1. Consideration of the morphology of a thrombus shows the presence of platelets and many confounding factors and processes.
2. This suggests that in vivo a balance is stuck between activation and inactivation.
3. Laboratory evidence of platelet involvement in thrombus will be reviewed. While a short platelet survival is proof of platelet involvement, “damage” can result in “exhausted” platelets which circulated for a normal time. The following tests will be considered; The non-specific heparin thrombin clotting time; serotonin release; malondialdehyde production and platelet factor 4 and β thromboglobulin and platelet aggregation.
4. The contribution of the vessel wall will be considered.
5. Successful therapeutic treatment of thrombosis if rightly understood should enlarge our knowledge of the mechanics involved.

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There are two forms of factor VIII (F VIII), the high molecular weight factor VIII (HMW F VIII) and the low molecular weight factor VIII (LMW F VIII), in rabbit plasma. Rabbit LMW F VIII was adsorbed by Al(OH)₃ and did not appear in the cryoprecipitate. By utilizing adsorption with Al(OH)₃, elution with 0.15M sodium citrate and concentration with polyethylene glycol, we isolated rabbit LMW F VIII from rabbit plasma. Finding that rabbit LMW F VIII could be adsorbed by Al(OH)₃, we obtained a procoagulant by utilizing this on the assumption that such a LMW F VIII was present in a small quantity in human plasma. This procoagulant activity eluted at the same elution volume where rabbit LMW F VIII eluted on the 6% agarose gel column and was inhibited by rabbit antihuman F VIII serum, indicating that it was human LMW F VIII. Rabbit LMW F VIII did not combine with rabbit HMW F VIII subcomponent which dissociated from purified HMW F VIII in the presence of 0.25M CaCl₂. Also, human HMW F VIII subcomponent which dissociated from purified HMW F VIII in the presence of 0.25M CaCl₂ did not combine with human LMW F VIII isolated by above method. These results indicated that LMW F VIII was also present in human plasma and that it had properties some what different from those of the LMW F VIII subcomponent dissociated from human purified F VIII under high ionic strength.

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To investigate the role of von Willebrand factor in the thrombogenesis, we measured plasma level of von Willebrand factor (VIII RCoF and VIII RAG) in 31 cases of chronic myeloproliferative diseases; chronic myelogenous leukemia (CML): 19, primary thrombocythemia (PT): 8 and polycythemia vera (PV): 4, who present haemorrhagic and/or thrombogenic manifestations. In several cases we also investigated the platelet von Willebrand factor and it's movement to extracellular medium in vitro. VIII RCoF was measured by Weiss' method, and VIII RAG by Laurell's method. Platelets of five cases and six normals were washed 4 times by tris buffer (pH 7.3) by Weiss' method and then suspended in the buffer. One part of the suspension was used to measure platelet von Willebrand factor, using a freezing and thawing technique. The other of the suspension was incubated 37°C 2hrs, then studied ristocetin induced aggregation. Supernatant was also collected from this incubated suspension for the measurement of VIII RCoF and VIII RAG. Plasma level of VIII RCoF in those patients was distributed from abnormal low level to extreme high level. Sometime the data were variable in same patients depend on the intervals of measurement. Plasma level of VIII RAG exhibited the same tendency. Platelets of 2 CML cases had very high value of VIII RCoF and VIII RAG and had strong aggregability to ristocetin after incubation at 37°C for 2hrs. The supernatant of these incubated suspension had the activity of both VIII RCoF and VIII RAG, the latter was not detected in the supernatant of normal, PT or PV. Platelets of 2 cases of PT and a case of PV had very low von Willebrand factor and after incubation aggregability to ristocetin was weak and these supernatant had low VIII RCoF and any VIII RAG was not detected.
Whether these results of platelet von Willebrand factor are characteristic to the diseases or not, it is necessary to separate and identify this platelet factor physicochemically.

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By employing Laurell's method, a sensitive and reproducible antigen assay for human Factor XII was carried out with purified Factor XII and monospecific rabbit anti-body.
It was noted that TAIYO-ID-1 agarose was most suitable material in the procedure.
Precise measurements of Factor XII were possible for concentrations as low as 3% of that in normal pooled plasma. A good correlation (correlation coefficient=0.82) existed between the titers of Factor XII measured by clotting activity and antigen assay among 40 normal adults. Factor XII antigens were absent in patients with congenital Factor XII deficiency, but other congenital deficient plasmas, including those of patients with hemoplilia A and B, Factor XI or Factor VII deficiency, von Willebrand disease and hereditary angioneurotic edema (C^-₁; inactivator deficiency), contained normal amount of Factor XII antigen. Both clotting activity and antigen of Factor XII were reduced in the plasmas of patients with liver cirrhosis or those of half cases with disseminated intravascular coagulation. They were in normal ranges in those of patients treated with Warfarin. Factor XII antigen was normal in spite of reduced clotting activity in the plasmas of patients suffered from SLE.

