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Serum inhibition of plasmin and of plasminogen activation in thrombotic diseases
Journal of 24therosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
SERUM I N H I B I T I O N OF PLASMIN A N D OF P L A S M I N O G E N A C T I V A T I O N IN T H R O M B O T I C D I S E A S E S
G. TSITOURIS, R. STATHOPOULOU AND I. TSEVRENIS Department of Cardiology, Second Department of Internal Medicine, University of 24thens Medical School and The Blood Bank of Hippokration General Hospital, Athens (Greece)
(Received March 7th, 1967)
SUMMARY The serum of five patients with acute myocardial infarction, nine patients with angina and recent electrocardiographic changes of myocardial ischemia and four patients with acute cerebral thrombosis was tested for antiplasmin (inhibitory) activity. In four patients with myocardial infarction, six patients with myocardial ischemia and three with cerebral thrombosis, a significantly higher than the normal inhibition of the plasmin activity was noted. In the remaining five cases, the inhibitory activity was within the normal range. The serum of ten patients with acute myocardial infarction was studied for inhibitory activity of the plasminogen activation b y streptokinase. Most of the patients were found to have increased inhibitory activity of the plasminogen activation. Our d a t a indicate that in the serum of a number of patients with t h r o m b o t i c diseases, there are factors inhibiting the fibrin clot lysis. Although similar changes were not seen in all the studied cases and particularly those with myocardial ischemia, more extensive studies might disclose that changes in the plasmin-plasminogen enzyme system play an important role in the production of these pathological conditions.
INTRODUCTION The reintroduction of the thrombogenetic theory of atherosclerosis b y DUGUID4 in 1946 and the recent appearance of purified proteolytic and fibrinolytic enzymes has greatly stimulated the interest of the investigators in the fibrinolytic phenomena. G. TSlTOURIS:Associate Professor of Cardiology, University of Athens Medical School. R, STATHOPOULOU: Assistant, Department of Medicine, University of Athens Medical School. I. TSEVRENIS: Associate Professor of Medicine, University of Athens Medical School, Director of Blood Bank, Hippokration General Hospital. j . Atheroscler. Res., 7 (1967) 425-433
426
G. TSITOURIS, R. STATHOPOULOU, I. TSEVRENIS
Some authors 1,5,1s have suggested that impaired fibrinolytic mechanism m a y underlie the pathogenesis of atherosclerosis. This concept has been based on ASTRUP'S assumption t h a t imbalance of a potential equilibrium between intravascular clotting and fibrinolysis could lead to mural thrombosis through a decreased fibrinolytic activity. Recently several authors2,9,1~,lT, 22 have reported a decreased fibrinolytic activity in the blood of patients with thrombo-embolic diseases and a high content of components inhibiting either plasmin or the activation of plasminogen b y streptokinase and urokinase. Nevertheless, the results were not in all instances conclusive6,11 and some investigators have been unable to find a b n o r m a l fibrinolytic activity in similar cases. The aim of the present work has been to investigate the plasmin inhibitors and the inhibitors of the activation of plasminogen b y streptokinase in the blood of patients with acute thrombotic episodes. MATERIAL AND METHODS
Material Anticoagulant. Double oxalate mixture of W i n t h r o p was used as naticoagulant. Buffer. Tris buffer, p H 7.4, ionic strengh 0.15; 24.2 g o t Tris were dissolved in 1000 ml H20, 25 ml of this solution + 17.0 ml 0.2 N HC1 was made up to 100 ml with H20. Fibrinogen. H u m a n fibrinogen ("Kabi") with a coagulability of 95 ~ (kindly supplied b y Kabi Ltd., Stockholm) was used in a concentration of 0.1 ~ in Tris buffer. The solution was stored at - - 2 0 ~. Streptokinase. "Varidase" (kindly supplied b y Lederle Laboratories, New York, U.S.A.) containing 20,000 units of streptokinase and 5000 units of streptodornase per vial. The material was dissolved and diluted to various concentrations (50, 25, 10 U / m l 0.9 ~ NaC1). Fresh solutions were prepared prior to the experiment. e-Aminocaproic acid, in powder (kindly supplied b y Lederle Laboratories, New York, U.S.A.), was m a d e up as a 0 . 1 % solution in 0.9 % NaC1. Thrombin. Bovine (Parke, Davis) containing 5000 units per vial. Two solutions were prepared. One containing 100 U/ml in Tris buffer a n d another containing 10 U in 0.9 ~o NaC1. Fresh solutions were prepared every week and stored at --20~ in concentration of 1000 U/ml. Human plasmin. "Actase" containing 1,000,000 factory units per vial (kindly supplied b y Ortho Pharmaceutical Corporation, Raritan, U.S.A.) was dissolved to a concentration of 50 U/ml in Tris buffer. Fresh solution was p r e p a r e d prior to experiment.
Subjects Twenty-eight patients from the wards of the Cardiology Department and of the Department of Internal Medicine of the Hippokration University Hospital, ranging in age from 42 to 74 years, formed two groups which were utilized as s t u d y groups. The t i m e from the onset of the episode did not exceed 4 days. G r o u p 1 consisted of 5
j. Atheroscler. Res., 7 (1967) 425-433
SERUM INHIBITION OF PLASMIN IN THROMBOTIC DISEASES
427
p a t i e n t s with acute m y o c a r d i a l infarction, 9 patients with angina pectoris a n d recent ECG changes of m y o c a r d i a l ischemia, a n d 4 patients with acute cerebral thrombosis. This group was studied for serum inhibition of plasmin activity. Group 2, consisting of 10 p a t i e n t s with acute m y o c a r d i a l infarction, was s t u d i e d for serum i n h i b i t i o n of the a c t i v a t i o n of plasminogen b y streptokinase. T w e n t y h e a l t h y individuals, ranging in age from 25 to 62 years, were selected from the non-professional donors of the Blood B a n k of the same hospital, a n d a pooled s t a n d a r d serum s a m p l e d e r i v e d from these subjects was used as the mean n o r m a l value. In order to h a v e the range of the n o r m a l vahles similar d e t e r m i n a t i o n s of serum inhibitions were also carried out in 40 n o r m a l individuals aged from 25 to 70 years. As controls two solutions were used in which saline 0.9 % and e-aminocaproic acid 0 . 1 % h a d replaced serum. The clot lysis t i m e with 50 U/ml actase (serum inhibition of plasmin a c t i v i t y ) was 743 + 136 sec. The clot lysis time using 50 U, 25 U, and 10 U/ml s t r e p t o k i n a s e (serum inhibition of plasminogen a c t i v a t i o n b y streptokinase) was 201 4- 26 sec, 267 :t: 52 sec, and 762 :J: 142 sec respectively with the serum concentration 100 ~ a n d i 9 2 4- 22 sec, 231 4- 45 sec, a n d 567 ! 131 sec with t h e serum concentration 25 %. Methods Collection o f blood. Blood was collected in o r d i n a r y glass tubes a n d allowed to clot. Two hours later the serum was s e p a r a t e d b y centrifugation at 2000 • g for 10 rain a n d then stored at - - 2 0 ~ until required. The sera of the controls were m i x e d and stored at - - 2 0 ~ in aliquots of 2 ml. S e r u m i n h i b i t i o n o f p l a s m i n activity. A test system was p r e p a r e d consisting of the following components in order of addition: 0.2 ml serum, 0.2 ml " A c t a s e " (50 U/ml), 0.4 ml 10//o fibrinogen, a n d 0.2 ml t h r o m b i n (100 U/ml). The tubes were placed in a
