A multi-center double blind study for the clinical evaluation of TCUK was carried out on 115 patients with cerebral thrombosis.
The administration of TCUK was done continuously in doses of 60, 000 I. U. for 7 days under comparison with negative placebo.
The study was performed according to the protocol, shown in Table 1-6 and the effects of TCUK were judged by the grade of improvement of apparent subjective complaints, disturbances of consciousness, motorius and sensorial disorders of extremities and comprehensive daily life activities. The total scoring of improvements in individual disorders and their comprehensive evaluation were made for judgement of the global improvement and availability of the drugs, obtaining the following conclusions:
(1) The availability, particularly the global improvement of TCUK were better than those of placebo in their statistical significance (Table 10).
(2) In the serial evaluations, the clinical effects were more significant in patient groups of mild or moderate grades in the severity of disorders, of ages between 40-69, without histories of brain damages and without simultaneous infusions of large amount of solutions.
(3) The side effects of TCUK were rare (Table 11) with only two exceptional cases with disturbance of liver functions (Table 18), but the relationship between this liver function disturbance and administration of TCUK was obscure and no cases of hemorrhagic infarction were found in connection with TCUK administration in this study.

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The clinical effect of thrombolytic therapy with urokinase (UK) at the daily dosis of 60, 000 units (high dosis group-H) on cerebral infarction was estimated by means of multiple center double blind tests in comparison with that of cases treated with its 6, 000 units (low dosis group-L).
The patients involved in this study were limited to those of cerebral infarction who were not caused by embolism coming from other organs such as endocardium, lung and deep vein of lower extremities, and started their treatment within two weeks after the onset. The cases with severer disorder of consciousness than Grade II in the International Classification of Sanity Disorder, were excluded from this study.
The general improvement after one week UK treatment was realized to be significantly more in H-group than in L-(38% to 24%), but that at three weeks after stopping this treatment turned to be not significant (58% to 40%) although H-group showed still some higher tendency of improvement than L-group.
The utility of UK judged comprehensively from the improvement and side effect was also calculated to be significantly more with high dosis than low (36% to 22%) after one week treatment and three weeks after stopping the treatment as well.
This difference between H- and L-group in utility was more obvious in cases with lower severity. The apparent side effect invited during this treatment with high or low dosis was almost none and no difference was observed between H- and L-groups.
From these findings it was supposed that high dosis administration of UK than 60, 000 units/day might be more effective in severe cases of Grade II.

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Plasma levels of prekallikrein and kallikrein in pregnancy and labor have been measured by the chromogenic substrate, S-2302. At the same time, fibrinolytic inhibitors such as α₁-AT, α₂-MG, AT III and Cl-inactivator as well as α₂-PI were determined by a radial immunodiffusion method.
1) Levels of plasma prekallikrein in normal pregnancy were significantly greater than those in non-pregnant state, and the mean prekallikrein level at 42 weeks of gestation was 201±22.12%, thereafter it decreased to 104±20.2% within 30 minutes after delivery. Prekallikrein levels in uterine blood were significantly higher than those in cord blood as well as retroplacental blood.
2) Plasma kallikrein concentrations in normal pregnancy were within non-pregnant levels until 42 weeks of gestation, but the level increased to 35.33±10.12% just before delivery and after delivery it raised to 65.33±13.22%.
3) The mean prekallikrein titer in twin pregnancy was 121.33±26.1%, whereas in cases with severe toxemia and hydatidiform mole, the levels were 68±8.96% and 77.16±28.1%, respectively.
4) After Cl-inactivator concentration showed over 20mg/dl in early pregnancy, it dropped below 20mg/dl until 40 weeks of gestation.
Therefore, it was suggested that circulating plasma prekallikrein and kallikrein system can work on the mechanism of labor pain and coagulation and fibrinolytic activity taking place in the utero-placental circulation.

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Although various chromogenic substrates have been developed for determination of proteases, there are few reports on the determination of glandular kallikrein (GK) in tissues using chromogenic substrates.
The authors have determined GK of the rat stomach by measuring its esterolytic activity on Pro-Phe-Arg-NE (PPA-NE) which is one of the naphthylester derivatives.
The extracted rat stomach was kept frozen for one week; then thawed, homogenized, and ultracentrifuged for sixty minutes at 105, 000G.
The supernatant was used as specimens and examined for the existence of contaminative proteases. The results showed that their amounts were so small that their effects on determining the activity of GK were negligible.
The authors determined the esterolytic activity of the specimen on PPA-NE and also the amount of kallidin produced from low molecular weight kininogen acted by the specimen, and studied correlation between the values of two measurments. The correlation coefficient for these two variables was:
r=0.953 [y=10.354x+0.051]
This high correlation implies that GK of the rat stomach can be estimated by its esterolytic activity on PPA-NE.