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The clotting time of experimental and clinical plasma specimens were investigated by addition of thrombin or snake venom (Defibrase).
I. In vitro studies:
(1) Thrombin time and snake venom clotting time were dependent on the concentrations of thrombin or snake venom, and when these concentrations were settled, the clotting times were dependent on the fibrinogen concentration in the plasma.
(2) Thrombin times were prolonged in the cases with 135γ of FDP, but snake venom times were not so makedly prolonged. The discrepancy between thrombin time and snake venom time were prominent with increase of FDP. Snake venom times were prolonged with decrease of fibrinogen, but not related with the amounts of FDP in the plasma.
(3) When the purified antithrombin III, α₁-antitrypsin and α₂-Macroglobulin were added to the plasma separately, only the antithrombin III made thrombin time prolonged in the case with 0.45mg/ml of antithrombin III in the final concentration.
II. Clinical studies:
In the liver diseases, thrombin times were dependent on the concentration of fibrinogen in the plasma, but snake venom times were not so much related to the concentration of fibrinogen. In the case with fulminant hepatitis showing 120mg/dl of fibrinogen, 10γ/ml of FDP and 11mg/dl of antithrombin III, snake venom time showed shorter clotting time than that of thrombin time. These results showed the existence of molecular abnormality of fibrinogen in the plasma.

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Blood coagulation-fibrinolysis system is of great importance on tumor growth and metastasis. Our previous studies on the inhibition of metastases by sulfated polysaccharides have shown that intravascular coagulation was a very important mechanism at an early stage of hematogenous metastasis.
For the purpose of investigating the significance of fibrinogen on tumor growth and metastasis, we studied effect of defibrinogenation with ancrod in mice. Lewis lung tumor (3LL) is a favorable model of pulmonary metastases in syngeneic C57BL/6 mice. Therefore, 3LL cells were inoculated via tail vein for intravenously induced pulmonary metastases or subcutaneously implanted into foot pad for those of spontaneity.
Primary tumor was amputated 9 or 12 days after tumor implantation. Mice were autopsied on day 14 after intravenous tumor inoculation or day 23 after tumor implantation into foot pad. Pulmonary metastases were assayed grossly by counting the number of pulmonary surface nodules and measuring dry weight of metastatic lungs.
Our experimental results for ancrod on tumor growth and metastases are as follows. Significant reduction of intravenously induced pulmonary metastases in mice treated with 200units/kg of ancrod once intraperitoneally, one hour before tumor inoculation (P<0.001). Plasma fibrinogen levels decreased to the extent of trace in the first 4 hours and was 55mg/dl in 12 hours after administration in mice treated with same dose of ancrod. Furthermore, significant reduction of spontaneous pulmonary metastases was obtained in mice treated with 200units/kg of ancrod twice daily for post-amputation period starting from day 15 to 22 after tumor implantation (P<0.01). However, no inhibition of spontaneous metastases was observed in mice treated for only pre-amputation, and pre-and post-amputation period. Plasma fibrinogen levels increased prominently after tumor amputation in mice. On the contrary, those were less than 71mg/dl in all mice bearing 3LL treated with ancrod. No inhibition of primary tumor was observed at the time of amputation. The significance of fibrinogen on tumor growth and metastasis was discussed.

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Tissue thromboplastin (T. Tbp) plays an important part in inducing the pulmonary microthrombo-embolism. We have studied the ultrastructures of T. Tbp to demonstrate how it changes during coagulation.
[Materials and Methods] T. Tbp from lungs of rabbits, lyoplastin (Mochida Ltd.), was used for these studies. Lyoplastin was injected into ear vein of rabbits. Lungs were resected at several seconds, 10sec, 1min, 5min, 24hrs, 48hrs or 1 week after the injection.
The specimens were fixed in phosphate buffered formalin, followed by post-fixation in osmium tetroxide solution. They were examined by transmission electron microscope.
[Results] Concentrically arranged membrane structures of the injected T. Tbp disappeared in extremely short time after the injection. 1 min after the injection, the fibrin fibers were seen between single sheet of membrane and endothelial cells of capillaries. In the rabbit which had died suddenly after the injection of T. Tbp, multiple pulmonary thrombi made of fibrin and platelets were seen in capillaries. The endothelial cells of capillaries were destroyed and interstitial tissues were edematous. At 24hrs after the injection, no fibrin-thrombi were seen in capillaries, while endothelial cells were degenerated in some capillaries. It was suggested that multiple pulmonary fibrin-thrombi had been resolved in 24hrs. Type B alveolar epithelial cells seemed to lose their lamellar inclusion bodies in rather short time after the injection and the vacuolization of the lamellar inclusion bodies progressed till 1 week or more after the injection.
Thus, the injection of T. Tbp in rabbits brought remarkable injuries in pulmonary capillaries and surrounding tissues.

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We have already reported on the measurement of urinary FPA. The purpose of this study is to clarify the origin of the urinary FPA.
1. Tracer study using ¹²⁵I-FPA gave the following results: 1) The disappearance curve of ¹²⁵I-FPA from blood showed the following equation,
X=78.8e^-0.531t+19.4e^-0.0175t
where: X is the FPA (%) remained in blood at the time t (min.). 2) The disappearance rate of ¹²⁵I-FPA from blood to extravascular space was very fast. The rapid component of this equation gave 1.8min. of half life of FPA. 3) The blood FPA appeared in urine 5min. after injection. The accumulated FPA in urine for 1hr reached to 40% of the injected FPA.
2. The study of organ distribution of ¹²⁵I-FPA at 1hr after injection showed less than 10% of total FPA mainly in liver and kidney.
3. The fibrinogen added to urine gave no increased level of FPA. The immunoreactivity of FPA in urine was not significantly changed by the incubation for 24hr at 37°C. The possibility for decomposition and generation of FPA in the urinary tract was, therefore, found to be negative.
In conclusion, although the determination of FPA level in plasma is a direct evidence for the generation of thrombosis, our results showed that the FPA level can not be a diagnostic proof for estimating the size of thrombi, since plasma FPA was excreted rapidly into urine. The measurement of urinary FPA may suggest a possibility for the detection of intravascular thrombosis.