TABLE 1 I N H I B I T I O N OF F I B R I N O L Y T I C A C T I V I T Y I N BIORMAL CONTROL A N D I N P A T I E N T S W I T H A C U T E M Y O C A R D I A L I N F A R C T I O N , M Y O C A R D I A L I S C H E M I A , AND C E R E B R A L T H R O M B O S I S ( E X P R E S S E D AS A N T I P L A S M I N L Y S I S T I M E S I N SEC A G A I N S T 5 0 U A C T A S E / M L )
.Normal controls a
Patients with acute myocardial infarction
Patients with recent changes of myocardial ischemia
Patients with acute cerebral thrombosis
720 750 780 790 690 750 750 750 870
1082 1245 870 1042 1290
1310 900 1020 720 1275 1310 1215 1215 760 960
1600 1105
1410 660
Reproducibility of the determination of the lysis time with the same pooled serum sample from twenty healthy subjects. J. Atheroscler. Res., 7 (1967) 425-433
428
G. TSITOURIS, R. STATHOPOULOU, I. TSEVRENIS
37 ~ w a t e r b a t h a n d t h e lysis t i m e d e t e r m i n e d b y i n v e r t i n g t h e t u b e s at r e g u l a r i n t e r v a l s u n t i l t h e clot failed to cling to t h e b o t t o m of t h e tube. T h e t i m e r e q u i r e d to lyse t h e fibrin clot was considered to b e a m e a s u r e of t h e i n h i b i t o r y a c i t v i t y of t h e t e s t s e r u m . T h e t i m e was recorded from t h e a d d i t i o n of t h r o m b i n . Serum inhibition of plasminogen activation by streptokinase. D e t e r m i n a t i o n of s e r u m i n h i b i t i o n was carried o u t a c c o r d i n g to t h e t e c h n i q u e of NILSSON et al. 18. T h e t e s t s y s t e m c o n s i s t e d of t h e following c o m p o n e n t s in o r d e r of a d d i t i o n : 0.5 m l serum, 0.2 m l 1 % fibrinogen, 0.1 m l s t r e p t o k i n a s e , a n d 0.2 m l t h r o m b i n (10 U/ml). T h e t u b e s were i n c u b a t e d at 37 ~ a n d c h e c k e d for lysis in t h e m a n n e r d e s c r i b e d a b o v e . T h e c l o t lysis t i m e was recorded from t h e m o m e n t t h e s t r e p t o k i n a s e was a d d e d . A s s a y s w e r e c o n d u c t e d w i t h two c o n c e n t r a t i o n s of s e r u m (100 % a n d 25 % d i l u t i o n in 0.9 % NaC1) a n d t h r e e d i l u t i o n s of s t r e p t o k i n a s e (50, 25, 10 U s k / m l 0 . 9 % NaC1). A s s a y s c o n d u c t e d w i t h a s t r e p t o k i n a s e s o l u t i o n 25 U / m l d e m o n s t r a t e d b e s t t h e difference in i n h i b i t o r y a c t i v i t y . A solution of 0.9 % saline a n d a s o l u t i o n of 0 . 1 % of e - a m i n o c a p r o i c a c i d were u s e d as blanks. RESULTS
Serum inhibition of plasmin activity T a b l e 1 shows t h e lysis t i m e s ( a n t i p l a s m i n a c t i v i t y ) of t h e " A c t a s e " w h e n i t was i n c u b a t e d w i t h t h e serum of t h e n o r m a l c o n t r o l a n d w i t h t h e s e r u m of t h e p a t i e n t s .
140012001000" (~ 800" .~ 600 "~ 4 0 0 o "~ 200-
tll,.ll
IH
Jlnl] r
ACute myocardial infarction
Myocardial ischemia
IllIn
Illll II li Ill ll I Control serum (reproducibility)
Cerebral thrombosis
Fig. 1. Inhibitionoffibfinolyticactivityinnormalsubjects, and in patients with acute myocardial infarction, recent changes of myocardial ischemia, and acute cerebral thrombosis. The antiplasmin lysis times against 50 U/inl actase, are shown by the height of the columns. The solid columns on the left represent repeated determinations (reproducibility) with the same batch of the control serum (pooled serum sample). Each of the empty columns on the right represent a different patient. Normal range is between the solid horizontal lines.
J. Atheroscler. Res., 7 (1967) 425-433
429
SERUM I N H I B I T I O N OF PLASMIN IN THROMBOTIC DISEASES
In 4 of the 5 tested patients with myocardial infarction, and in 6 patients of the 10 with myocardial ischemia, and in 3 of the 4 patients with acute cerebral thrombosis, the clot lysis time was longer than the mean normal value and the normal range. The remaining cases showed values within the normal range and very close to the mean value. The lysis times of nine determinations on the same b a t c h of the control pooled serum were satisfactorily constant. The differences in the lysis time are graphically shown in Fig, 1. The height of the solid columns on the left represents the antiplasmin lysis times of the control pooled serum (mean normal value) and the height of the e m p t y columns on the right the values of the patients. The normal range of the normal values is between the horizontal lines. TABLE 2 R E P R O D U C I B I L I T Y O F T H E D E T E R M I N A T I O N OF T H E I N H I B I T I O N O F P L A S M I N O G E N A C T I V A T I O N B Y S T R E P T O K I N A S E (Sk) I N T H E S E R U M OF NORMAL CONTROL ( P O O L E D S E R U M S A M P L E ) , ( E X P R E S S E D AS A N T I P L A S M I N L Y S I S T I M E S IN S E C )
Serum concentrations 25 %
700 % 50 U
25 V
10 U
aO U
25 U
10 U
sk/ml
sk/ml
sk/ml
sk/ml
sk/ml
sk/ml
240 210 210 210 225 195 195 225
315 345 270 270 330 360 285 300
630 690 750 690 690 675 675 645
150 150 165 210 210 210 180 165
225 255 270 285 300 300 300 255
570 570 570 555 690 660 630 585
TABLE 3
(sk)
I N H I B I T I O N OF P L A S M I N O G E N A C T I V A T I O N B Y S T R E P T O K I N A S E IN N O R M A L C O N T R O L A N D I N P A T I E N T S W I T H A C U T e : M Y O C A R D I A L I N F A R C T I O N ( E X P R E S S E D AS A N T I P L A S M I N L V S I S T I M E S I N S]~C)
Serum concentration too %
Normal control (pooled s e r u m s a m p l e ) Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 P a t i e n t I0
25 %
50 U sk/ml
25U sk/ml
10U sk/ml
50U sk/ml
25 U sk/ml
IO U sk/ml
210 330 255 180 165 210 280 210 225 510 300
300 675 435 465 865 345 750 285 285 555 435
690 960 630 1320 1080 985 1040 900 1350 ~ 60 m i n 500
180 240 240 210 225 170 240 225 205 345 200
255 630 375 300 300 300 330 300 345
570 840 990 845 1710 810 1365 695 675 1290 570
435 230
j . Atheroscler. Res., 7 (1967) 4 2 5 - 4 3 3
430
G. TSITOURIS, R. STATItOPOULOU, I. TSEVRENIS
._E
Serum conce~tr Streptokir
concentrc Fig. 2. I n h i b i t i o n of plasminogen activation b y streptokinase in n o r m a l controls and in patients
with acute myocardial infarction. • mean normal value; Q, patient. Normal range is between the solid horizontal lines.