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Fibrinogen derivatives (FD) in sera from normal children and from patients with coagulopathy were characterized using a method of immunoabsorption followed by SDS-polyacrylamide gel electrophoresis. Electrophoretic bands with antigenic capacity against anti-fibrinogen serum were identified by their molecular weights, and classified as follows: high molecular weight fibrinogen complexes (HMWFC), fragments X, Y, D, E, D-dimer and others. Their concentrations and proportions to total serum FD in normal children (N=20) were shown in Table 2. D-dimer, derived from crosslinked fibrin, and HMWFC were markedly elevated in patients with disseminated intravascular coagulation (DIC). Proportions of D-dimer and HMWFC were raised in Henoch-Schönlein purpura and in rapidly progressive glomerulonephritis, and concentrations of D-dimer and HMWFC were increased in nephrotic syndrome. Using this technique, it may be possible to discriminate between DIC and primary fibrinolysis, and to detect changes in the blood coagulation and fibrinolysis systems in patients with various forms of coagulopathy.

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Patterns of VIII R: AG in the plasma, cryoprecipitate and cryosupernatant fractions from normal subject and von Willebrand's disease variants were investigated by SDS 1.5% polyacrylamide gel electrophoresis-crossed immunoelectrophoresis (SDS PAGE-CIE). Neutralization of SDS after PAGE was performed by inserting an intermediate agarose layer imprignated with Triton X-100. Molecular weight of VIII R:AG in normal plasma ranged from 8×10⁵ to 8×10⁶ daltons. Normal cryoprecipitate showed several precipitin peaks with a molecular weight range of 8×10⁵-1×10⁶ daltons. Normal cryosupenatant had two low peaks with 8×10⁵-2×10⁶ daltons.
VIII R:AG in the plasma and cryoprecipitate fractions from von Willebrand's disease variants, showing fast anodal mobility in the conventional CIE, gave 2-3 peaks with low molecular weights of 8×10⁵-3×10⁶ daltons. Plasma VIII R:AG in these patients were suggested to stay in low levels of polymerization/aggregation of subunit and not to show VIII R:WF similar to VIII R:AG in normal cryosupernatant.

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Changes in serum enzyme activities and in the coagulation-fibrinolytic system in drug-induced hepatic injury were studied in rats. Hepatic injury was induced in rats by single dose administration of carbon tetrachloride (CCl₄: 200 or 600μl/kg), D-galactosamine (D-Gal: 150 or 450mg/kg) and α-naphthyl isothiocyanate (ANIT: 60 or 180mg/mg). All determinations were performed at 1 to 48 hours after administration of CCl₄ or D-Gal, and at 7 to 72 hours after administration of ANIT.
The results were follows.
In the rats treated with D-Gal, the activities of GPT and GOT in the serum were found to correlate closely with the hepaplastin test (HPT) and prothrombin time (PT) results.
In rats dosed with CCl₄, serum enzyme activities clearly elevated at 1 hour, while the serum GPT, GOT, and LDH activities maximized at 24 hours. On the other hand, HPT values, activated partial thromboplastine time (APTT) values and antithrombin III (AT III) levels were slower to change and earlier to recover than the serum enzyme activities. Thus, there appeared to be almost no relationship between serum enzyme activities and coagulation-fibrinolytic activities.
In rats dosed with ANIT, No regular tendency was observed in the variation of the coagulation-fibrinolytic system in comparison with serum enzyme activity variation.
These results suggested that the degree and patterns of variation in serum enzyme activity and in the coagulation-fibrinolytic system differed considerably with differences in the type of hepatic injuries.

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In order to analyze the relationship between autoimmunological phenomena and coagulofibrinolytic reactions in systemic lupus erythematosus (SLE), we studied 20 patients with SLE, 9 of whom were active (CH₅₀<30U/ml), 11 were inactive (CH₅₀>30U/ml), about thrombotic episodes and coagulofibrinolytic functions. The results obtained were as follows:
1) 5 thrombotic episodes were complicated in active SLE, and 1 in inactive SLE.
2) In active SLE β-thromboglobulin level was increased, fibrinogen turnover was increased, and levels of plasminogen and antithrombin-III were low as compared with those in inactive SLE.
3) CH₅₀ and level of antithrombin-III showed a positive correlation (r=0.77) and a clear parallelism in a case of SLE.
It was postulated that with the progress of disease activity in SLE it was inclined toward hypercoagulable state, and so thrombotic tendency was accelerated.