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In-vitro and in-vivo studies of the relationship between the doses of urokinase and inhibitory activities in plasma were performed.
On the in-vitro determinations of “one dimensional diffusion method”, the following relationship of the doses of urokinase to the inhibitory activities in plasma was recognized:
log y=ax+b…Formula (1)
where y is the minimal effective dose of urokinase, x is the level of the inhibitory activity of the plasma in fibrinolytic process.
The correlation between the doses of urokinase to the levels of the inhibitory activity in the plasma was not shown on the assay system of “urokinase-induced olasma clot lysis time method”.
We investigated the relationship between the doses of the urokinase added in plasma and the levels of the anti-plasmin activities of the plasma by the method of lysis time. Immediately after the addition of urokinase to the plasma, the following relationship between the doses of urokinase and the suppressed levels of anti-plasmin activities was observed over the range more than 100IU/ml of urokinase.
log y′=cx′+d…Formula (2)
where y′ is the dose of urokinase, x′ is the suppressed level of anti-plasmin.
In addition, when the urokinase was kept in plasma at 37°C for several hours, the slope of Formula (2) changed, that is to say, the anti-plasmin activity was greatly suppressed when small amounts of urokinase was added to plasma.
Furthermore, we measured the levels of the anti-plasmin activities following the administration of urokinase to two patients. It was shown that the level of anti-plasmin activity was decreased about 60% of the pretreatment level following the administration of 300, 000 IU of urokinase.
It is concluded that the thrombolytic activity is probably induced from the administration at least 300, 000 IU of urokinase.

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A new method for detecting fibrinogen related proteins such as fibrin polymer, soluble fibrin complexes (SFC), fibrinogen, early FDP (mainly X), α, β, γ and γ-dimer, was developed by using the technique of adsorption and elution from staphylococci. The procedure of staphylococcal elution technique (SET) was as follows: One tenth ml of sodium citrate plasma was mixed with 2mg of staphylococci and reacted for 10min, washed three times by saline. After washing 0.3ml of 0.05M Tris buffer (pH 11.6) was added and centrifuged. The pH of the resultant supernatant was adjusted to 8.0 by adding 0.05M Tris buffer (pH 6.0). This supernatant (0.2ml) was electrophoresed by SDS-PAGE. After staining and destaining the density of protein band was measured by scanning densitometer.
In vitro experiments using purified fibrinogen, FDP-D and FDP-E revealed that fibrinogen, SFC which was made by adding low concentration of thrombin to fibrinogen, and early FDP (mainly X) which was made from the digestion of fibrinogen by plasmin were eluted by SET. Moreover, D and E were also eluted by SET, though the amount of eluted D and E were fairly low.
In vitro experiments using normal plasma revealed that eluted normal plasma contained 5 protein bands corresponding to fibrin polymer, fibrinogen and γ-globulin, and probably corresponding to D and E or albumin.
Eluted plasma with DIC showed the bands of fibrin polymer, SFC and fibrinogen. Eluted plasma with large dosage of UK therapy showed the bands of fibrin polymer, fibrinogen, partly degraded fibrinogen and FDP-X. The reduction of the eluted normal plasma demonstrated 3 bands which were coincided with α, β, and γ-chain. The reduction of the eluted plasma with DIC demonstrated the bands of γ-dimer (7.1%), α-chain (44.7%), β-chain (27.2%), γ-chain (18.9%) and another bands.
Although problems as to precise quantification and as to adsorption and elution of D or E are unsettled, SET would be available for clinic as well as the study of the relation between fibrinogen and its structure.

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Fibrin polymers formed from fibrinogen with thrombin in the presence of EDTA were suspended in a medium containing glucose, arabic gum and imidazole-HCl buffer and were sonicated at 20kHz for 20min to make a suspension containing fibrin particles of small size. The fibrin suspension was used as a substrate for determining the enzymic activity of plasmin and plasminogen activated with urokinase. The kinetic study on the reaction of the fibrin particles with plasmin in the presence and the absence of fibrinogen revealed that the Km value of fibrin for plasmin is 4.2×10^-7M and the Ki value of fibrinogen is 1.2×10^-5M.

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Comparison of three synthetic substrates, S-2251 (H-D-Val-Leu-Lys-pNA), TLMe (tosyl lysine methyl ester), TAMe (tosyl arginine methyl ester) was done with respect to substrate specificity for thrombin, urokinase (UK), UK-activated plasmin, and kaolin-activated plasma (kallikrein). TLMe was hydrolyzed by thrombin and to a lesser extent by plasmin, and TAMe was hydrolyzed by thrombin and to a lesser extent by plasmin and kaolin-activated plasma. S-2251 was very specific to plasmin. The hydrolysis of S-2251 by plasma in the presence of UK and SK (streptokinase) was higher when clot was formed by the addition of thrombin than in the absence of clot. The hydrolysis of S-2251 by euglobulin in the presence of UK was also higher when clot was formed, thus, inhibitors may not be related to the better activation of plasminogen in the presence of fibrin clot. It may be suggested that plasminogen was better activated by activators in the clot than in its absence. Acid treated plasminogen (mainly Lys-plasminogen) was not better activated by UK in the clot compared to the activation in the plasma.