Serum inhibition of the activation of plasminogen by streptokinase T a b l e 2 shows t h e lysis t i m e s a n d t h e r e p r o d u c i b i l i t y in series of d i l u t i o n s of t h e c o n t r o l s e r u m w h e n it is i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s of s t r e p t o k i n a s e . T e n d e t e r m i n a t i o n s on t h e s a m e b a t c h of t h e c o n t r o l p o o l e d s e r u m show f a i r l y cons t a n t values. T h e v a l u e s of ten p a t i e n t s w i t h a c u t e m y o c a r d i a l i n f a r c t i o n are shown in T a b l e 3 a n d F i g . 2. I t can r e a d i l y be seen t h a t t h e lysis t i m e s in m o s t of t h e s t u d i e d p a t i e n t s were l o n g e r t h a n t h e c o n t r o l p o o l e d s e r u m a n d t h e n o r m a l r a n g e a n d t h e clot lysis r e t a r d e d . I n a few p a t i e n t s , t h e lysis t i m e w a s w i t h i n , or v e r y close, t h e n o r m a l values. T h e difference in t h e values b e t w e e n t h e p a t i e n t s a n d the c o n t r o l was m o s t clear w h e n t h e s e r u m was i n c u b a t e d w i t h 10 U s k / m l . I n t w o cases t h e lysis t i m e s were s h o r t e r t h a n t h e m e a n value. T h e c h a n g e s of t h e lysis t i m e w h i c h were p r o d u c e d b y t h e d i l u t i o n of t h e s e r u m or b y t h e i n c u b a t i o n w i t h different concent r a t i o n s of s t r e p t o k i n a s e d i d n o t show l i n e a r r e l a t i o n s h i p . T h e 1 : 4 (25 %) d i l u t i o n of s e r u m g a v e t h e b e s t c o n c e n t r a t i o n of values. DISCUSSION I t h a s b e e n k n o w n for m o r e t h a n half a c e n t u r y 8 t h a t n o r m a l s e r u m possesses
J. Atheroscler. Res., 7 (1967) 425-433
SERUM INHIBITION
O F P L A S M I N IN T H R O M B O T I C D I S E A S E S
431
factors which inhibit the action of proteolytic enzymes but controversy still surrounds the nature and n u m b e r of components capable of inhibiting plasmin and plasminogen activators. A lot of work dealt with trypsin inhibitors and their possible identity with the plasmin inhibitors. However, SHULMAN19,20 using 1311 labeled fibrinogen as a substrate and JACOBSSON1~ working with electrophoresis found different characteristics for the trypsin and the plasmin inhibitors. RATNOFF et al. 16 in 1954 suggested t h a t there m a y be at least three plasmin inhibitors in the h u m a n serum b u t NORMAN15 using a caseinolytic assay found only two. One rapidly acting and heat stable which migrates as an a2-globulin during starch electrophoresis and a second slowly acting and heat labile migrating as an al-globulin. MflLLERTZ 12 in the meantime discovered t h a t crude bovine globulin inhibited the activation of bovine plasminogen b y h u m a n streptokinase a c t i v a t e d globulin, and he considered it as a strong evidence for the existence of an inhibitor of plasminogen activation. NORMAN14 found t h a t normal serum contains an excess of antiplasmin over the plasminogen capable of being converted into plasmin. The problem of the inhibitors in the disease states is even more controversial. Proteolytic inhibition has been demonstrated in m a n y conditions of tissue necrosis 21. Increased antifibrinolytic activity has been described in patients with pneumonia, cirrhosis of the liver, coronary thrombosis, acute bacterial endocarditis, acute streptococcal infections, as well as in diabetes, cholecystitis, gangrene, arteritis, etc. 10. HUlVIE9 in 1958 found decreased fibrinolysis in patients with acute m y o c a r d i a l infarction and SANDBERG et at. 17 and TSlTOORIS et al. 22 described an a p p a r e n t increased antiplasmin activity in the plasma of patients with acute thrombo-embolic diseases and postoperatively. Similar findings were reported b y others 3 also, b u t there has been no general agreement 11. NILSSON et al. a3 in 1960 described a case of a young patient who had since childhood progressive s y m p t o m s of massive venous t h r o m bosis and it was associated with a high content of components in the serum, inhibiting the activation of plasminogen b y streptokinase and urokinase. GORMSEN6 determined the concentration of these serum inhibitors in patients with acute t h r o m botic episodes and atherosclerosis and after extensive surgery, but his findings were not conclusive. He found high content of inhibitors in some acute t h r o m b o t i c cases and postoperatively b u t no increase in patients with atherosclerosis. Recently CHAKRABARTIet al.Z found an incidence of low fibrinolytic activity of 33 ~ a m o n g patients of occlusive vascular disease, particularly to patients below the age of 60. The d a t a presented in this s t u d y show t h a t fixed concentrations of plasmin when they were incubated with the serum of patients with acute myocardial infarction, recent changes of myocardial ischemia and acute cerebral thrombosis, require in most of the cases longer times to dissolve standard fibrin clots than with the serum of normal control subjects. In addition, the activation of plasminogen b y streptokinase in a m e d i u m containing serum from patients with acute myocardial infarction was not as high as when the medium contained serum of the normal control subjects. I t is then a p p a r e n t t h a t in the serum of these patients there were factors which inhibited j . Atheroscler. Res., 7 (1967) 425-433
432
G. TSITOURIS, R. STATHOPOULOU, I. TSEVRENIS
t h e lysis of a fibrin c l o t b y p l a s m i n a n d t h e a c t i v a t i o n of p l a s m i n o g e n b y s t r e p t o k i n a s e . H o w e v e r , in 1 case of 5 w i t h m y o c a r d i a l i n f a r c t i o n , in 4 c a s e s of 9 w i t h m y o c a r d i a l i s c h e m i a , a n d in 1 case w i t h a c u t e c e r e b r a l t h r o m b o s i s of 4, t h e a n t i p l a s m i n lysis t i m e w a s w i t h i n t h e n o r m a l r a n g e . L i k e w i s e , in s o m e of t h e t e s t e d p a t i e n t s w i t h m y o c a r d i a l i n f a r c t i o n no i n c r e a s e d i n h i b i t o r y a c t i v i t y of t h e a c t i v a t i o n of p l a s m i n o g e n b y s t r e p t o k i n a s e w a s f o u n d . S i m i l a r d i f f e r e n c e s h a v e b e e n o b s e r v e d b y o t h e r s also a n d t h e y m i g h t b e of i m p o r t a n c e f r o m t h e p r o g n o s t i c p o i n t of viewS. T h e p l a s m i n u s e d for t h e a n t i p l a s m i n a s s a y c o n s i s t e d of s t r e p t o k i n a s e a c t i v a t e d plasminogen. For this reason, this method does not distinguish between antiplasmin a c t i v i t y a n d i n h i b i t i o n of p l a s m i n o g e n a c t i v a t i o n a n d t h e v a l u e s g i v e n for a n t i p l a s m i n m a y t h e r e f o r e h a v e b e e n i n f l u e n c e d b y t h e p r e s e n c e of a n i n h i b i t o r of p l a s m i n o g e n activation. A l t h o u g h t h e d a t a p r e s e n t e d b y m o s t a u t h o r s g i v e i n d i c a t i o n s for t h e p r e s e n c e of a r e t a r d e d f i b r i n o l y s i s in t h r o m b o - e m b o l i c diseases, t h e p r o b l e m of t h e f i b r i n o l y t i c a c t i v i t y a n d a t h e r o s c l e r o s i s is still u n e l u c i d a t e d .