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Coagulation and fibrinolysis was investigated in 14 patients with ulcerative colitis, 3 patients with colonic tuberculosis, 2 patients with Crohn's disease, 3 patients with ischemic colitis, 5 patients with aphtoid colitis and 4 patients with radiation-induced proctitis.
The results were as follows: (1) Hypercoagulability was found in the most patients with ulcerative colitis and radiation-induced proctitis. (2) Platelet count was strikingly increased in the patients with active ulcerative colitis. (3) Level of antithrombin III was decreased in the patients with ischemic colitis, ulcerative colitis and radiation-induced proctitis. Moreover, serum plasminogen activity was decreased in the patients with active ulcerative colitis and radiation-induced proctitis and it returned to normal value in patients with ulcerative colitis in remission. On the other hand, fibrin/fibrinogen degradation products of serum was increased in the patients with active ulcerative colitis and Crohn's disease.
The results indicate that hypercoagulability and increased fibrinolytic activity exist in the patients with inflammatory bowel diseases.

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Fibrin-Fibrinogen degradation products (FDP) have been found in the serum and urine of many patients with renal disease.
To clarify the role of intraglomerular coagulation in renal disease, serum and urine FDP D-dimer was examined in reference to histology on 82 patients with several renal disease.
The results were as follows:
1. D-dimer in serum was found in 14 of 64 cases and that in urine in 14 of 34 cases.
2. Serum or urinary D-dimer positive cases had severer renal histological damages on light microscopy and more massive intraglomerular fibrin deposits by immunofluorescence in comparison with D-dimer negative cases.
3. The ratio of D-dimer to D fraction R_D-D in urine FDP was higher than that in serum. This result suggests that there is acceleration of intrarenal coagulation and fibrinolysis in urinary D-dimer positive cases.
4. D-dimer in arterial blood, renal vein blood, and systemic vein blood of 14 cases were also examined, but any significant difference was revealed among the blood specimens.
5. There was a case which had more amount of fragment of γ-γ dimer in SDS-PAGE pattern of 2-mercaptoethanol reduced FDP in renal vein blood than that in arterial vein blood.

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It is well known that thrombosis and/or embolism have been complicated with mitral stenosis (MS). To evaluate thrombotic state in cases of MS, the coagulofibrinolytic examination was carried out, e. g., the change of plasma antithrombin III (AT III) and α₂-plasmin inhibitor (α₂-PI).
Hypercoagulable state (low plasma AT III level) and normal state of fibrinolysis (normal plasma α₂2-PI level) were found in cases MS. The tendency of hypercoagulability was marked in cases with atrial fibrillation (af) and chronic congestive heart failure (CHF). As the blood flow disturbance due to af or CHF could be regarded as the hypercoagulability, a comparison was made between MS and heart diseases without rheumatic heart disease (non RHD). Cases of non-af and af-CHF showed the comparable decrease in plasma AT III but at the same time the decreased level of plasma α₂-PI, that is, there was the acceleration of both coagulation and fibrinolysis.
The significant difference could not be detected between cases of MS-af with and without thrombosis and/or embolism.
It was concluded that MS was in hypercoagulable state especially in cases with of and CHF and that the hypercoagulability in MS might play an important role on the thrombi formation but it was not the cases in non RHD.

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We conducted the present study in the belief that changes in the coagulation-fibrinolysis system during normal pregnancy have a sufficiently great clinical importance.
With 30 non-pregnant women as the control, total 270 normal gravidas were observed during pregnancy.
Laboratory tests performed were those on the activities of coagulation factors XII, IX, VIII, VII, X, V, II and XIII, platelet counts, Fbg, PLg-Act, pro-Act, PLg, SFMC, FDP, AT-III, C₁-INA, α₂-PI, α₂-M, and α₁-AT.
The activity of the intrinsic coagulation system was markedly accelerated since the middle stage of pregnancy, while that of the extrinsic system tended to be slightly accelerated in late pregnancy. Factor XIII was diminished, C₁-INA decreased, SFMC and FDP elevated. From the above findings, chronic DIC pattern was recognized.
In the fibrinolytic system, PLg-Act was markedly diminished pro-Act and PLg gradually elevated, α₂-PI and α₂-M did not change, and α₁-AT increased. These indicated that the fibrinolytic system was depressed on the whole.

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In the familial AT III deficiency, the levels of AT III of propositus and her mother measured immunologically and biologically were below normal. And AT III of her brother measured immunologically was half in normal in spite of normal biological antithrombin activity.
Progressive antithrombin activity and anti-Xa activity of familial AT III deficiency were within normal range. Although the rates of thrombin neutralization activity of propositus and her mother were below normal, the rate of her brother's plasma remained in normal range. And the rates of thrombin neutralization activity calculated as mg protein of AT III were highest in the plasma of her brother, and became lower in her mother, propositus and normal plasmas in this order.
It is concluded that her brother has no episode of thrombosis because the antithrombin of her brother reacts as normal in neutralization to thrombin.

