Ca antagonists, Nifedipine, Diltiazem and Verapamil enhanced PGI₂ production human vascular endothelial cells derived from umbilical veins via activation of phospholipases. Their mode of action was analyzed with reference to Ca⁺⁺ mobilization and cyclic nucleotides. PGI₂ pioduction by Diltiazem, A23187, arachidonic acid (AA) or PGH₂ was remarkably decreased by the pretreatment with TMB-8. And its inhibition rate was increased in addition of Diltiazem or A23187. The enhancement of PGI₂ production by Diltiazem or A23187 was almost diminished in Ca⁺⁺ free medium. When the endothelial cells were pretreated with MIX, intracellular cAMP and cGMP concentration was increased, and PGI₂ producton was decreased. Addition of dibutyryl cAMP or 8-bromo cGMP increased intracellular cAMP or cGMP concentration, respectively, and simultaneously suppressed PGI₂ production. Addition of PGI₂ or AA increased the concentration of PGI₂ of the medium and intracellular cAMP concentration, but had no effect on cGMP concentration. While in the presence of 1mM MIX, Diltiazem had no effect on intracelluler cAMP concentration but decreased cGMP concentration. Through these results, it is speculated that 1) PGI₂ production is depend upon mainly intracellular Ca⁺⁺, 2) and this dependency is dominant in the early step of AA cascade. 3) The enhancemt of PGI₂ production by Diltiazem or A23187 was also depend upon extracellular Ca⁺⁺. 4) The enhanced PGI₂ production is suppressed by increased cAMP due to increased PGI₂ production, as an autoregulation mechanism. 5) PGI₂ production is regulated by not only cAMP but also cGMP concentration. 6) Enhancement of PGI₂ production induced by Diltiazem may be derived from extra to intracellular mobilization of Ca⁺⁺ or decreased cGMP concentration.

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The fibrinolytic activity was examined before and after intracoronary thrombolysis with urokinase in 11 patients with acute myocardial infarction. Urokinase was administered as a solution of 24×10⁴IU in 20ml into the obstructed coronary artery and this therapy was repeated till recanalization was achieved or maximum dose of 96×10⁴IU was reached. In two cases the dose of urokinase was increased to 120×10⁴IU and in another case to 144×10⁴IU; in this patient cardiac rupture occurred 9 hours after the procedure (Table 1). The mean dose of urokinase administered was 103×10⁴IU. Fibrinolytic activity was determined every 8 hours for 24 hours and at 48 hours after intracoronary thrombolysis.
The result of intracoronary thrombolysis was observed in 8 of the 11 patients; recanalization was achieved in 75% (6 patients) (Table 1).
The fibrinolytic activity before and after intracoronary thrombolysis is summarized in Table 2. Euglobulin lysis time and the fibrinolytic activity of euglobulin fraction by fibrin plate method showed markedly increased fibrinolytic activity soon after urokinase administration (Figures 1, 2).
Plasminogen, fibrinogen and α₂-plasmin inhibitor were decreased markedly (Figures 3, 4, 6), and fibrin or fibrinogen degradation products (FDP) were increased after urokinase administration (Figure 5). α₂-Macroglobulin decreased gradually to the steady state level of 83% of the control value after 16 hours, α₁-Antitrypsin was rather, increased significantly 48 hours after intracoronary thrombolysis (Figure 8).
In conclusion, the decrease in α₂-plasmin inhibitor, plasminogen or fibrinogen may indicate enhanced fibrinolytic activity, though they are not direct indicators. α₁-Antitrypsin may not act as an anti-plasmin in vivo, although it has been thought to be an anti-plasmin. The dose of 103×10⁴IU of urokinase seems to be sufficient to activate fibrinolysis, because plasminogen, fibrinogen and α₂-plasmin inhibitor decreased and FDP increased markedly and significantly after the administration of this amount of urokinase. Intracoronary thrombolysis with simultaneous measurement of fibrinolytic activity in vitro is a good way to evaluate laboratory measurements of fibrinolytic activity, because the changes in the intracoronary thrombus during the administration of urokinase can be seen directly by coronary arteriogaphy.