Greater numbers
of p a t i e n t s a t e
n e e d e d a n d m o r e p h y s i o l o g i c a l w o r k s h o u l d b e d o n e in o r d e r t o d e t e r m i n e t h e n a t u r e a n d t h e r o l e of t h e i n h i b i t o r s w h i c h f u n d a m e n t a l l y r e m a i n s u n k n o w n . REFERENCES 1 ASTRUP, T. In: I. H. PAGE (Ed.), Connective Tissue, Thrombosis and Atherosclerosis, Academic Press, New York, 1959, p. 223. 2 CHAKRABARTI,R., G. R. FEARNLEY, E. D. HOCKING, A. DELITHEOS AND G. M. CLARKE, Fibrinolytic activity related to age in survivors of myocardial infarction, Lancet, 1966, i: 573. 3 DEWIT, D., Investigations on the inhibitors of the fibrinolytic system, Thromb. Diath. Haemorrhag., 1964, 22: 105. 4 DUGUID, J. B., Thrombosis as a factor in the pathogenesis of coronary atherosclerosis, J . Pathol. Bacteriol., 1946, 58: 207. 5 DUGUID, J. B., In: I. H. PAGE (Ed.), Connective Tissue, Thrombosis and Atherosclerosis, Academic Press, New York, 1959, p. 13. 6 GORMSEN, J., Fibrinolytic activity inhibition of plasminogen activation, ,4cta med. scand., 1962, 172: 657. 7 GUEST, M. M., B. M. DALY, A. G. WARE, AND W. H. SEEGERS Antifibrinolysin activity in human plasmas during pathological states, J. Clin. Invest., 1948, 27: 793. 8 HILDEBRANDT, H., Weiteres fiber hydrolische Fermente, deren Schicksal und Xu sowie fiber Fermentfestigkeit und Hemmung der Fermentationen im Organismus, Virchows Arch. Pathol. Anat., 1893, 131: 5. 9 HUME, R., Fibrinolysis in cardiac infarction, Brit. Heart ]., 1958, 20: 15. 10 JACOBSSON, K., Trypsin and plasmin inhibitors in human blood serum, Scand. J. Clin. Lab. Invest., 1955, 7: Suppl. 11 ~,~ERSKEY, C., H. GORDON, H. LACKNER, V. SCHRIRE, B. J. I~APLAN, SOUGIN-MIBASHAN, H. L. NOSSEL AND A. MOODIE, Blood coagulation and fibrinolysis in relation to coronary heart disease, Brit. med. J., 1960, i: 219. 12 Mt3LLERTZ, S. In: T. KOELLER AND W. R. MERZ (Eds.), Proceedings of the 1st International Conference on Thrombosis and Embolism, Benno Schwabe, Basle, 1954, p. 79. 13 NILSSON, I. M., H. KROOK, N. H. STERNBY, E. SODERBERG AND N. SODERSTR()M, Severe thrombotic disease in a young man with bone marrow and skeletal changes and with a high content of an inhibitor in the fibrinolytic system, Acta med. scand., 1961, 169: 323. 14 NORMAN, P. S., Studies of the plasma system, Part 2 (Inhibition of plasmin by serum or plasma), dr. Exptl. Med., 1958, 108: 53. 15 NORMAN, P. S. AND B. HILL, Physical properties of the two plasmin inhibitors in plasma, J. Exptl. Med., 1958, 108: 639. 16 RATNOFF, O. D., I. H. LEPOW AND L. PILLEMER, The multiplicity of plasmin inhibitors in human serum, demonstrated by the effect of primary amino compounds, Bull. Johns Hopkins Hosp., 1954, 94: 169. J. Atheroscler. Res., 7 (1967) 425-433
SERUM INHIBITION OF PLASMIN IN THROMBOTIC DISEASES
433
17 SANDBERG, H., G. TSITOURIS, A. C. DELEON, JR. AND S. BELLET, Studies on inhibition to t h e p l a s m i n - p l a s m i n o g e n fibrinolytic e n z y m e s in p a t i e n t s with myocardial infarction, A m . J . Cardiol., 1960, 5: 666. 18 SHERRY, S., A. 1~. FLETCHER AND W. ALKJAERSIG, Fibrinolysis a n d fibrinolytic a c t i v i t y in m a n , Physiol. Rev., 1959, 39: 343. 19 SHULMAN, N. R., M e c h a n i s m of t h e inhibition of trypsin, p l a s m i n a n d c h y m o t r y p s i n b y s e r u m using fibrin t a g g e d w i t h I TM as a substrate, J. Exptl. Med., 1952, 95: 571. 2o SHULMAN, N. R., D e m o n s t r a t i o n t h a t separate proteolytic inhibitors exist in serum; their distinctive properties a n d t h e specificity of their action, J. Exptl. Med., 1952, 95: 593. Zl SHULMAN, N. R.~ Physiological aspects of variations in proteolytic inhibition, J. Exptl. Med., 1952, 95: 605. 22 TSITOURIS, G., H. SANDBERG, A. C. DELEON, JR., L. LECKS AND S. BELLET, Studies of t h e plasm i n - p l a s m i n o g e n s y s t e m in t h r o m b o - e m b o l i c diseases. I t s modifications b y t h r o m b o l y s i s t h e r a p y , Am. J. Cardiol., 1960, S: 680.