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The effect of heparin infusion on the antithrombin III (AT III) metabolism was studied on the rat using radioimmunoassay (RIA) method and ¹²⁵I labelled AT III as a tracer. Rat AT III was purified from rat-defibrinated plasma by heparin-Sepharose 4B affinity chromatography and gel filtration using Sephadex G-200. This purified AT III was used for RIA and metabolism studies. Labelling of AT III with ¹²⁵I was performed according to Hunter and Greenwood.
Plasma levels of AT III were significantly decreased in the group treated with heparin infusion for 6 hours, although no significant differences were observed in AT III content in various organs.
The behavior of i. v. injected ¹²⁵I-AT III was studied on the normal control rat and heparinized groups. The half life of ¹²⁵I-AT III in plasma 2.13 days in normal male rats. After heparin infusion for 6 hours, the percent dose radioactivity per milliliter plasma was 1.16±0.51 and 2.01±0.38 in the control group. Significant difference (p<0.05) was observed between both groups. On the contrary, the percent dose radioactivity per gram tissue wet weight was significantly increased in liver, lung and large intestine in the heparin treated group. The decreased amount in plasma of i. v. injected ¹²⁵I-AT III by heparin infusion appears to be trapped and metabolized by organs, mainly in the liver.

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Protamine has been used as the principal neutralizing agent of heparin but there are few reports on the interaction of antithrombin III (AT III), heparin, and protamine and this neutralizing mechanism was analyzed by means of column chromatography, crossed immunoelectrophoresis, and Heparin Sepharose affinity chromatography.
Sephadex G-200 chromatography of heat defibrinated plasma with ³H-heparin revealed radioactivity in all protein fractions and protein free heparin fraction. Immediate antithrombin activity was detected in AT III fractions. When ³H-heparinized plasma was fractionated on Sephadex G-200 after neutralization by protamine, most of radioactivity was eluted in the void volume and the radioactivity of other fractions was markedly decreased and immediate antithrombin activity was lost in any fraction but progressive antithrombin activity was seen in the third protein peak.
Electrophoretic mobility of heparin treated AT III was increased depending on the heparin concentrations, but when heparinized plasma was neutralized with protamine, electrophoretic movility of AT III was recovered. Meanwhile protamine treatment did not affect the electrophoretic movility of untreated AT III.
AT III bound Heparin Sepharose was eluted with protamine and eluted fractions were chromatographed on Sephadex G-75 and AT III fraction was separated from protamine.
These results indicate that protamine separates AT III from AT III-heparin complex with binding to heparin and that separated AT III recovers original activity.

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Plasma antithrombin III (AT III) activity was determined in 50 patients with various liver diseases, comparing with immunological activity (antigen), hepaplastintest (HPT) and other liver function tests.
Plasma AT III activity was determined by the method of Nakamura using chromogenic substrate and antigen was measured by SRID.
1) In acute hepatitis, AT III activity (67±38.6 %, normal: 100±20) varied from significant low value in the patients with severe hepatocellular damage to high value in the patients of convalescence and cholestatic type (125±19.2). Marked reduction of AT III activity was observed in fulminant hepatitis (16.2±12.0) and decompensated liver cirrhosis (22.3±9.8). In hepatoma, AT III activity (62.8±31.9) varied widely from normal to low levels and showed rather higher value than that of decompensated cirrhosis.
2) There was a significant positive correlation between AT III activity and antigen in whole liver diseases (r=0.87, p<0.01, n=50), however the AT III activity tended to be lower than that of antigen in severe parenchymal damage, e. g. fulminant hepatitis, decompensated cirrhosis and some patients of acute hepatitis.
In some case, there was observed definite dissociation between AT III activity and antigen. This fact may suggest the possible hypercoagulable state followed by release of a large amount of tissue thromboplastin, endotoxin or appearance of abnormal protein.
3) Both determinations showed a similar lower value in cases with severe parenchymal damage, but AT III activity tended to show a higher value than HPT in cases with cholestasis, convalescence and hepatoma.
The determination of AT III activity seems to provide a clinical significant information different from AT III antigen or HPT in liver diseases.

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It was well defined that antithrombin III (AT III) could neutralize thrombin, activated factor X and plasmin. partial deficiencies of AT III have been shown to be associated with recurrent venous thromboembolism.
This report presents the variations of AT III and heparin resistance (HR) of patients with ilio-femoral vein thrombosis, superficial vein thrombophlebitis or leg varicosis, compared with collagen disease or Buerger' s disease. AT III was measured by immunodiffusion method.
The results were as follows:
1. Plasma AT III of all patients with venous diseases maintained almost normal values (26.2±7.1mg/dl), but out of them, ilio-femoral vein thrombosis showed low levels of AT III (24.7±7.3mg/dl), especially in acute phase (19.4±4.7mg/dl), and superficial vein thrombophlebitis retained within normal range (27.7±7.2mg/dl).
2. Two clinical cases demonstrated here showed the relative correlation between clinical feature and plasma AT III.
3. In a series of patients with or without liver cirrhosis who had undergone major surgery, plasma AT III decreased during 3-14 days after operation and recovered to preoperative value. But they did not suffer fromdeep vein thromboembolism.
From these investigations, we suppose that plasma AT III decrease in iliofemoral vein thrombosis is due to the increased metabolism of AT III which combined with thrombin and that hypo-AT III-emia may cause venous thromboembolism together with other hypercoagulable factors.

