in Expt lC, the number of animals used were too few to make any real judgment on significant differences except with the two animals injected with high dosesof SK which showed SO-100% lysis. In Expt lD, little lysis is produced by plasmin when compared to the activator (Table 1, Expt A) in the combined O-60‘A and 60-100 ‘A lysis categories. In these animal experiments, the question as to whether circulating dog plasminogen or clot human plasminogen, or both plasminogens, are activated by the activator has not been answered. The human activator was found to be a much better activator of bovine plasminogen than the dog activator (equimolar dog plasmin-SK complex), in vitro4. The mechanismsof lysis of artificially produced human clots in the dog by the activator and by SK could be different (Table 1, Expt B) even though higher dosesof SK did produce an excellent fibrinolytic effect (Table 1, Expt C). In a similar animal model in which clots were produced in the jugular vein by electrical means,the activator was found to be an excellent fibrinolytic agent and more effective than the individual components5 Since circulating SK inhibitor and antibody levels were not determined in our experiments, it is also possible that the differencesin fibrinolytic effectsbetween activator and SK may be due to these substances.Studies of levels of circulating plasminogen, plasmin, and activator could also be critical in evaluating the experiments. The question of spontaneous lysis is difficult to answer and may be important in evaluating these, and future, experiments. These preliminary observations in an experimental dog model will require more extensive and detailed experiments, as described above, to validate the conclusions drawn. This animal model may be useful in the development of the activator as a possible therapeutic agent in human subjects. In the animal model, the activator is fibrinolytically effective at low doses,produces a rapid response, and loses its activity very quickly thus preventing extensive activation of circulating plasminogen. REFERENCES M., BACK, N., SOKAL, J. E., AND COLLINS, G. L. (1957). Clinical and experimental studies on fibrinolytic enzymes. Ann. N. Y. Acud. Sci. 68,97-135. AMBRUS, J. L., BACK, N., MIHALYI, E., AND AMBRUS, C. M. (1956). Quantitative method for the in-vivo testing of fibrinolytic agents; effect of intravenous trypsin on radioactive thrombi and emboli. Circ. Res. 4,430-439. BACK, N., AMBRUS, J. L., GOLDSTEIN, S., AND HARRISSON, J. W. E. (1956). In vivo fibrinolytic activity and pharmacology of various plasmin (fibrinolysin) preparations. Circ. Res. 4,440443. BACK, N., AMBRUS, 3. L., SIMPSON, C. L., AND SHULMAN, S. (1958). Study on the effect of streptokinaseactivated plasmin (fibrinolysin) on clots in various stages of organization. J. Clin. Znoest.37,864871 BARLOW, G. H., SUMMARIA, L., AND ROBBINS, K. C. (1969). Molecular weight studies on human plasminogen and plasmin at the microgram level. J. Biol. Chem. 244,1138-l 141. AMBRUS,

J. L., AMBRUS,

C.

4 L. Summaria and K. C Robbins, unpublished results. 5 G. H. Barlow, D. Martin, L. Summaria, and K. C. Robbins, unpublished results.

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CLIFFTON,E. E., AND MOOTSE,G. (1967). Species specificity in fibrinolytic activity. Thromb. Diath. Haemorrh. 18,291-293. DERENZO,E. C., SIITERI,P. K., HUTCHINGS,B. L., AND BELL, P. H. (1967). Preparation and certain properties of streptokinase. J. Biol. Chem. 242,533-542. FREIMAN,A.H.,BANG,N. U.,G~0~~1,C.E.,~~~C~1~~0~,E.E.(1960).Factorsaffectingtheformation and dissolution of experimental thrombi. Amer. J. Cardiol. 6,426-429. LING, C.-M., SUMMARIA,L., AND ROBBINS,K. C. (1967). Isolation and characterization of bovine plasminogen activator from a human plaminogen-streptokinase mixture. J. Biol. Chem. 242, 14191425. RABINER,S. F., AND ROBBINS,K. C. (1962). Studies on Fibrinolysis: I. A circulating substance capable of converting plasmin-sensitive to plamin-resistant clots. Proc. Sot. Exp. Biol. Med. 111,701-706. ROBBINS,K. C., SUMMARIA, L., ELWYN, D., AND BARLOW,G. H. (1965). Further studies on the purification and characterization of human plasminogen and plasmin. J. Biol. Chem. 240,541-550. ROSCHLAU, W. H. E., AND PAINTER,R. H. (1962). Human fibrinolysin with and without urokinase: Its effect on experimental arterial thrombi in dogs. Can. J. Biochem. Physiol. 40,1819-1838. TSAPOGAS, M. J., AND FLUTE, P. T. (1964). Experimental thrombolysis with streptokinase and urokinase. Brit. Med. Bull. 20,223-227. WULF, R. J., AND MERTZ, E. T. (1969). Studies on plasminogen VIII. Species specificity of streptokinase. Can. J. Bioehem. 47,927-93 1.