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A 27-year-old male was investigated in the St. Marianna Hospital because of recurrent thrombosis which first appeared at his 25 years of age. Since he had the first episode of thrombosis as myocardial infarction, he had an attack of femoral vein thrombosis and subsequently he had a surgery of colon resection due to the superior mesenteric venous thrombosis.
The plasminogen activity assay by an amidolytic method revealed 40%, 47.8%, 57.2%, and 108% of normal in the propositus, his brother, father and mother, respectively. The antigen level of plasminogen was within normal range in both propositus and his family.
No discrete difference was noted between the abnormal and normal plasminogens by SDS PAGE under non-reducing and reducing conditions. SDS PAGE in reduced system for cleavage of plasminogen by urokinase, while the H chain and the L chain of plasminogen appeared more slowly in present abnormal plasminogen in the cleavage of plasminogen by streptokinase.
By the procedure of preparative isoelectric focusing method, present abnormal plasminogen demonstrated additional bands for each of corresponding bands equivalent to those of normal plasminogen.
The reason why only the propositus developed recurrent thrombosis, while no other members did not, is not easily explained, although obesity which had progressively appeared during 6 months prior to the attack might have contributed to thrombosis as a causative risk factor.

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Protein C was measured by means of an enzyme-linked immunosorbent assay (ELISA) in plasmas from 46 normal subjects, 34 patients with disseminated intravascular coagulation (DIC) and 4 patients with thrombotic thrombocytopenic purpura (TTP). Normal range in the assay was 73.5-155.0% (95% confidence limits) of a normal pooled plasma. In patients with DIC, plasma protein C concentrations were significantly decreased, with a geometric mean value of 43.1%. The protein C concentration was positively correlated with plasma prothrombin, antithrombin III and serum pseudocholinesterase, and was negatively correlated with von Willebrand factor antigen (vWF:Ag) and vWF:Ag/factor VIII ratio. These findings suggest that low protein C concentrations in DIC mean a consumption of protein C probably due to its activation by thrombin and/or impaired liver synthetic function. In patients with TTP, plasma protein C levels were normal with a geometric mean value of 125.3%, indicating that the pathophysiology of TTP is quite different from that of DIC.

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Plasma levels of prothrombin, abnormal prothrombin and protein C were measured in liver diseases.
Concentration of protein C was determined by Enzyme immunoassay. Concentration of Prothrombin and abnormal prothrombin were determined by Echis carinatus venom-chromogenic substrate (S-2238) method. Concentration of urinary γ-carboxyglutamic acid (Gla) was determined using HPLC by Kuwata, et al.
Plasma sapmles were collected from 10 healthy normal adults, 8 patients with acute hepatitis, 11 patients with chronic hepatitis, 17 patients with liver cirrhosis, 7 patients with hepatoma and 9 patients with liver metastatic carcinoma.
The results obtained were as follows.
1) Concentration of plasma prothrombin, and protein C were significantly decreased in liver diseases, and these two parameters were significantly correlated. (γ-=0.72)
2) Plasma abnormal prothrombin levels were markedly increased in most patients with hepatoma and another carcinomas, and this levels were slightly increased in several cases with liver cirrhosis.
In these cases, Fibrin monomer complex were appeared and protein C levels were significantly decreased.
These patients were treated with parental VK₂, and concentration of urinary γ-Gla were increased in these patients.
These datas were suggested that decreasing Protein C levels indicated disorders of hepatic protein synthesis as well as hepatic thrombus formation in liver diseases and hepatic thrombosis may be associated with the premature release of intra cellualr uncarboxylated prothrombin precursers into the plasma in liver diseases.