j . Atheroscler. Res., 7 (1967) 42S-433
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
SERUM I N H I B I T I O N OF PLASMIN A N D OF P L A S M I N O G E N A C T I V A T I O N IN T H R O M B O T I C D I S E A S E S
G. TSITOURIS, R. STATHOPOULOU AND I. TSEVRENIS Department of Cardiology, Second Department of Internal Medicine, University of 24thens Medical School and The Blood Bank of Hippokration General Hospital, Athens (Greece)
(Received March 7th, 1967)
SUMMARY The serum of five patients with acute myocardial infarction, nine patients with angina and recent electrocardiographic changes of myocardial ischemia and four patients with acute cerebral thrombosis was tested for antiplasmin (inhibitory) activity. In four patients with myocardial infarction, six patients with myocardial ischemia and three with cerebral thrombosis, a significantly higher than the normal inhibition of the plasmin activity was noted. In the remaining five cases, the inhibitory activity was within the normal range. The serum of ten patients with acute myocardial infarction was studied for inhibitory activity of the plasminogen activation b y streptokinase. Most of the patients were found to have increased inhibitory activity of the plasminogen activation. Our d a t a indicate that in the serum of a number of patients with t h r o m b o t i c diseases, there are factors inhibiting the fibrin clot lysis. Although similar changes were not seen in all the studied cases and particularly those with myocardial ischemia, more extensive studies might disclose that changes in the plasmin-plasminogen enzyme system play an important role in the production of these pathological conditions.
INTRODUCTION The reintroduction of the thrombogenetic theory of atherosclerosis b y DUGUID4 in 1946 and the recent appearance of purified proteolytic and fibrinolytic enzymes has greatly stimulated the interest of the investigators in the fibrinolytic phenomena. G. TSlTOURIS:Associate Professor of Cardiology, University of Athens Medical School. R, STATHOPOULOU: Assistant, Department of Medicine, University of Athens Medical School. I. TSEVRENIS: Associate Professor of Medicine, University of Athens Medical School, Director of Blood Bank, Hippokration General Hospital. j . Atheroscler. Res., 7 (1967) 425-433
426
G. TSITOURIS, R. STATHOPOULOU, I. TSEVRENIS
Some authors 1,5,1s have suggested that impaired fibrinolytic mechanism m a y underlie the pathogenesis of atherosclerosis. This concept has been based on ASTRUP'S assumption t h a t imbalance of a potential equilibrium between intravascular clotting and fibrinolysis could lead to mural thrombosis through a decreased fibrinolytic activity. Recently several authors2,9,1~,lT, 22 have reported a decreased fibrinolytic activity in the blood of patients with thrombo-embolic diseases and a high content of components inhibiting either plasmin or the activation of plasminogen b y streptokinase and urokinase. Nevertheless, the results were not in all instances conclusive6,11 and some investigators have been unable to find a b n o r m a l fibrinolytic activity in similar cases. The aim of the present work has been to investigate the plasmin inhibitors and the inhibitors of the activation of plasminogen b y streptokinase in the blood of patients with acute thrombotic episodes. MATERIAL AND METHODS
Material Anticoagulant. Double oxalate mixture of W i n t h r o p was used as naticoagulant. Buffer. Tris buffer, p H 7.4, ionic strengh 0.15; 24.2 g o t Tris were dissolved in 1000 ml H20, 25 ml of this solution + 17.0 ml 0.2 N HC1 was made up to 100 ml with H20. Fibrinogen. H u m a n fibrinogen ("Kabi") with a coagulability of 95 ~ (kindly supplied b y Kabi Ltd., Stockholm) was used in a concentration of 0.1 ~ in Tris buffer. The solution was stored at - - 2 0 ~. Streptokinase. "Varidase" (kindly supplied b y Lederle Laboratories, New York, U.S.A.) containing 20,000 units of streptokinase and 5000 units of streptodornase per vial. The material was dissolved and diluted to various concentrations (50, 25, 10 U / m l 0.9 ~ NaC1). Fresh solutions were prepared prior to the experiment. e-Aminocaproic acid, in powder (kindly supplied b y Lederle Laboratories, New York, U.S.A.), was m a d e up as a 0 . 1 % solution in 0.9 % NaC1. Thrombin. Bovine (Parke, Davis) containing 5000 units per vial. Two solutions were prepared. One containing 100 U/ml in Tris buffer a n d another containing 10 U in 0.9 ~o NaC1. Fresh solutions were prepared every week and stored at --20~ in concentration of 1000 U/ml. Human plasmin. "Actase" containing 1,000,000 factory units per vial (kindly supplied b y Ortho Pharmaceutical Corporation, Raritan, U.S.A.) was dissolved to a concentration of 50 U/ml in Tris buffer. Fresh solution was p r e p a r e d prior to experiment.
Subjects Twenty-eight patients from the wards of the Cardiology Department and of the Department of Internal Medicine of the Hippokration University Hospital, ranging in age from 42 to 74 years, formed two groups which were utilized as s t u d y groups. The t i m e from the onset of the episode did not exceed 4 days. G r o u p 1 consisted of 5
j. Atheroscler. Res., 7 (1967) 425-433
SERUM INHIBITION OF PLASMIN IN THROMBOTIC DISEASES
427
p a t i e n t s with acute m y o c a r d i a l infarction, 9 patients with angina pectoris a n d recent ECG changes of m y o c a r d i a l ischemia, a n d 4 patients with acute cerebral thrombosis. This group was studied for serum inhibition of plasmin activity. Group 2, consisting of 10 p a t i e n t s with acute m y o c a r d i a l infarction, was s t u d i e d for serum i n h i b i t i o n of the a c t i v a t i o n of plasminogen b y streptokinase. T w e n t y h e a l t h y individuals, ranging in age from 25 to 62 years, were selected from the non-professional donors of the Blood B a n k of the same hospital, a n d a pooled s t a n d a r d serum s a m p l e d e r i v e d from these subjects was used as the mean n o r m a l value. In order to h a v e the range of the n o r m a l vahles similar d e t e r m i n a t i o n s of serum inhibitions were also carried out in 40 n o r m a l individuals aged from 25 to 70 years. As controls two solutions were used in which saline 0.9 % and e-aminocaproic acid 0 . 1 % h a d replaced serum. The clot lysis t i m e with 50 U/ml actase (serum inhibition of plasmin a c t i v i t y ) was 743 + 136 sec. The clot lysis time using 50 U, 25 U, and 10 U/ml s t r e p t o k i n a s e (serum inhibition of plasminogen a c t i v a t i o n b y streptokinase) was 201 4- 26 sec, 267 :t: 52 sec, and 762 :J: 142 sec respectively with the serum concentration 100 ~ a n d i 9 2 4- 22 sec, 231 4- 45 sec, a n d 567 ! 131 sec with t h e serum concentration 25 %. Methods Collection o f blood. Blood was collected in o r d i n a r y glass tubes a n d allowed to clot. Two hours later the serum was s e p a r a t e d b y centrifugation at 2000 • g for 10 rain a n d then stored at - - 2 0 ~ until required. The sera of the controls were m i x e d and stored at - - 2 0 ~ in aliquots of 2 ml. S e r u m i n h i b i t i o n o f p l a s m i n activity. A test system was p r e p a r e d consisting of the following components in order of addition: 0.2 ml serum, 0.2 ml " A c t a s e " (50 U/ml), 0.4 ml 10//o fibrinogen, a n d 0.2 ml t h r o m b i n (100 U/ml). The tubes were placed in a