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This report was undertaken to study the direct effects of endotoxin on the in vitro fibrinolytic system which has not been fully understood.
Shartening of both euglobulin lysis time and partial thromboplastin time was observed by the activation of normal plasma with endotoxin proportionally to its dose above 0.1mg per ml. And it was suggested that contact system was activated and surface mediated fibrinolysis was induced with endotoxin. Endotoxin had no direct effect on the plasmin activity, but it has accelelating effect on the plasminogen activator activity of urokinase and streptokinase measured by the fibrin agar plate method.
Endotoxin detected in various gram-negative bacterial infections is extremely little as compared with the in vitro effective dose in the present study. Thus it was suggested that the direct effect of endotoxin on the fibrinolytic system is not caused in vivo. Therefore the fibrinolytic system is mainly influenced by the secondary effects of platelet, leucocyte and vascular endothelium damage caused by endotoxin.

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Fibrinolytic effects of Defibrase (Batroxobin) administered into a patients with Barger's disease and four normal adults are obserbed in this paper.
On the cases for single injection with Batroxobon (0.8-1.6BU/kg) into four normal adults, the fibrinogen in plasma is not detected by usual method between 8 and 24hrs after injection, and then it increases gradually. However, the concentration of FDP including X, Y, D and E fragments indicated by immunoelectrophoretic technique, with plasmin activity measures more than 1, 000μg/ml between 4 and 48hrs after injection and the levels of plasminogen and α₂-plasmin inhibitor show the lowest values between 8 and 13hrs and decrease to 40-50% and 20-30% respectively.
On the case of Barger's disease treated with continuous administration of Batroxobin (0.5-0.9BU/kg), the factors of fibrinolytic system shows the same tendencies as single shot data of normal cases and those tendencies are kept continuously during the treatment with Batroxobin. Furthermore, in general the plasma after the injection with Batroxobin clots spontaneously, when it has been collected in the test-tubes, and it is analyzed that the clot consists of des-A fibrinogen which was digested into X fragment of FDP and crosslinked of γ-chain of des-A fibrinogen in paraclot. Therefore, it is presumed that this phenomenon might be one kind of enzymatic paracoagulation of plasmin-digested des-A fibrinogen with or without fibrinogen by the exsisting of the very small amount of snake venom and activated factor XIII.

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Vascular endotherial cells release plasminogen activator (plg-act) which is thought to play a key role in maintaining the fluidity of circulatory blood. It was reported that endotherial cells had thrombin receptors, so the present study was performed to evaluate the effect of purified thrombin on plg-act release from the vascular wall of the perfused hind leg of dog with physiological solution. Plg-act activity was measured using a standard fibrin plate. When 5units/ml thrombin (5ml) was administered to the perfused hind leg, plg-act was released within 5 sec. and reached to the peak activity at 15-30 sec. A dose response of plasminogen activator activity to thrombin was also detected to a certain extent (1units/ml-50units/ml). The peak activity of released plg-act after the administration of thrombin (5units/ml) was in the range of 0.38 CTA units/ml-0.44 CTA units/ml. The effects of DFP-thrombin and TLCK-thrombin, which are inactive in fibrinogen-fibrin conversion, on plg-act release were examined. The two sorts of chemically modified thrombin did not release plg-act. Furthermore, the effect of acetylated thrombin was also examined. The acetylated thrombin did not cause plg-act release. These results obtained indicate that thrombin releases plg-act from the vascular wall so long as thrombin maintains the intact active center of fibrinogen-fibrin conversion.

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The role of tissue fibrinolysis, lysosomal enzymes and lipid peroxidation on the hemorrhagic necrosis of digestive mucosa was investigated using experimental models of DIC (disseminated intravascular coagulation), hemorrhagic shock and mesenteric occlusion in the dogs. The experimental model of DIC was produced by intravenous administration of bacterial lipopolysaccharide (endotoxin), hemorrhagic shock by withdrawing blood from the femoral artery through cannula, keeping the arterial pressure between 50 and 60mmHg, and mesenteric occlusion by ligating the intestinal artery.
The fibrinolytic activity and the lysosomal enzymatic activity, beta-glucuronidase and acid phosphatase, in the biopsied digestive mucosa revealed marked elevation following the administration of endotoxin, withdrawal of blood and ligation of the intestinal artery, to which the changes of the lipid peroxide value were found to be correlated.
These data led us to conclude that the lipid peroxidation due to the ischemic change damages the lysosomal membrane followed by the release of an array of hydrolytic enzymes including plasminogen activator to bring about cell damage. The results allow to conclude that the lipid peroxidation plays an important role in the pathogenesis of ischemic colitis.