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The effect of daily oral administration of 50-100mg aspirin was evaluated in 8 patients (Table 1). Platelet aggregation and platelet malondialdehyde (MDA) production were significantly inhibited after administration of aspirin for a week. Plasma thromboxane-B₂ (TXB₂) levels showed a tendency to decrease (0.1<p<0.05). Plasma β-thromboglobulin (β-TG) and platelet factor 4 (PF₄) were not decreased. In order to detect intracardiac thrombi and evaluate the effect of aspirin therapy, platelet survival and accumulation of radioactivity in the thrombus using In-111-labeled autologous platelets were observed in two patients wite heart diseases before and after the aspirin therapy. A case of mitral stenosis with left atrial thrombi showed that the radioactivity on the thrombus was slightly decreased and platelet survival prolonged from 6.1 to 6.6 days during the administration of 50mg aspirin. In this case, plasma thromboxane B₂ level was decreased from 130 to 50pg/ml and platelet aggregation was markedly inhibited during the therapy. Another case of myocardial infarction with left ventricular thrombi showed that the radioactivity on the thrombus was slightly decreased and platelet survival prolonged from 7.6 to 8.2 days during the administration of 50mg aspirin. In this case, platelet aggregation induced by ADP and epinephrine were markedly inhibited, but plasma thromboxane B₂ level was not changed. Daily low-dose of aspirin inhibited platelet aggregation and platelet MDA production, but did not inhibit plasma TXB₂, β-TG and PF₄. Platelet survival after aspirin administration slightly prolonged and accumulation of labeled platelets on the thrombi supressed in slight degree in comparison with these before aspirin administration.

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Platelet survival time by MDA method, platelet functions and some coagulation parameters were examined in 45 patients with prosthetic valves. These parameters were also measured after administration of aspirin (40mg/day) with or without dilazep (300mg/day) for 4 weeks, in 22 of these 45 patients. All patients except one were under warfarin treatment, and the mean value of thrombo-test was 20.9% (8.5-85%).
The following results were obtained;
1) Platelet survival time was decreased in 26 (57.8%) of these patients.
2) After administration of aspirin with or without dilazep, platelet aggregability was suppressed significantly.
3) Platelet survival time was prolonged after 4 weeks of administration of aspirin with dilazep, as compared with that obtained before treatment.
These results suggest that small dose of aspirin with dilazep may prevent thrombo-embolic complications in these patients with prosthetic heart valves.

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We studied ex vivo and in vitro effects of elastase on platelet aggregation and its arachidonic acid (AA) metabolism in normal aubjects. Aggregation of platelets in citrated plasma (PRP aggregation) and of washed platelets were nephelometrically studied and whole blood aggregation by the impedance method was also determined. Elastase showed the significant inhibitory effects on PRP aggregation induced by collagen (0.5μg/ml) and AA (increase in its threshold concentration) as well as on whole blood aggregation by collagen (2.0μg/ml) after its oral administration of 10, 800 units per day for a week in 8 volunteers. Plasma prostacyclin-regenerating activities were not affected throughout. In in vitro experiments, elastase inhibited washed platelet aggregation induced by AA, collagen and thrombin, and PRP aggregation induced by ADP, epinephrine and STA2 (a stable analog of thromboxane A2) was also inhibited. Elastase decreased TX B2 production by platelets incubated with collagen or thrombin as measured by a radioimmunoassay, but failed to inhibit its production by exogenous AA.
Thus, it was demonstrated that elastase inhibited platelet aggregation in certain conditions ex vivo as well as in vitro and its mechanism seemed to be due to some inhibitory effects on the platelet membrane rather than through the inhibition of cyclo-oxygenase activities.

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It has been reported by a number of authors that such monoclonal immunoglobulins as IgA, IgG, IgM may interfere with the polymerization of fibrin in multiple myeloma. Few have reported on the other immunoglobulin, IgE.
We studied in the case of a 4-year-old boy patient with bronchial asthma who showed the increased IgE (4900IU/ml), and the low fibrinogen concentration (85mg/dl). He presented the response like an anaphylactic shock when he was infused the fibrinogen (1.5g) to prevent the hemorrhagic disorder at the operation of transuretheral resection to stenosis of the urethra.
At that time, some considerable discrepancies were found in the patient between the fibrinogen levels estimated by immunological method and clotting method, and between those of thrombin time and reptilase time.
The above discrepancies were also obtained from the fibrinogen of a 60-year-old IgE myeloma patient. However, neither anti-thrombin activity nor anti-reptilase activity was revealed in his plasma by chromogenic substrate method (S-2238). The prolongation of fibrin polymerization induced by thrombin and reptilase found in the IgE myeloma patient plasma suggests that IgE might inhibit the fibrinogen-fibrin conversion.