TABLE 1 I N H I B I T I O N OF F I B R I N O L Y T I C A C T I V I T Y I N BIORMAL CONTROL A N D I N P A T I E N T S W I T H A C U T E M Y O C A R D I A L I N F A R C T I O N , M Y O C A R D I A L I S C H E M I A , AND C E R E B R A L T H R O M B O S I S ( E X P R E S S E D AS A N T I P L A S M I N L Y S I S T I M E S I N SEC A G A I N S T 5 0 U A C T A S E / M L )
.Normal controls a
Patients with acute myocardial infarction
Patients with recent changes of myocardial ischemia
Patients with acute cerebral thrombosis
720 750 780 790 690 750 750 750 870
1082 1245 870 1042 1290
1310 900 1020 720 1275 1310 1215 1215 760 960
1600 1105
1410 660
Reproducibility of the determination of the lysis time with the same pooled serum sample from twenty healthy subjects. J. Atheroscler. Res., 7 (1967) 425-433
428
G. TSITOURIS, R. STATHOPOULOU, I. TSEVRENIS
37 ~ w a t e r b a t h a n d t h e lysis t i m e d e t e r m i n e d b y i n v e r t i n g t h e t u b e s at r e g u l a r i n t e r v a l s u n t i l t h e clot failed to cling to t h e b o t t o m of t h e tube. T h e t i m e r e q u i r e d to lyse t h e fibrin clot was considered to b e a m e a s u r e of t h e i n h i b i t o r y a c i t v i t y of t h e t e s t s e r u m . T h e t i m e was recorded from t h e a d d i t i o n of t h r o m b i n . Serum inhibition of plasminogen activation by streptokinase. D e t e r m i n a t i o n of s e r u m i n h i b i t i o n was carried o u t a c c o r d i n g to t h e t e c h n i q u e of NILSSON et al. 18. T h e t e s t s y s t e m c o n s i s t e d of t h e following c o m p o n e n t s in o r d e r of a d d i t i o n : 0.5 m l serum, 0.2 m l 1 % fibrinogen, 0.1 m l s t r e p t o k i n a s e , a n d 0.2 m l t h r o m b i n (10 U/ml). T h e t u b e s were i n c u b a t e d at 37 ~ a n d c h e c k e d for lysis in t h e m a n n e r d e s c r i b e d a b o v e . T h e c l o t lysis t i m e was recorded from t h e m o m e n t t h e s t r e p t o k i n a s e was a d d e d . A s s a y s w e r e c o n d u c t e d w i t h two c o n c e n t r a t i o n s of s e r u m (100 % a n d 25 % d i l u t i o n in 0.9 % NaC1) a n d t h r e e d i l u t i o n s of s t r e p t o k i n a s e (50, 25, 10 U s k / m l 0 . 9 % NaC1). A s s a y s c o n d u c t e d w i t h a s t r e p t o k i n a s e s o l u t i o n 25 U / m l d e m o n s t r a t e d b e s t t h e difference in i n h i b i t o r y a c t i v i t y . A solution of 0.9 % saline a n d a s o l u t i o n of 0 . 1 % of e - a m i n o c a p r o i c a c i d were u s e d as blanks. RESULTS
Serum inhibition of plasmin activity T a b l e 1 shows t h e lysis t i m e s ( a n t i p l a s m i n a c t i v i t y ) of t h e " A c t a s e " w h e n i t was i n c u b a t e d w i t h t h e serum of t h e n o r m a l c o n t r o l a n d w i t h t h e s e r u m of t h e p a t i e n t s .
140012001000" (~ 800" .~ 600 "~ 4 0 0 o "~ 200-
tll,.ll
IH
Jlnl] r
ACute myocardial infarction
Myocardial ischemia
IllIn
Illll II li Ill ll I Control serum (reproducibility)
Cerebral thrombosis
Fig. 1. Inhibitionoffibfinolyticactivityinnormalsubjects, and in patients with acute myocardial infarction, recent changes of myocardial ischemia, and acute cerebral thrombosis. The antiplasmin lysis times against 50 U/inl actase, are shown by the height of the columns. The solid columns on the left represent repeated determinations (reproducibility) with the same batch of the control serum (pooled serum sample). Each of the empty columns on the right represent a different patient. Normal range is between the solid horizontal lines.
J. Atheroscler. Res., 7 (1967) 425-433
429
SERUM I N H I B I T I O N OF PLASMIN IN THROMBOTIC DISEASES
In 4 of the 5 tested patients with myocardial infarction, and in 6 patients of the 10 with myocardial ischemia, and in 3 of the 4 patients with acute cerebral thrombosis, the clot lysis time was longer than the mean normal value and the normal range. The remaining cases showed values within the normal range and very close to the mean value. The lysis times of nine determinations on the same b a t c h of the control pooled serum were satisfactorily constant. The differences in the lysis time are graphically shown in Fig, 1. The height of the solid columns on the left represents the antiplasmin lysis times of the control pooled serum (mean normal value) and the height of the e m p t y columns on the right the values of the patients. The normal range of the normal values is between the horizontal lines. TABLE 2 R E P R O D U C I B I L I T Y O F T H E D E T E R M I N A T I O N OF T H E I N H I B I T I O N O F P L A S M I N O G E N A C T I V A T I O N B Y S T R E P T O K I N A S E (Sk) I N T H E S E R U M OF NORMAL CONTROL ( P O O L E D S E R U M S A M P L E ) , ( E X P R E S S E D AS A N T I P L A S M I N L Y S I S T I M E S IN S E C )
Serum concentrations 25 %
700 % 50 U
25 V
10 U
aO U
25 U
10 U
sk/ml
sk/ml
sk/ml
sk/ml
sk/ml
sk/ml
240 210 210 210 225 195 195 225
315 345 270 270 330 360 285 300
630 690 750 690 690 675 675 645
150 150 165 210 210 210 180 165
225 255 270 285 300 300 300 255
570 570 570 555 690 660 630 585
TABLE 3
(sk)
I N H I B I T I O N OF P L A S M I N O G E N A C T I V A T I O N B Y S T R E P T O K I N A S E IN N O R M A L C O N T R O L A N D I N P A T I E N T S W I T H A C U T e : M Y O C A R D I A L I N F A R C T I O N ( E X P R E S S E D AS A N T I P L A S M I N L V S I S T I M E S I N S]~C)
Serum concentration too %
Normal control (pooled s e r u m s a m p l e ) Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 P a t i e n t I0
25 %
50 U sk/ml
25U sk/ml
10U sk/ml
50U sk/ml
25 U sk/ml
IO U sk/ml
210 330 255 180 165 210 280 210 225 510 300
300 675 435 465 865 345 750 285 285 555 435
690 960 630 1320 1080 985 1040 900 1350 ~ 60 m i n 500
180 240 240 210 225 170 240 225 205 345 200
255 630 375 300 300 300 330 300 345
570 840 990 845 1710 810 1365 695 675 1290 570
435 230
j . Atheroscler. Res., 7 (1967) 4 2 5 - 4 3 3
430
G. TSITOURIS, R. STATItOPOULOU, I. TSEVRENIS
._E
Serum conce~tr Streptokir
concentrc Fig. 2. I n h i b i t i o n of plasminogen activation b y streptokinase in n o r m a l controls and in patients
with acute myocardial infarction. • mean normal value; Q, patient. Normal range is between the solid horizontal lines.
