Study on the Effect of Streptokinase-Activated Plasmin (Fibrinolysin) on Clots in Various Stages of Organization Nathan Back, … , C. Lenore Simpson, Sidney Shulman J Clin Invest. 1958;37(6):864-871. https://doi.org/10.1172/JCI103677. Research Article Find the latest version: https://jci.me/103677/pdf STUDY ON THE EFFECT OF STREPTOKINASE-ACTIVATED PLASMIN (FIBRINOLYSIN) ON CLOTS IN VARIOUS STAGES OF ORGANIZATION 1.2 By NATHAN BACK, JULIAN L. AMBRUS, C. LENORE SIMPSON, AND SIDNEY SHULMAN (F1roni. The Roswell Park Memorial Institute and the Departmnents of Pharmnacology, Bacteri- ology and Iimmunology, and Pathology, University of Buffalo Medical School, Buffalo, N. Y.) (Submitted for publication July 25, 1956; accepted February 20, 1958) In previous studies (1, 4), a method was de- disappearance of radioactivity and by histopatho- veloped for the quantitative recording of the lysis logic study. of thrombi and emboli produced with I'31-labeled MATERIALS AND METHODS fibrinogen. Using this method, the in vivo fibri- nolytic activity of human and bovine plasmin was Materials and methods were similar to those de- demonstrated (2). Other proteolytic enzymes scribed in previous studies (1). Under aseptic condi- tions, I3"-labeled purified bovine fibrinogen was intro- (crude pancreatic protease, trypsin, carboxypep- duced through side branches into isolated segments of tidase, papain and ficin) were ineffective in maxi- veins of dogs and clotted with the aid of thrombin. mal tolerated doses (1, 3, 4). Literature on the Semi-constricting aluminum wire ligatures were placed therapeutic use of proteolytic enzymes has been proximal to the clots and artery clamps isolating the segment were released one-half hour after clot formation. reviewed (1, 2). In all previous experiments, Radioactivity over the area was recorded with the aid treatment was started one-half hour after forming of a specially constructed lead shield, a scintillation labeled clots in the experimental animals. The counter, an analytical radiation rate meter, and an Ester- question arose whether plasmin treatment would line recorder. be effective in dissolving organized clots and Mongrel dogs, 10 to 16 Kg. in body weight, were used. At the time of the beginning of treatment, the clots varied whether there is a correlation between degree of in age from 30 minutes to 10 days. In most dogs two organization and plasmin-induced lysis. The clots of the same age were present; one was removed preparations referred to as "plasmin" in this study before institution of plasmin treatment. All other clots actually contain a variety of enzymatic activities; were removed after the last treatment. Sections from at they are fibrinolytic, proteolytic and are able to least two areas of each clot were examined histologically. Five daily intravenous doses of human plasmin, 30 activate the fibrinolysin system. Presumably they plasmin units (Loomis) per Kg.,3 were given. One mg. represent a mixture of plasmin, plasmin-activator of the plasmin preparation used was found to contain 3 and free streptokinase. It is difficult to ascribe fibrinolytic plasmin units (Loomis) or 0.098 proteolytic casein unit (6). The specific activity was 0.28 casein therapeutic activity to any one of these compo- unit per mg. protein. This dose was previously found nents. The purpose of these experiments is to (2) to cause lysis of fresh clots but not to alter fibrino- study therapeutic indications and conditions for gen and prothrombin levels. In most previous studies the dose was administered in four hour infusions; in the the preparations currently undergoing clinical present experiments, rapid intravenous inj ections were trial. Labeled clots were produced in several veins given. In the previous studies, human plasmin was pre- of dogs at various time intervals. At the time of pared from Cohn's Fraction III according to the method these clots were of various ages. Dif- of Kline (7). In the present study a simple streptokinase- treatment, activated acid extract of Fraction III was used, prepared ferences in lysis were established by recording by Dr. Werner Baumgarten, Sharp and Dohme Division of Merck and Co., West Point, Pa. The following in- 1 Preliminary report presented before the Federation formation was made available on the method of prepara- of American Societies of Experimental Biology, Atlantic tion of this material. Fraction III, prepared by the City, 1956 (Fed. Proc. 1956, 15, 396). 2 Supported by grants from The American Heart As- 3A plasmin unit (Loomis) is defined as the quantity sociation, Parke Davis and Co., The Sharp and Dohme of enzyme needed to dissolve 0.6 ml. of a 0.3 per cent Division of Merck and Co., and the United States Public purified bovine fibrin clot at 450 C. in two minutes at pH Health Service. 7.2 (5). 