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A case of Hepatosplenomegaly associated with Pancytopenia, bleeding tendency and markedly shortened Euglobulin Clot Lysis Time (ELT) was presented. The patient was undergone Splenectomy aiming of recovery from Pancytopenia. Histologically spleen showed Splenitis-like changes. It is of interest ELT recovered to normal range after Splenectomy. Therefore, the results of fibrinolytic data obtained were as followed.
1) In vitro, ELT of normal plasma was shortened in addition of patient's plasma to normal plasma.
2) t-AMCHA and Trasylol prolonged ELT of patient's plasma in vitro and in vivo.
3) Patient's plasma and euglobulin did not so strongly hydrolyze TAME, AGLME, S-2251, S-2238 and RM601-1 (Pentapharm).
4) A supernatant of homogenized spleen digested Bovine Standard Fibrin Plate, but not Bovine Plasminogen free Fibrin Plate and also this supernatant did not inhibit Plasmin and Urokinase.
From these results, fibrinolytic system of this case was specific, and it might be suggested that the spleen of this case with decreased liver function and inflammation of spleen released high amount of Plasminogen Activator. Furthermore, we discussed the spleen might play some rule in the fibrinolytic system.

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For the evaluation of fibrinolytic activity of the glomerulus, we employed the isolated glomeruli of the rat and compared with that of Urokinase. The fibrinolytic activities were examined by the fibrin agar plate, auther's original method (S-P disc method) and the method using the chromogenic substrate, S-2444 which is specific for Urokinase. By S-P disc method fibrinolytic activities were determined by counting the radioactivities of the degradated products of ¹²⁵I-fibrinogen by means of fractionation with SDS-polyacrylamide gel disc electrophoresis.
The results obtained were as follows;
(1) Most of the fibrinolytic factors in the rat glomerulus were plasminogen activator.
(2) By S-P disc method the difference of the fibrinolytic activity between isolated glomeruli and Urokinase were detected in the course of the degradation of ¹²⁵I-fibrinogen, i. e. isolated glomeruli produced mainly Fragment X, however Urokinase produced all of the Fg. D. P. fragments.
(3) The homogenate of renal medulla and cortex had the amidolytic effect on S-2444 and the former showed higher activity than the latter. However isolated glomeruli showed neither enzyme activity on this specific substrate for Urokinase nor inhibitory effect on this system.
These findings suggest that in the fibrinolytic mode of action the plasminogen activator in the glomerulus is different from Urokinase which is reported to be synthesized in the renal tubular cells. And from these point of view glomerulus and renal tubulus may act the different part respectively in the fibrinolytic system of the kidney.

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In order to clarify the mechanism of the release of the plasminogen activator (Plg. Act.) in the tracheobronchial secretion of rat, the effect of some vasoactive drugs to this activity in the secretion was studied in this experiment.
The results obtained from this experiment were as follows: It was observed that after the injection of noradrenalin the blood pressure was temporarily elevated and the fibrinolytic activity was too elevated. Following these changes the activity of Plg. Act. in the secretion was increased in comparison with the injection of atropine sulfate.
From these results it was suggested that the release of Plg. Act. into the respiratory lumen might be partly regulated by neurogenic system.

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We have investigated plasma kallikrein system in various diseases. This study was undertaken to elucidate the mechanism changing the level of these factors in various disorders.
Factors of plasma kallikrein system were measured with TAMe hydrolysis method introduced by R. W. Colman. Factors in fibrinolytic system were measured with plasminogen free fibrin plate. Protease inhibitors (α₁ antitrypsin, α₁ antichymotrypsin C-1 inactivator, Inter α₁ trypsin inhibitor, antithrombin III, α₂ Macroglobulin) were measured by paltigen plates.
In 42 cases of hematological diseases, although the value of prekallikrein and enzyme inhibitor changed according to the patient's condition, only prekallikrein and plasminogen showed high correlation suggestive of the hypothesis that prekallikrein level changed due to the consumptive or degradation process. Enzyme inhibitor paralleled with α₂ macroglobulin but not with C₁ inactivator. This result was differ from in vitro investigation with purified materials.
In 89 cases of liver diseases, the value of prekallikrein showed parallel changes with that of enzyme inhibitor. They showed good correlation with antithrombin III, C₁ inactivator. In 25 cases of liver cirrhosis, it was objous that prekallikrein and enzyme inhibitor correlated well with TP, A/G, Proth. T, HPT, antithrombin III, C₁ inactivator.
This study clearly defined that in liver diseases, the prekallikrein and enzyme inhibitor changed the value according to the state of synthetic function of the liver, while in hematological diseases, the value changed according to the consumptive mechanism of the factors.

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A young Japanese male with multiple thromboembolism was found to have antithrombin III deficiency. A family survey spanning 4 generations revealed a total of 4 members with AT III deficiency. Two of these 4 affected members, including the propositus, had experienced thrombotic problems. Besides the reduced AT III levels, hyperlipidemia was observed in all the affected members, particularly in the levels of triglyceride and phospholipid. Treatment with warfarin led to an increasing tendency in AT III level. The mode of inheritance of the defect was found to be autosomal dominant. A discussion of the significance of AT III deficiency is given together with a brief review of previously reported cases.