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The method of plasma membrane isolation using polylysine beads which was developed by Jacobson and Branton was applied to human platelets. The method is based on the electrostatic binding of positively charged polylysine beads and negatively charged cells, followed by disruption of the cells. The portion of the surface membrane attached on the beads remains bound there after disruption and is separated from other cellular components by washing. Purity of the platelet membrane preparation obtained by This method was estimated by enrichment of the ¹²⁵I specific activity of the bead membrane preparation isolated from the platelets whose surface was iodinated with lactoperoxidase and H₂O₂. Marker enzymes, bis (p-nitrophenyl) phosphate and Na, K-ATPase for membranes, cytochrome oxidase for mitochondria and beta-glucuronidase for granules, were also assayed. These studies showed that the membrane preparation isolated on beads was satisfactorily pure as compared with the preparation isolated by widely used sucrose gradient centrifugation method. Analysis by polyacrylamide gel electrophoresis revealed that almost all species of the surface glycoproteins were present on the membrane preparation isolated with polylysine beads. The polylysine beads technique is a rapid, reproducible and efficient method for preparation of considerably pure platelet plasma membranes.

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The authors have reported an activation of platelets associated with an increase in plasma von Willebrand factor (vWF) level after isometric exercise in patients with ischemic heart disease (IHD). In this study, glass beads columns were employed as an experimental model to exclude the influence of vWF from intima of blood vessels.
MATERIALS and METHODS: 0.38% citrated blood samples obtained from 13 healthy subjects were filtrated through glass beads column (glass surface: 25cm²) two times with the column tenewed each time. Platelet count (Coulter Counter ZBI), platetet size (Coulter Channelyzer), platelet sensitivity to ADP-aggregation (Sano, et al.: Thrombos. Haemostas., 37: 329, 1977), plasma vWF level (Sano, et al.: Thrombos. Haemostas., 42: 1589, 1979 (Fig. 1) and plasma factor VIII related antigen (F VIII R:AG) (Partigen immunoplate) were measured before and after the filtrations.
RESULTS: Platelet count and size did not change significantly, while platelet sensitivity, vWF and F VIII R:AG increased significantly after filtration comparing to the non-filtrated original samples (Table 1, Fig. 2). The increments after the second filtrations comparing to the original values were significantly greater than the increments after the first filtrations.
CONCLUSION: An interaction between platelets and rough surface of glass beads indued an activation and release reaction of platelets in vitro.
This finding suggests the significance of such kind of interaction in the activation of platelets associated with release reaction in vivo, which was observed in IHD patients after isometric exercise.

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Plasma β-thromboglobulin (β-TG) and platelet factor 4 (Pf-4) levels were assayed in 34 healthy controls and 67 patients suffering from cardiovascular or cerebrovascular diseases. Both were significantly increased in 4 acute myocardial infarctionn patients and 4 cerebral infarction patients compared to controls. In contrast, the mean levels in old myocardial infarction and angina pectoris patients were almost within normal limits. In 38 patients with atrial fibrillation with or without heart valvular disease, assay of β-TG gave a range of 10-175ng/ml (mean±SD, 55.0±43.0ng/ml) and that of Pf-4 gave a range of 3-99.5ng/ml (mean±SD, 26.0±34.6ng/ml). These were significantly greater (p<0.005, p<0.025, respectively) than the levels of controls. These results indicated that the assay of plasma β-TG and Pf-4 levels may be useful for the diagnosis of acute thromboembolic diseases and for assessing the participitation of platelets in prethrombotic states like atrial fibrillation in heart diseases.

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EG-626 (7-ethoxycarbonyl-4-hydroxymethyl-6, 8-dimethyl-1 (2H)-phthalazinone) is a newly synthesized anti-atherosclerotic and anti-aggregatory agent. The mode of inhibitory action of EG-626 on arachidonic acid (AA)-induced rabbit platelet aggregation was studied in relation to its antagonistic effect on the action of thromboxane (TX) A₂, and the following results were obtained.
EG-626 in a dose of 3 to 100μg/ml inhibited sodium arachidonate (SAA)-, ADP-, or collagen-induced rabbit platelet aggregation in a concentration dependent manner, 8 and 50μg/ml inhibited rabbit platelet aggregation induced by TXA₂ generated by prostaglandin (PG) H₂ and platelet microsomes and 10 and 60μg/ml markedly inhibited rabbit platelet aggregation induced by labile aggregation-stimulating substance (LASS). However, a dose of 100μg/ml had no discernible inhibitory effect on the conversion of AA to its metabolites using washed platelet suspension. In addition, EG-626 inhibited the contractile response of aortic strips to TXA₂, without affecting TXA₂-formation.
These results suggest that EG-626 inhibits AA-induced rabbit platelet aggregation primarily through an antagonistic effect on the actions of TXA₂ and other active metabolites of AA, without affecting their biosynthesis in platelets.