Serum inhibition of the activation of plasminogen by streptokinase T a b l e 2 shows t h e lysis t i m e s a n d t h e r e p r o d u c i b i l i t y in series of d i l u t i o n s of t h e c o n t r o l s e r u m w h e n it is i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s of s t r e p t o k i n a s e . T e n d e t e r m i n a t i o n s on t h e s a m e b a t c h of t h e c o n t r o l p o o l e d s e r u m show f a i r l y cons t a n t values. T h e v a l u e s of ten p a t i e n t s w i t h a c u t e m y o c a r d i a l i n f a r c t i o n are shown in T a b l e 3 a n d F i g . 2. I t can r e a d i l y be seen t h a t t h e lysis t i m e s in m o s t of t h e s t u d i e d p a t i e n t s were l o n g e r t h a n t h e c o n t r o l p o o l e d s e r u m a n d t h e n o r m a l r a n g e a n d t h e clot lysis r e t a r d e d . I n a few p a t i e n t s , t h e lysis t i m e w a s w i t h i n , or v e r y close, t h e n o r m a l values. T h e difference in t h e values b e t w e e n t h e p a t i e n t s a n d the c o n t r o l was m o s t clear w h e n t h e s e r u m was i n c u b a t e d w i t h 10 U s k / m l . I n t w o cases t h e lysis t i m e s were s h o r t e r t h a n t h e m e a n value. T h e c h a n g e s of t h e lysis t i m e w h i c h were p r o d u c e d b y t h e d i l u t i o n of t h e s e r u m or b y t h e i n c u b a t i o n w i t h different concent r a t i o n s of s t r e p t o k i n a s e d i d n o t show l i n e a r r e l a t i o n s h i p . T h e 1 : 4 (25 %) d i l u t i o n of s e r u m g a v e t h e b e s t c o n c e n t r a t i o n of values. DISCUSSION I t h a s b e e n k n o w n for m o r e t h a n half a c e n t u r y 8 t h a t n o r m a l s e r u m possesses
J. Atheroscler. Res., 7 (1967) 425-433
SERUM INHIBITION
O F P L A S M I N IN T H R O M B O T I C D I S E A S E S
431
factors which inhibit the action of proteolytic enzymes but controversy still surrounds the nature and n u m b e r of components capable of inhibiting plasmin and plasminogen activators. A lot of work dealt with trypsin inhibitors and their possible identity with the plasmin inhibitors. However, SHULMAN19,20 using 1311 labeled fibrinogen as a substrate and JACOBSSON1~ working with electrophoresis found different characteristics for the trypsin and the plasmin inhibitors. RATNOFF et al. 16 in 1954 suggested t h a t there m a y be at least three plasmin inhibitors in the h u m a n serum b u t NORMAN15 using a caseinolytic assay found only two. One rapidly acting and heat stable which migrates as an a2-globulin during starch electrophoresis and a second slowly acting and heat labile migrating as an al-globulin. MflLLERTZ 12 in the meantime discovered t h a t crude bovine globulin inhibited the activation of bovine plasminogen b y h u m a n streptokinase a c t i v a t e d globulin, and he considered it as a strong evidence for the existence of an inhibitor of plasminogen activation. NORMAN14 found t h a t normal serum contains an excess of antiplasmin over the plasminogen capable of being converted into plasmin. The problem of the inhibitors in the disease states is even more controversial. Proteolytic inhibition has been demonstrated in m a n y conditions of tissue necrosis 21. Increased antifibrinolytic activity has been described in patients with pneumonia, cirrhosis of the liver, coronary thrombosis, acute bacterial endocarditis, acute streptococcal infections, as well as in diabetes, cholecystitis, gangrene, arteritis, etc. 10. HUlVIE9 in 1958 found decreased fibrinolysis in patients with acute m y o c a r d i a l infarction and SANDBERG et at. 17 and TSlTOORIS et al. 22 described an a p p a r e n t increased antiplasmin activity in the plasma of patients with acute thrombo-embolic diseases and postoperatively. Similar findings were reported b y others 3 also, b u t there has been no general agreement 11. NILSSON et al. a3 in 1960 described a case of a young patient who had since childhood progressive s y m p t o m s of massive venous t h r o m bosis and it was associated with a high content of components in the serum, inhibiting the activation of plasminogen b y streptokinase and urokinase. GORMSEN6 determined the concentration of these serum inhibitors in patients with acute t h r o m botic episodes and atherosclerosis and after extensive surgery, but his findings were not conclusive. He found high content of inhibitors in some acute t h r o m b o t i c cases and postoperatively b u t no increase in patients with atherosclerosis. Recently CHAKRABARTIet al.Z found an incidence of low fibrinolytic activity of 33 ~ a m o n g patients of occlusive vascular disease, particularly to patients below the age of 60. The d a t a presented in this s t u d y show t h a t fixed concentrations of plasmin when they were incubated with the serum of patients with acute myocardial infarction, recent changes of myocardial ischemia and acute cerebral thrombosis, require in most of the cases longer times to dissolve standard fibrin clots than with the serum of normal control subjects. In addition, the activation of plasminogen b y streptokinase in a m e d i u m containing serum from patients with acute myocardial infarction was not as high as when the medium contained serum of the normal control subjects. I t is then a p p a r e n t t h a t in the serum of these patients there were factors which inhibited j . Atheroscler. Res., 7 (1967) 425-433
432
G. TSITOURIS, R. STATHOPOULOU, I. TSEVRENIS
t h e lysis of a fibrin c l o t b y p l a s m i n a n d t h e a c t i v a t i o n of p l a s m i n o g e n b y s t r e p t o k i n a s e . H o w e v e r , in 1 case of 5 w i t h m y o c a r d i a l i n f a r c t i o n , in 4 c a s e s of 9 w i t h m y o c a r d i a l i s c h e m i a , a n d in 1 case w i t h a c u t e c e r e b r a l t h r o m b o s i s of 4, t h e a n t i p l a s m i n lysis t i m e w a s w i t h i n t h e n o r m a l r a n g e . L i k e w i s e , in s o m e of t h e t e s t e d p a t i e n t s w i t h m y o c a r d i a l i n f a r c t i o n no i n c r e a s e d i n h i b i t o r y a c t i v i t y of t h e a c t i v a t i o n of p l a s m i n o g e n b y s t r e p t o k i n a s e w a s f o u n d . S i m i l a r d i f f e r e n c e s h a v e b e e n o b s e r v e d b y o t h e r s also a n d t h e y m i g h t b e of i m p o r t a n c e f r o m t h e p r o g n o s t i c p o i n t of viewS. T h e p l a s m i n u s e d for t h e a n t i p l a s m i n a s s a y c o n s i s t e d of s t r e p t o k i n a s e a c t i v a t e d plasminogen. For this reason, this method does not distinguish between antiplasmin a c t i v i t y a n d i n h i b i t i o n of p l a s m i n o g e n a c t i v a t i o n a n d t h e v a l u e s g i v e n for a n t i p l a s m i n m a y t h e r e f o r e h a v e b e e n i n f l u e n c e d b y t h e p r e s e n c e of a n i n h i b i t o r of p l a s m i n o g e n activation. A l t h o u g h t h e d a t a p r e s e n t e d b y m o s t a u t h o r s g i v e i n d i c a t i o n s for t h e p r e s e n c e of a r e t a r d e d f i b r i n o l y s i s in t h r o m b o - e m b o l i c diseases, t h e p r o b l e m of t h e f i b r i n o l y t i c a c t i v i t y a n d a t h e r o s c l e r o s i s is still u n e l u c i d a t e d .