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In the estimation of anti-thrombin III (AT III) in the plasma, biological or immunological methods have generally been employed so far. However, certain limitations such as the intricate and time-consuming nature of the procedures can be raised against their practical use.
Recently conventional methods using chromogenic peptide substrate have been devised, although inasmuch as the reaction time is extremely short with this method it is inevitable that technical failure occures and the method is not convenient for examining large member of specimens. Also, due to the presence of inhibitors other than AT III in the plasma, the dilution effect should be considered.
The above difficulties have been overcome in the following way. Citrated plasma was diluted 71-folds with Tris-HCl buffer (pH 8.8) containing appropriate amounts of heparin and aprotinin. The diluted specimen (0.05ml) was then added to the same buffer (0.2ml), thrombin solution (0.1ml), and allowed to stand at 37°C for 10min. This was followed by addition of the substrate (Tos-Gly-Pro-Arg-pNA: purchased from Pentapharm) and then further incubation at 37°C for 10min. The reaction was stopped with 2.5ml of acetic acid (10%) then absorbancy at 405nm was measured.
The results of our experiment will be summarized as follows:
1) The reaction rate of AT III for anti-thrombin action is accelerated by heparin.
2) The activity of plasma enzymes having amidase activity such as trypsin and plasmin except thrombin, was inhibited by aprotinin.
3) Anti-thrombin action was not affected by any inhibitor other than AT III.
4) Measurements of AT III activity in a series of diluted specimens showed a linearity up to 125%.
5) A good correlation with the immunological method was observed (r=0.988).
6) The procedure was well suited to the measurement of AT III in relation to the estimation of hemorrahgic diathesis.

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One of the serine protease inhibitors, Antithrombin III (AT III) plays an important roles on the development of hypercoagulable state and this experimental studies were designed to investigate its metabolic kinetics from aspect of AT III biosynthesis in rat.
AT III was purified by heparin Sepharose affinity chromatography and as a precursor of AT III 14C-Glycine was utilized. From the radioactivity into AT III fractions of rat tissues, its biosynthesis was evaluated on the effect of some experimental conditions.
In order to investigate the effect of the ischemia in liver on AT III synthesis, one lobe of liver was ligated for 2 or 4 hours after intraportal injection of ¹⁴C-Glycine, and compared with non-ligated liver lobes. The biosynthesis of AT III was inhibited 64% for 2 hour-ligated liver lobe and 79% for 4 hour-ligated one, while the biosynthesis of tissue proteins was inhibited 56% for 4 hour-ligated liver lobe but there was no difference for 2 hour-ligated one.
Intraperitonially injected Ethionine, ATP depleting agent, inhibited the incorporation of ¹⁴C-Glycine into both AT III fractions and tissue proteins but the biosynthesis of AT III was more remarkably inhibited than tissue proteins in liver. The ratio of ¹⁴C-Glycine incorporation into AT III/tissue proteins was reduced in liver (p<0.01).
It can be said that ischemia and/or ATP depletion in liver inhibit the biosynthesis of AT III more prominently than other tissue proteins.
These results suggest a possibility of vicious cycle between the inhibition of AT III biosynthesis in ATP depletion induced by ischemia and the development of hypercoagulable state due to circulating disorders.

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This study was carried out to know how to play Antithrombin III (AT III) had taken in thromboembolic disorders, and which of two methods that consisted of immunological method by single radial immunodiffusion and quantitative clotting assay for AT III, showed in more significant and useful for clinical survey to thromboembolic disorders.
1) AT III normal healthy persons (n=300) were decreasing to low level according to age in both sex. In sex difference, AT III of male group was higher value than female until third decade. And female group showed in higher value of AT III than male after fourth decade. Thus, AT III in normal have been influenced significantly by the difference of sex and age.
2) In thromboembolic disorders, it was presumed that increase and decrease of AT III would be evaluated in comparison with strict age and sex matched control. 24 cases to be chosen from within DIC group corresponded to the cases of same age and sex from other groups with liver cirrhosis, myocardial infarction and malignant tumor. The lowest mean value of 18.2±5.4mg/dl was found in DIC group (n=24) and second in low AT III was the death group with myocardial infarction of 20.7±2.9mg/dl (n=24). Third group was liver cirrhosis of 21.6±6.1mg/dl (n=24).
3) In hereditary AT III deficiency, the propositus had both of low levels of AT III obtained from immunological concentration and doting assay. The discrepancy between two methods were found in her young brother, and child. Those two blood relatives were shown in normal clotting activity and in pathological low level of immunological concentration. The same discrepancy was found in the clinical condition of secondary decrease of AT III such as liver cirrhosis.
It is suggested that clotting activity and immunological concentration in AT III were found to have good correlation in normal, and pathological conditions in low level of AT III would appeared to be the discrepancy between the results from two methods.

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Tritiated heparin prepared from porcine intestinal mucosa was fractionated by gel chromatography using Sephadex G-200. The radioactivity of fractions was widely distributed. The obtained fractions were processed for metachromasia of Azur A and anticoagulant activity. These fractions were devided into three main groups designated as high, middle and low molecular heparins. Mean molecular weight, sulfate content, heparin activity and complex formation with plasma protein were investigated on these three groups. These three groups were intravenously injected into rats respectively and their anticoagulant activity, radioactivity in the blood, urinary excretion and tissue distribution were studied in comparison with the whole heparin.
The high molecular heparin had high anticoagulant activity, high sulfate content and great metachromasia, complex with antithrombin III, and was neutralized combining with protamine sulfate. On the other hand, the low molecular heparin had no anticoagulant activity and did not combine with plasma protein or protamine sulfate. When high molecular heparin was administrated into rats, it provided the higher and longer lasting anticoagulant activity and radioactivity in the blood than the whole heparin. 43.8% of radioactivity of injected high molecular heparin was excreted into the urine in three hours and much more of the low molecular heparin was excreted in the same condition, while more radioactivity of injected high molecular heparin accumulated in organs, especially in the liver and kidneys.
Through these experimental results, it is concluded that high molecular heparin would be the most potent and effective form for anticoagulant therapy.