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In order to investigate the effect of fibrinolytic activity on viscoelasticity of blood clot, the lytic phase of clotting curve was annalyzed by use of a viscoelastorecorder.
As parameters of clottability, the maximum rigidity modulus G′m and the maximum rigidity loss modules G″m were measured. As parameters of fibrinolysis, the degree of decrease in rigidity modulus for 60min following G′m, that is D′, and the degree of decrease in rigidity loss modulus for 60min following G″m, that is D″, were measured. Then D′/G′m, D″/G″m and G″m/G′m were calculated.
When urokinase (UK) was added to the normal blood in vitro (concentration: 100U/ml blood), no significant difference was observed in G′m but there was a significant increase in both D′/G′m and G″m/G′m compared with those of controls.
On the contrary, when tranexam acid (AMCHA) was added to the normal blood (concentration: 1.7mg/ml blood), D′/G′m had a decreasing tendency and D″/G″m had a increasing tendency.
These data suggest that the lytic phase of clotting curve is accerelated by not only the fatigueing of platelet contractility but also the fibrinolytic activity, since it is known that the relaxation after G′m is attained is caused by a fatigueing of platelet contractility.

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Cannulation of the thoracic duct and femoral vein in dogs was performed so that samples could be taken at intervals. Lymph fibrinolytic factors (especially plasminogen activator, plasminogen and antiplasmin) and the influence of urokinase administered intravenously by one shot on thoracic duct lymph fibrinolysis were studied in dogs weighing 10kg.
Results were as follows. Plasminogen activator, plasminogen and antiplasmin are presented in lymph. Levels of plasminogen in thoracic duct lymph were lower than in blood, but there was no significant difference in antiplasmin levels between in blood and lymph. Lymph plasminogen activator levels were variable and changes of lymph fiibrinolytic activity were not definitive after the administration of 120, 000 units of urokinase. Antiplasmin levels, however, reduced transiently in lymph as well as in blood. The highest radio activity in lymph was seen 30 minutes after administration of ¹²⁵I-labeled urokinase. Administration of urokinase seemed to increase thoracic duct lymphatic flow, but there was no statistical difference.

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Boosting effect of ultrasonic vibration on fibrinolytic activity of urokinase has been investigated. Fibrinolysis was studied grouply in (1) experimental fibrin clot with urokinase (2) clot with urokinase preceded expose of clot to ultrasonic waves, (3) clot exposed to ultrasonic waves and (4) clot with saline solution. Scanning electron microscopic examinations were made on the clots in each four series. Fibrinolysis was not evident in the clots (3) and (4), though fine cracks were seen in the clots (3). The four hour rate of fibrinolysis was 50% in the clots (1) where as 100% in clots (2).
Procedures (1) (2) (3) and (4) were also emplyed in the Chandler's Loop Technique clot of four hour rotation. The rate of fibrinolysis was 50% in the clot (1) when 60 IU of the urokinase was used, where as 86% in the clot (2), which scanning electron microscopically gave a feature of decreased fiber structure. Fibrinolysis was accererated by ultrasonic vibration in the clot with certain amount of urokinase added, breaking up of the clot being reached in shorter time than the series without ultrasonic wave application.
Ultrasonic vibration mechanically might cause microstream in the clot architechture, which could help urokinase contact directly with the fiber net. Scanning electron microscopic examinations revealed morphological representation of enhanced fibrinolysis by ultrasonic vibration. It was suggested hat physical ultrasonic vibration could be applied to boost a chemical fibrinolytic effect of urokinase.

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Although studies of UK have been developed, it is still uncertain as to the presence of UK in the circulating blood. In addition, the relation between UK antigen and its inhibitor was not fully examined. The present study was carried out to elucidate these problems.
Anti-UK serum used in this experiment was obtained from rabbit, immunized by low molecular weight UK and showed a single arc on Ouchterlony's immunodiffusion or immunoelectrophoresis. This anti-serum neutralized UK activity. The relationships between UK antigen, UK activity and α₂PI antigen in the healthy adults or patients with thrombosis were examined. The correlation between UK antigen and UK activity was r=0.36 (p<0.05), UK activity and α₂PI antigen was r=-0.1 (p<0.05), and UK antigen and α₂PI antigen was r=0.23 (p>0.05).
In the experiment of gel filtration of UK and plasma on Sepharose 2B, UK activity was decreased and UK antigen was migrated more anode region on Laurell electrophoresis than UK only, when UK was incubated with plasma.
Normal plasma or plasma with thrombosis were eluted on anti-UK Sepharose column chromatography, electrophoresed on SDS polyacrylamide. Several protein bands were migrated on polyacrylamide gel. These results suggest the possibility that UK is present in the circulating blood, forming a complex with inhibitor. However, further investigation is required to resolve this possibility.