Greater numbers
of p a t i e n t s a t e
n e e d e d a n d m o r e p h y s i o l o g i c a l w o r k s h o u l d b e d o n e in o r d e r t o d e t e r m i n e t h e n a t u r e a n d t h e r o l e of t h e i n h i b i t o r s w h i c h f u n d a m e n t a l l y r e m a i n s u n k n o w n . REFERENCES 1 ASTRUP, T. In: I. H. PAGE (Ed.), Connective Tissue, Thrombosis and Atherosclerosis, Academic Press, New York, 1959, p. 223. 2 CHAKRABARTI,R., G. R. FEARNLEY, E. D. HOCKING, A. DELITHEOS AND G. M. CLARKE, Fibrinolytic activity related to age in survivors of myocardial infarction, Lancet, 1966, i: 573. 3 DEWIT, D., Investigations on the inhibitors of the fibrinolytic system, Thromb. Diath. Haemorrhag., 1964, 22: 105. 4 DUGUID, J. B., Thrombosis as a factor in the pathogenesis of coronary atherosclerosis, J . Pathol. Bacteriol., 1946, 58: 207. 5 DUGUID, J. B., In: I. H. PAGE (Ed.), Connective Tissue, Thrombosis and Atherosclerosis, Academic Press, New York, 1959, p. 13. 6 GORMSEN, J., Fibrinolytic activity inhibition of plasminogen activation, ,4cta med. scand., 1962, 172: 657. 7 GUEST, M. M., B. M. DALY, A. G. WARE, AND W. H. SEEGERS Antifibrinolysin activity in human plasmas during pathological states, J. Clin. Invest., 1948, 27: 793. 8 HILDEBRANDT, H., Weiteres fiber hydrolische Fermente, deren Schicksal und Xu sowie fiber Fermentfestigkeit und Hemmung der Fermentationen im Organismus, Virchows Arch. Pathol. Anat., 1893, 131: 5. 9 HUME, R., Fibrinolysis in cardiac infarction, Brit. Heart ]., 1958, 20: 15. 10 JACOBSSON, K., Trypsin and plasmin inhibitors in human blood serum, Scand. J. Clin. Lab. Invest., 1955, 7: Suppl. 11 ~,~ERSKEY, C., H. GORDON, H. LACKNER, V. SCHRIRE, B. J. I~APLAN, SOUGIN-MIBASHAN, H. L. NOSSEL AND A. MOODIE, Blood coagulation and fibrinolysis in relation to coronary heart disease, Brit. med. J., 1960, i: 219. 12 Mt3LLERTZ, S. In: T. KOELLER AND W. R. MERZ (Eds.), Proceedings of the 1st International Conference on Thrombosis and Embolism, Benno Schwabe, Basle, 1954, p. 79. 13 NILSSON, I. M., H. KROOK, N. H. STERNBY, E. SODERBERG AND N. SODERSTR()M, Severe thrombotic disease in a young man with bone marrow and skeletal changes and with a high content of an inhibitor in the fibrinolytic system, Acta med. scand., 1961, 169: 323. 14 NORMAN, P. S., Studies of the plasma system, Part 2 (Inhibition of plasmin by serum or plasma), dr. Exptl. Med., 1958, 108: 53. 15 NORMAN, P. S. AND B. HILL, Physical properties of the two plasmin inhibitors in plasma, J. Exptl. Med., 1958, 108: 639. 16 RATNOFF, O. D., I. H. LEPOW AND L. PILLEMER, The multiplicity of plasmin inhibitors in human serum, demonstrated by the effect of primary amino compounds, Bull. Johns Hopkins Hosp., 1954, 94: 169. J. Atheroscler. Res., 7 (1967) 425-433
SERUM INHIBITION OF PLASMIN IN THROMBOTIC DISEASES
433
17 SANDBERG, H., G. TSITOURIS, A. C. DELEON, JR. AND S. BELLET, Studies on inhibition to t h e p l a s m i n - p l a s m i n o g e n fibrinolytic e n z y m e s in p a t i e n t s with myocardial infarction, A m . J . Cardiol., 1960, 5: 666. 18 SHERRY, S., A. 1~. FLETCHER AND W. ALKJAERSIG, Fibrinolysis a n d fibrinolytic a c t i v i t y in m a n , Physiol. Rev., 1959, 39: 343. 19 SHULMAN, N. R., M e c h a n i s m of t h e inhibition of trypsin, p l a s m i n a n d c h y m o t r y p s i n b y s e r u m using fibrin t a g g e d w i t h I TM as a substrate, J. Exptl. Med., 1952, 95: 571. 2o SHULMAN, N. R., D e m o n s t r a t i o n t h a t separate proteolytic inhibitors exist in serum; their distinctive properties a n d t h e specificity of their action, J. Exptl. Med., 1952, 95: 593. Zl SHULMAN, N. R.~ Physiological aspects of variations in proteolytic inhibition, J. Exptl. Med., 1952, 95: 605. 22 TSITOURIS, G., H. SANDBERG, A. C. DELEON, JR., L. LECKS AND S. BELLET, Studies of t h e plasm i n - p l a s m i n o g e n s y s t e m in t h r o m b o - e m b o l i c diseases. I t s modifications b y t h r o m b o l y s i s t h e r a p y , Am. J. Cardiol., 1960, S: 680.
j . Atheroscler. Res., 7 (1967) 42S-433