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Serum lipids, pulse wave velocity (PWV) and α₂ plasmin inhibitor (α₂ PI) of 21 cases with von Willebrand's disease (VWD) were compared with those of normal control subjects.
10 adult VWD patients (average age 40 years) showed significantly lower (p<0.05-0.01) serum cholesterol, triglyceride, free cholesterol and free fatty acid levels than the age matched 20 controls. PWV of 70 normal controls was 4.57±1.34m/sec and the correlation coefficient between age and PWV was 0.297 (p<0.05). Significant difference (p<0.05) was observed in PWV between male (4.72±0.49m/sec) and female (4.40±38m/sec). 12 of the 15 patients with VWD (8-79 years old) had lower value of PWV as compared with the control. α₂ PI level of VWD was apparently lower than the control but not significant.
Theses results might support the hypothesis of resistarce to arteriosclerosis in patients with von Willebrand's disease.

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Factor IX antigen (IX:Ag) was studied by immunoradiometric assay (IRMA) in 24 cases of Hemophilia B using monoclonal anti-human Factor IX antibody (65-10). Precise measurements of Factor IX:Ag were possible for concentration as low as 0.01U/ml (1%) of that in normal pooled plasma. A good correlation (correlation coefficient=0.77) existed between the titers of Factor IX measured by clotting assay (IX:C) and IX:Ag assay among 15 normal adults. There was also a good correlation between the titers of IX:Ag measured by polyclonal anti-human IX antibody (Rabbit) and monoclonal antibody (65-10) in 15 normal adults (r=0.78). IX:Ag were absent (less than 0.01U/ml) with this assay in 15 Hemophilia B^- patients. In 5 cases of Hemophilia B⁺ patients, the titers of IX:Ag with polyclonal antibody were 0.47±0.22U/ml, and that with monoclonal antibody (65-10) were 0.48±0.20U/ml.
However, in 4 B_M patients, the dose responce curve was not parallel to normal pooled plasma. In these B_M patients, prolonged Ox-brain prothrombin time was corrected by addition of monoclonal antibody (65-10).
These results suggest that the epitope detected by the monoclonal antibody (65-10) might be the abnormal part of the B_M molecule which prolongs Oxbrain prothrombin time.

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A new fractionation method is proposed to produce an intermediately purified and highly potent factor VIII concentrate. This method is based on the removal of fibrinogen by heat, which is a major coexisting protein in the conventional AHF products. In this process, VIII:C slightly decreased when 90% of fibrinogen was removed from non heated material. The test-products, prepared from 370ml of fresh frozen plasma, contained 150-180 units of VIII:C, and the ratios of VIII:C/VIIIR:Ag, VIIIR:RCo/VIIIR:Ag and VIII:C/VIIIR:RCo were 0.7-1.1. The contents of fibrinogen and total protein were about 0.3mg and 2mg per one unit of VIII:C, respectively. These results suggest the usefulness of the heat removal of fibrinogen for the production of the factor VIII concentrate with intermediate purity and high potency.

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Since high-concentrate thrombin preparations have excellent effectiveness for local hemostasis in patients with coagulopathies, the blood coagulation mechanism in the presence of high-concentrate thrombin, using purified thrombin, and plasminogen-free fibrinogen was investigated. The blood coagulation analyzer, Coag-Stat was utilized in the kinetic study of fibrin transformation. The first stage of fibrin precipitation speed was accelerated as the concentration increased and amounts of fibrin were gradually decreased in relation to the amount of sodium heparin given. The general result of this investigation was that anti-thrombin III and a large amount of heparin caused neutralization of dosages of 100 units/ml thrombin with neutralization increasing with heparin dosage. In the investigation of thrombin diffusion, high-concentrate thrombin was diffused on an agarose plate, containing plasminogen-free fibrinogen, and was seen to precipitate an increasing amount of fibrin.
From the result of this study, the accelerative function of thrombin in blood coagulation was confirmed again. High-concentrate thrombin penetrated and diffused into the fibrin network during scores of hours. We presume that this function brings about continual hemostatic efficacy. In a practical clinical application of thrombin, it seems that plasma inhibitor and released heparinoid affect the preparation. From this experiment about 100 units/ml of thrombin was neutralized under AT-III and extra doses of heparin. Moreover, taking many kinds of plasma inhibitors into consideration, this approach showed that clinically more than 1000 units/ml thrombin was more effective.