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To find out whether the depressed fibrinolytic activity in pregnancy can be ascribed to an increased content of inhibitors in the blood or to a release of inhibitors from the placenta, we studied the variation of the fibrinolytic inhibitors such as α₁-AT, α₂-MG, AT-III and C₁-inactivator during pregnancy. However, we did not find any significant increase in inhibitors. In this paper, the levels of antiplasmin including α₂-PI devised by Aoki and UK inhibitors extracted from the placenta by Kawano during pregnancy and delivery were examined.
Methods:
The material consisted of citrated plasma from pregnant women and extracts from the placenta. α₂-PI was assessed with a single radial immunodiffusion method using antiserum made by Aoki. Antiplasmin activity was measured with chromogenic substrate S-2251 and then UK inhibitor was calculated from a standard curve prepared from placental UKI using the chromogenic substrate S-2444. The results were expressed relative to the content of normal standard as %, iu/ml and u/g.
Results:
1) The levels of α₂-PI with a single radial immunodiffusion method correlated well to those of antiplasmin using the chromogenic substrate S-2251.
2) Antiplasmin activity including α₂-PI remained unchanged throughout pregnancy within 80-130%, and followed a similar patern after delivery. How-ever, it relatively decreased in retroplacental blood as well as uterine venous blood.
3) In extracts of the placenta the concentration of antiplasmin was low.
4) On the other hand, extract of the placenta contained a high concentration of UKI.
5) The concentration of UKI was extremely low in each trimester, but the increased values were noted at term and in labor as well as in retroplacental blood, and gradually decreased after delivery.
Discussion: Antiplasmin activity might directly or indirectly act upon the depression of the fibrinolytic activity in pregnancy, but UK inhibitor might be found intensively during placental separation.

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The relation between the characteristic variations of the serum protease inhibitors and clinical course were examined in patients with various blood diseases.
Materials and Methods;
Sixty-nine patients with various blood diseases (25 patients of AML, 4 of ALL, 11 of CML, 4 of APLA, 18 of iron dificiency anemia (IDA) and 7 of ITP were selected in this study. Thirty control normal subjects were also picked out. Serum protease inhibitors were measured by use of the single radial immunodiffusion method. Alpha₁-antitrypsin (α₁AT), α₂-macroglobulin (α₂M), inter-α-trypsin inhibitor (IαI), α₁-antichymotrypsin (α₁X), antithrombin III (AT-III) and plasminogen (PLg) were respectively determined in this examination.
Results and Conclusions;
The serum protease inhibitors in the blood diseases were compared with those in the normal subjects. Normal levels were shown as follows.
Alpha₁AT; 251±22mg/dl, α₂M; 213±47mg/dl, IαI; 58±5mg/dl, α₁X; 45±5mg/dl, AT-III; 23±4mg/dl and PLg; 14±2mg/dl.
Alpha₁ AT and α₁X markedly increased, α₂M, IαI and PLg decreased in AML and ALL.
Alpha₁ AT and AT-III increased in CML. Alpha₁ AT, α₁X and AT-III significantly increased in APLA. Alpha₂M and α₁X increased, PLg decreased in ITP. Alpha₂M markedly increased in IDA. When the patients with leukemia changed to remission stage from acute stage, serum α₁AT and α₁X elevating in acute stage decreased, on the other hand serum IαI and α₂M transfered to normal range from low level. In the complete remission stage of the clinical course, the α₁AT and α₁X levels returned to the normal levels but α₂M, IαI and PLg continued slightly elevated in AML.
It was suggested that these hematologic findings would be available as indication of prognosis and therapeutic effect in various blood diseases.

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Effect of ticlopidine administration, a new inhibitor of platelet aggregation, at daily dose of 300mg was compared with aspirin at daily dose of 500mg in double blind basis. The suppression of platelet aggregations induced with ADP, collagen and epinephrine were compared in 38 cases of thrombotic tendency or enhanced platelet functions.
Overall judgement of the inhibitory efficacy of ticlopidine on the aggregations induced with collagen or epinephrine was shown to be as potent as that of aspirin, but the inhibitory efficacy of ticlopidine on the aggregation induced with ADP was apparently superior to that of aspirin. The maximum aggregation rate was significantly decreased and aggregation profile was improved in the ticlopidine-treated group when compared with the aspirin-group. Maximum aggregation rates induced with collagen or epinephrine were significantly decreased in both groups to the same extent.
Blood filtration velocity through Nuclepore filter of 5μm pore size was significantly enhanced in the ticlopidine-group but not in the aspirin-group. Other coagulation parameters and laboratory data were unchanged in both groups, except bleeding time and prothrombin time which were slightly prolonged in the aspirin group.
From above results, it was expected that ticlopidine may exert superior clinical effect to aspirin on thrombotic diseases and microcirculatory disorders.