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Problems in the Urokinase (UK) therapy, were marked difference of the sensitivity against UK in case by case, and hypercoagulability induced by UK administration in some cases. Then, utilizing Urokinase resistant time (UKRT) that indicates the sensitivity against UK, we deviced the method for determination of the optimal administration dose. Furthermore, we used UKRT to monitor the blood coagulation fibrinolysis after UK administration. Twenty three cases of cerebral thrombosis were treated by rapid venous drip infusion of calculated UK doses. All cases were obtained optimal fibrinolytic state without bleeding. Eighteen cases (78%) showed excellent or good clinical results, in which UKRT were shortened between 6 to 10min. at 1 to 3 hours after UK administration. Only increased fibrinolysis might be induced in these cases. Five cases (22%) showed no change or poor clinical results, in which UKRT was prolonged, and then occurrence of hypercoagulability and increased fibrinolysis were suspected.
In conclusion, UKRT may be practically available for the determination of optimal UK doses and monitoring the fibrinolytic therapy.

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A fibrinolysis inhibitor was extracted and partially purified from human cornified cells. The inhibitor activity was quantitatively measured for plasmin and urokinase in amydolytic reactions of chromogenic tri-peptide substrates, H-D-Val-Leu-Lys-pNA and PyroGlu-Gly-Arg-pNA. Human cornified cells scraped from heels were homogenized in 0.1M Tris-HCl+0.14M NaCl, pH 8.0, and supernatant was separated by centrifugation at 24, 000g. The extract did not show inhibition for plasmin. One mg of protein of the extract, whereas, inactivated urokinase that hydrolyzes 3.0 nmole of the substrate per minute (3.0mIU/mg protein). The inhibitory effect was found to increase with time for 30 minutes in a enzyme-inhibitor mixture, illustrating “time dependent inhibition”. Sephacryl S-200 gel chromatography and subsequent DEAE Sepharose ion exchange chromatography yielded the urokinase inhibitor fraction that exhibited 254mIU/mg protein in specific activity. SDS-polyacrylamide 10% gel electrophoresis showed seven protein bands in this fraction. Molecular weight of the urokinase inhibitor was estimated to be approximately 35, 000 in the gel chromatography.
A proteinase inhibitor that progressively inactivates urokinase was demonstrated in human cornified cells. We suggest that a regulatory function of the urokinase inhibitor possibly participates in physiological and pathological proteolytic mechanisms in the epidermis.

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The immunological properties of Bilokinase (BK) were elucidated by comparing them to those of Urokinase (UK). Highly purified BK and UK were prepared from bovine bile and bovine urine, respectively, and both activators were administered to rats or rabbits with Freund's complete adjuvant for 2 months. The antisera obtained showed a specific reaction in the precipitin formation against their own antigen, but did not show a cross reaction to the other antigen. It was also demonstrated that the BK activity was suppressed only by the anti-BK antiserum and not by the anti-UK antiserum which inhibited UK activity.
In addition, anti-human UK antiserum which was prepared by immunizing rabbits with commercial human UK, further purified in our laboratory showed a strong inhibitory effect on the activity of human UK, whereas the activity of human BK which was extracted from the bile of patients with cholelithiasis was not inhibited by the anti-human UK antiserum.
These results strongly suggest that these two activators are antigenically distinct proteins.

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A high molecular form of human kidney cultured urokinase (T HMW-UK) was highly purified by affinity chromatography on (N^α-(ε-aminocaproyl)-DL-homoarginine hexylester)-Sepharose, followed by isoelectric focusing and Sephadex G-100 gel filtration. The molecular weight (53, 400 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), amino acid composition, and kinetic parameters for Glu-plasminogen activation (K_m=0.92μM, k^cat=28.7min^-1) of the enzyme resembled those of the high molecular form of urinary urokinase (U HMW-UK). However, some differences were found as follows. 1) T HMW-UK was more resistant to reduction, and the molecule did not completely dissociate into two protein bands (heavy and light chains) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2) The isoelectric point of T HMW-UK (pI=9.3) by isoelectric focusing was more alkali than that of U HMW-UK (pI 8.9). 3) The specific activity of T HMW-UK (63, 500IU/mg protein) was approximately half of that of U HMW-UK (100, 000-120, 000IU/mg protein).
The results indicate certain difference in physicochemical properties between T HMW-UK and U HMW-UK, and that some T HMW-UK is composed of single polypeptide chain or that some differences exist in the sugar composition of T HMW-UK and U HMW-UK.