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Levels of VIIIR; AG and PA in the venous blood before and after 5 minutes tourniquet and fbg metabolism were determined in 19 patients with arterial thromboembolic disease and 4 age-matched healthy volunteers and the following results were obtained;
patient group control group p value
mean±SD mean±SD
VIIIR; AG before tourniquet (%) 206.1±60.7 117.5±14.4 p<0.025
VIIIR; AG after tourniquet (%) 243.7±58.3 145.0±12.9 p<0.01
PA before tourniquet (IU/ml) 0.084±0.049 0.139±0.032 p<0.05
PA after tourniquet (IU/ml) 0.136±0.073 0.545±0.497 p<0.005
fbg T/2 (day) 2.57±0.31 3.25±0.34 p<0.005
fbg J₃x (mg/kg/day) 70.5±20.4 27.6±9.3 p<0.005
Correlations among these values were investigated, and significant relationship was obtained between VIIIR; AG after tourniquet and fbg J₃x (r=0.425, p<0.05), PA after tourniquet and fbg T/2 (r=0.550, p<0.01), and increase in PA after tourniquet and fbg T/2 (r=0.463, p<0.05).
It is suggested from these results that the functionally changed endothelial cells of patients with thromboembolic disease participate in accelation of fbg metabolism.

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Fibrin high-affinity urokinase (UK) differed from single chain UK was purified with fibrin-Sepharose chromatography and gel filtration. The molecular weight of the purified fibrin high affinity UK (Fib-UK) was 95-100K. This Fib-UK was similar in immunological nature to UK but was different from it by molecular weight, its binding affinity for fibrin and lower specific activity (1865IU/mg). The plasminogen activator activity of Fib-UK was quenched by anti Fib-UK serum and anti UK serum. Zymogram after SDS-PAGE revealed that Fib-UK lyzed plasminogen rich fibrin plate at the molecular weight of 95-100K. Immunoelectrophoresis of Fib-UK and UK revealed that Fib-UK had an antigenicity against UK, while UK had not an antigenicity against Fib-UK.
It was concluded that Fib-UK was a protein having an activator activity, UK antigen and fibrin high affinity, and its molecular weight was 95-100K.

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After human plasminogen activated by SK was partially reduced by dithioerythritol in the presence of plasmin-inhibitor leupeptin, a functionally active light (B)·SK complex was isolated by affinity chromatography with Lysine-Sepharose.
This LB·SK complex shows a molecular weight of 60, 000 composed of LB 22, 000 and modified SK: 37, 000 by SDS-polyacrylamide gel electrophoresis.
Results were as follows.
1) Binding site to SK was located in the part of LB chain of human plasmin.
2) Making complex with SK, amidolytic activity of LB·SK was increased twice to three times than that of LB itself.
3) LB·SK complex showed both fibrinolytic and plasminogen activator activities and activated human, bovine and rabbit plasminogen to plasmin in spite of race difference.
4) Thrombolytic activity of LB·SK complex had the most powerfull effect compared with SK, UK and human plasmin.

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Stability of antithrombin III concentrate was studied. Antithrombin III prepared from plasma (or Cohn P_IV-1) was bottled and lyophilized in the presence of 0.1M NaCl and 22mM phosphate (pH 7.3). Measured loss of activity in the lyophilized process was about 0-10% of the initial activity in either presence or absence of such stabilizer as albumin, sodium citrate or mannitol. The antithrombin III concentrate produced was stored at various temperatures and the first-order rate constant of the loss of activity was measured. From the rate data, the activation energy was calculated to be 38.9kcal·mol^-1 for antithrombin III purified from plasma and 37.4kcal·mol^-1 from P_IV-1.