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Ticlpidine, a new inhibitor of platelet aggregation, has been administered to 80 patients with ischemic ulcer due to chronic arterial occlusion, at daily dosis of 300mg or 500mg orally for 6 weeks. Overall judgement on its usefullness revealed that daily 500mg of the drug is superior to 300mg daily dose in the treatment of ulcer. Administration with 500mg daily dose of ticlopidine were judged as useful for the treatment of ischemic ulcer. Ticlopidine was also found to be effective in improving severe pain without showing any serious side effects. In this regard, the drug can be characterized as an useful drug for the treatment of severe ischemic ulcer due to chronic arterial occlusion.
Based on the pharmacological property of ticlopidine, inhibition on platelet adhesion and aggregation, it is speculated that enhanced platelet function may be closely correlated to the aggravation of these occlusive diseases.

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Ticlopidine (300mg/day) was administered to five patients with primary thrombocythemia of which one had been ingesting aspirin (10mg/kg) and has shown occult blood of feces, and four had no clinical signs of either hemorrhage or thrombosis. Their platelet functions were compared between before and after (1-3 weeks) the administration. Changes in the values of platelet functions more than twice or less than half were judged to be significant.
Ticlopidine was proved to suppress the platelet functions in primary thrombocythemia as reported in normal subjects: Although prolongation of bleeding time (Duke's method) and decrease of retention rate (Hellem-II method) were found in only half cases, aggregation by ADP (10^-6, 10^-5M at final concentration), adrenaline (10^-6M) or collagen (1μg/ml) using PRP with platelet count of 3×10¹¹/l was almost completely suppressed. Spontaneus platelet aggregation using undiluted (original) PRP was also suppressed. Clinically a patient who digested both Ticlopidine and aspirin had melena two days after the begining of administration of Ticlopidine, whereasthe other patients did not show any sign of hemorrhage during the administration.
In conclusion Ticlopidine can be employed as an antiplatelet drug in patients with primary thrombocythemia, especially who show relatively high aggregability of the platelets in ADP aggregation and show positive spontaneous platelet aggregation.

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The effect of ticlopidine on experimental thrombosis in femoral artery was investigated in the rabbits.
Ticlopidine was found to be an inhibitor of rabbit platelet aggregation induced by ADP, collagen or Na-arachidonate. The inhibitory effect lasted for at least 24 hours after a single administration of 100mg or 200mg/kg of ticlopidine. The persistent inhibition of ADP, collagen or Na-arachidonate aggregation of rabbit platelets was obtained by daily administration of 200mg/kg of ticlopidine. Ticlopidine also inhibited the in vivo aggregation of platelets induced by i. v. injection of ADP.
And finaly, the frequency of occluding thrombus formation at the site of stenosing ligature of the femoral artery of rabbits was significantly reduced by the administration of 200mg/kg of ticlopidine.
From these results, it is concluded that ticlopidine might be one of useful antithrombotic agents.

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We have long investigated the development and improvement of the artificial vessels. Recently, we tried to examine the substitution for the vena cava, the shunt between aorta and pulmonary artery as well as femoral artery substitution have also been examined in dogs using Gore-Tex grafts.
In the preliminary study where canine femoral artery was substituted with Gore-Tex artificial blood vessels (inner diameter, 2.8mm), the graft was kept patent to some degree for three weeks after the substitution but all grafts were obstructed six weeks after.
To obtain the better results in the small artery grafting, we used modified Gore-Tex graft of inner diameter of 3.0mm. In addition, a large dose of ticlopidine, a new platelet aggregation inhibitor, or aspirin was administered to dog before and after the surgery.
In the control group without drug treatment, the graft patency for a long period was hardly observed, while all grafts were patent when ticlopidine or aspirin in a large amount was used.
The histological and electron scanning microscopic findings of the grafts confirmed strong inhibitory action of ticlopidine and aspirin against blood platelet aggregation. Especially, in the ticlopidine-treated group, aggregated erythrocytes on the surface of the graft were fewer and the fibrin layer was thinner as compared to the aspirin-treated group.

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A polyethylene tubing was installed between the right carotid artery and the jugular vein of the rat to make a loop-shaped arterio-venous shunt. In most of the rats, the vascular shunt was obstructed by white thrombi together with blood clot within 4 hours after installation of the vascular shunt.
Oral administration of ticlopidine at doses above 5mg/kg markedly prevented the shunt obstruction through strong inhibition of platelet aggregation by this agent. In the ticlopidine-treated rats, only small numbers of single platelets scattered on the inner surface of the shunt were observed.
On the other hand, acetylsalicylic acid did not show any antithrombotic effect in this vascular shunt model, despite of its inhibitory action on collagen-induced platelet aggregation. Such ineffectiveness of acetylsalicylic acid seems to be probably related to almost complete inhibition of PGI₂ generation in the carotid artery by this agent.
Dipyridamole did not exert any effect on platelet aggregation at doses ranging from 10 to 300mg/kg, but increased the generation of PGI₂-like activity in the vascular tissue in a dose-dependent manner. This effect of dipyridamole, however, was too short in duration to give a significant antithrombotic effect in the present shunt model, although a tendency to prevent shunt obstruction was observed only at an extremely high oral dose or frequently repeated intraperitoneal doses of this agent.

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