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Tissue distribution of radioactive carbon yielded from ¹⁴C-Urokinase (¹⁴C-UK) in the mouse was studied by whole-body and micro-autoradiography. The mice were frozen with acetone-dry-ice at 30min, 1hr and 6hrs after an intraduodenal administration of ¹⁴C-UK. Whole-sagittal sections of the frozen mouse, made by a large microtome in a cryostat, dried in a cryostat, were contacted with X-ray film to obtain autoradiogram. For microautoradiography, pieces of tissues were fixed in 10% formalin solution for 24hrs and the material was embedded in paraffin to make 5μm thick sections. These sections were dipped in emulsion (NR-H2 type, Kodak), and the preparation were exposed for 2 weeks. After developing these sections were post-stained with hematoxylin-eosin. The radioactivity of ¹⁴C-UK remained at least 6hrs in the intestinal wall of mice. In histological microautoradiograms, the radioactivity of ¹⁴C-UK was mainly distributed in intravascular space.
Besides them, the radioactivity of ¹⁴C-UK also appeared in thymus, spleen and lymph nodes at 1hr after intraduodenal administration, and then it seemed to be transferred into the lymphoid channels as well as into the intravascular space.

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Urokinase (UK), a plasminogen activator, was immobilized on six different kinds of polymer tubes and the antithrombogenicity evaluated.
89 UK-immobilized tubes were used in cases of thyroid and breast surgery and 65 non-treated PVC tubes served as the controls. Patency of the former was 93.3%, while, that of the latter was only 4.6%. Seventeen PVC-UK drains were applied to patients surgically treated for cardiac disorders and 55 usual non-treated ones were used as the controls. Milking and/or thrombectomy with Fogarty's balloon catheters was rarely necessary in the former, while, that was frequently required in the latter.

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Plasmin inhibitor was obtained from a marine alga, Monostroma nitidum, in partially purified form after gel filtration and chromatography at pH 7.4 on ion exchange column packed DEAE-Sephacel. Partially purified protease inhibitor from M. nitidum, inhibited plasmin and trypsin activities, but did not inhibit kallikrein.
A plasmin inhibitor fraction was obtained from a marine red alga, Porphyra yezoensis. It was in purified by gel filtration on Sepharose CL6B and by ion exchange chromatography on DEAE-Sephacel at pH 7.4 and then purified by affinity chromatography on immobilized plasmin. The inhibitor fraction on gel filtration was eluted in the region of M. W. 10, 000-12, 000, which inhibits competitively the plasmin catalyzed hydrosis of H-D-Valyl-L-leucyl-L-Lysine-p-nitroanilide (S-2251). Purified plasmin inhibitor from P. yezoensis inhibited Cl esterase activity, but did not inhibit the alternative pathway.
Four types of purified protease inhibitors were obtained from a marine red alga, Schizymenia dubyi. Inhibitor I inhibited plasmin, kallikrein and plasma complement activities, but did not inhibit trypsin activity. Inhibitor II inhibited kallikrein activity, but did not inhibit plasmin and complement activities. Inhibitor III inhibited kallikrein, plasmin and trypsin activities, but did not inhibit complement activities. Inhibitor IV inhibited kallikrein and trypsin activities, but did not inhibit complement activities.
We investigated the distribution of the pharmacologically active polymer of marine algae and these compounds to have a proteinaceous nature.

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A conventional method for the estimation of plasma α₂-plasmin inhibitor (α₂-PI) was proposed upon introduction of a novel chromogenic substrate, yet procedures were still incommoded as routine assay method. We present a improved procedure so as to put this method in running order.
Procedure: Citrated plasma to be assayed is diluted first 101 folds with Tris-HCl buffer (pH 7.5) and 0.2ml of aliquot is incubated for 10 minutes at 37°C with 0.05ml of anti α₂-macroglobulin (anti α₂M) 0.1ml of plasmin solution is then added and stand for 10 minutes at the same temperature, followed by the addition of substrate solution (Tos-Gly-Pro-Lys-pNA) and incubate whole reaction mixture for further 10 minutes. Reaction is stopped by adding 2.5ml of acetic acid (10%) and read absorbancy at 405nm.
Results: Plasmin inhibiting activity of α₂-macroglobulin in specimen sera appears to be completely blocked by the addition of anti α₂M under the conditions provided. The fact that the estimated values with specimen sera are apparently the same as those of reference sera deprived of α₂-macroglobulin by affinity chromatography, is revealing of the specificity of this method for the α₂-PI assay. This assay method shows linearity up to 125% and the correlation coefficient of this method vs. the immunological method is 0.97.

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