864 EFFECT OF PLASMIN ON CLOTS OF VARIOUS AGES 865 alcohol fractionation method of Cohn, was extracted with Blood samples were taken before, and at various time acid, adjusted to pH 7.8, and to each equivalent of 40 mg. intervals after, plasmin injections for hematological and of Fraction III, 13,000 Christensen units of streptokinase biochemical analyses as in previous studies (14). Pro- was added (U. S. Patent 2,666,729). We found that when thrombin time is reported as clotting index: the ratio of lyophilized samples of the above material were diluted the prothrombin time prior to treatment to the prothrom- with increasing volumes of physiological saline, fibrino- bin time during and after plasmin administration. In lytic activity first increased, then decreased. Such bell- most experiments as in previous studies (1-4) prothrom- shaped dilution versus activity curves could be duplicated bin time was also determined after adding bovine fibrino- by adding excessive amounts of streptokinase to plasmino- gen, so as to eliminate the effect of fibrinogenopenia on gen prepared by the Kline method. This was interpreted prothrombin time determinations. as indicating that the preparation used in this study con- tained a considerable amount of free streptokinase. It RESULTS is known that most batches of streptokinase are poor activators of dog plasminogen. On the other hand Sherry, Clot lysis Titchener, Gottesman, Wasserman, and Troll have shown Figure 1 shows an experiment in which a dog (8) that streptokinase-activated human plasmin prepara- with one-half hour, one, two and three day old tions may activate dog plasminogen. It is presently a matter of controversy whether this effect of streptokinase- clots was treated with daily injections of 30 plas- activated human plasmin is due to the presence of "acti- min units (Loomis) per Kg. of human plasmin. vator" (9), or to the activating ability of a hypothetical The first point on the graph indicates radioactivity plasminogen-streptokinase complex (10, 11), or a plasmin- over the thrombus at the time of clot formation; streptokinase complex (4, 11). the second represents radioactivity immediately FIG. 1. EFFECT OF PLASMIN ON ONE-HALF HOUR, ONE, TWO AND THREE DAY OLD CLOTS, CLOTTING INDEX, FIBRINOGEN LEVEL AND WHITE BLOOD CELL COUNT 866 N. BACK, J. L. AMBRUS, C. L. SIMPSON, AND S. SHULMAN FIG. 2. EFFECT OF PLASMIN ON ONE-HALF HOUR AND 10 DAY OLD CLOTS, CLOT- TING INDEX, FIBRINOGEN LEVEL AND WHITE BLOOD CELL COUNT before initiation of treatment. It can be seen that three day clot. It should be remembered, how- 1131-labeled clots up to the age of two days com- ever, that statistical analysis of lysis of all three day pletely disappeared during the five day treatment old clots versus control clots revealed no significant period. Only slight lysis was evidenced in the difference. Figure 2 illustrates an experiment in which a dog with two 10 day old clots and one-half TABLE I hour old clot was treated. Radioactivity over the Effect of clot age on lysis half-hour old clot disappeared as in the previous experiment, but the 10 day old clots showed no Mean of % lysis to clots in untreated and standard error significant lysis compared Age of No. of Treatment of mean at end of with five old clots were clots clots of dogs treatment period dogs (2). Results day identical with those of 10 day old clots. Fresh i hr. 23 saline 23.54 ± 3.17 was before each (control) 1131-labeled fibrinogen prepared series of experiments. Thus, clots of various ages 10 days 4* 30 plasmin 23.72 3.75 5 days 4 units (Loomis) 22.43 ± 2.63 produced in the same dog had various starting 3 days 10 per Kg. body 20.49 3.55 levels of radioactivity. No relationship was found 2 days 6 weight 80.17 4.26 1 day 6 80.53 4.79 between base radioactivity levels and degree of 2hr. 6t 97.42 1.10 lysis. I summarizes the results of all clots in- * Eight additional clots gave similar results but are not Table included since somewhat different methods were used. vestigated in this study. It appears that under the t Three additional clots gave similar results after treat- ment with more purified plasmin preparations. conditions of these experiments and at the dose EFFECT OF PLASMIN ON CLOTS OF VARIOUS AGES 867 k * . A X FIG. 3. THREE DAY OLD CLOT WITH FNDOTHELIAL COVERING OVER THE ENDS (HEMATOXYLIN EosIN 420 X) level used, clots three days of age or older can not cells gradually disappeared. Van Giesen stain effectively be dissolved with human plasmin. revealed no collagen formation before the tenth day. Histopathologic findings It was not possible to distinguish the clots which had received plasmin therapy after three or more The histological findings were not uniform days of existence from the control clots of similar throughout the length of a clot. Despite this it age. Where treatment was given earlier, the clot was possible to grade the clots histologically and appeared to be less advanced than expected at the it was found that the histological picture correlated time of removal.