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Heterogeneity of antithrombin III (AT III) in plasma has been shown on isoelectric focusing (IEF). In this paper, interaction between heparin and each band of AT III separated by IEF was studied by means of Heparin Sepharose affinity chromatography.
IEF was carried out with pH 4.0-6.5 carrier ampholytes and AT III was detected by immunofixation. AT III in plasma was separated in five main bands with different isoelectric points (pls) between pH 4.82 and 5.12, and they were designated Band-1 to Band-5 on IEF. The quantitative composition of each band was evaluated by densitometric scan of the Coomassie Brilliant Blue R-250 stained band. Band-3 accounts for more than 60% of the total AT III. Plasma AT III bound on Heparin Sepharose was eluted with salt concentration gradient. The eluted AT III fractions were analyzed by IEF and immunofixation. Band-1, 2 was emerged in the lowest salt concentration and Band-3 tended to elute in lower salt concentration than Band-4. Furthermore, basic additional bands which were not detected in original plasma, were observed in eluted fraction. These results indicate each band of plasma AT III is proved to have different affinity to heparin and plasma AT III seems to be denaturated by passing through the Heparin Sepharose columm.

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Acceleration effects of various sulfated polysaccharides toward the anticoagulant activities of antithrombin III (AT III) and heparin cofactor II (HC II) were investigated in the purified system. And the neutralizing effects of histidinerich glycoprotein (HRG) toward these acceleration were also investigated. Heparin, chondroition polysulf ates-1, -5 and dextran sulfate (DS)-I (M. W.; 500, 000, S-contents; 18%) accelerated the antithrombin activities of both AT III and HC II, while dermatan sulfate, DS-II (M. W.; 4, 000, S-contents; 18%) and DS-III (M. W.; 4, 000, S-contents; 4.5%) accelerated only that of HC II. By the addition of HRG, the accelerating effects of heparin and chondroitin polysulfates toward the anticoagulant activities of AT III and HC II were neutralized in a dose-dependent manner resulting in the restoration of the thrombin activity. Therefore, it seems likely that HRG is a regulating factor of the anticoagulant activity of heparin in plasma. On the contrary, HRG showed a little neutralization activity for dermatan sulfate and DS-III, and it did not show any neutralizing effect toward DS-I and DS-II. These results indicate that the activities of AT III, HC II and HRG depend not only on the species but on the molecular weights and S-contents of the sulfated polysaccharides.

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AT III concentrate (Behringwerke) was administered to approximate the activity up to 80-120% on 4 congenital AT III deficient patients. AT III antigen was assayed by single radial immunodiff usion and activity was by S-2238 as heparin cofactor activity.
From the determined values of antigen and activity of infused AT III, regression line of time course was obtained for each case by computor analysis using the two compartment open model. Biological half life (T 1/2) was 72.3±35.8hrs for antigen and 75.6±18.0hrs for activity. Distribution volume (Vd) for antigen was 7.2±2.43L. Initial half disappearance time was about 24hrs. Mean maximal increase of AT III level was 0.27±0.04(mg/dl)/U/kg for antigen and 0.82±0.20%/U/kg for activity.
Based on the data of single dose experiments, the simulation curves of multiple administrations were calculated on the different intervals of infusion, and the reasonable steady state of concentration was considered to be achieved on the administration of 24hr- and 12hr-interval.
One of the deficient patients was received AT III concentrate every 24hrs for 3 days, and the simulation curve of 24hr-interval infusion was well correlated with the actually determined value, and the steady state of concentration was achieved in 120hrs after starting of the infusion.

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Levels of α₂-plasmin inhibitor (α₂ PI) activity and antigen were measured in normal infants and children from 1 month to 15 years of age. In 100 normal adults, the mean values of α₂ PI activity were 102.2±11.9% in a fibrinolytic method improved by author, 104.9±11.9% in an amidolytic method devised by author and 103.7±12.4% in Matsuda's amidolytic method. The levels of α₂PI antigen was 103.0±12.3%. In 50 infants at 1 month of age, the mean values of α₂ PI activity were 109.5±14.8% in the fibrinolytic method, 107.6±14.3% in the author's method and 110.2±12.8% in Matsuda's method. The levels of α₂ PI antigen was 109.4±13.9%. These values were increased with age and reached peak at 6-9 months and then decreased gradually with increasing age. The values of 11-15 years group reached almost the same values as that of adult group. There was no significant difference in α₂ PI activity and α₂ PI antigen or between male and female in each age group. This chronological change of α₂ PI was similer to that of α₂-globulin and that of α₂-macroglobubin.
The increase of both α₂ PI activity and antigen in the latter half of infancy period suggests the increased synthesis in the liver, but the essential cause is unknown.

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