It has been observed recently that highly concentrated human fibrinogen could be used as an adhesive for various human tissues. Tisseel^® kit was developed for this purpose, and is composed of Tisseel, a highly purified human fibrinogen fraction and aprotinin solution, thrombin 500 or 4U and calcium solution. In this article, we report effectiveness of the Tisseel^® kit and its several components in adhesive or wound-healing capacity by means of rat's skin adhesion system.
Our results suggest that there is an optimum concentration and dose for fibrinogen and factor XIII in the Tisseel^® kit for adhesion. Among them, the thrombin concentration has to be appropriately selected since a small dose of thrombin: 4U/ml, was found to be more favorable for wound-healing than a large dose of 500U/ml thrombin.
These results may imply that in clinical application, Method A with rapid adhesion effect is suitable for adhesion of internal organs, nerves and blood vessels, and Method B with high affinity for tissue and good wound-healing effect is appropriate for transplantation of skin and adhesion of bones, tendons and muscles. We suggest that the Tisseel^® kit can be properly used clinically with respect to both adhesive force and wound-healing.

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Aminobenzamidine was immobilized via a spacer moiety to a micro-particle hydrophilic gel (Asahipak; Asahi Chemical Co.) and this affinity adsorbent was used for the separation of plasmin and plasminogen by high-performance affinity chromatography. The adsorbent was packed in a stainless steel column. When Glu-Plg was applied to the column equilibrated with phosphate buffer (pH 7.4), it was slightly retarded. Both Lys-Plg and Pl were retained on the column. Glu-Plg II was eluted more slowly than Glu-Plg I. The similar tendency was observed in Lys-Plg I and Lys-Plg II. Lys-Plg was eluted with ε-aminocaproic acid (EACA). For the elution of Pl, the coexistence of EACA and urea was necessary. This result indicates a two-site interaction of Pl with the immobilized ligand, i. e., at the lysine-binding sites and the catalytic site. Fluorometric detection of eluted protein and on-line assay of Pl activity using a fluorogenic substrate (Boc-Glu-Lys-Lys-MCA) revealed that effective separation of the enzyme could be achieved with high sensitivity (<10μg) within 1h. When the mixture of Glu-Plg, Lys-Plg and Pl was applied to the system, each of them could be eluted with the stepwise change of eluents. This system should be applicable in clinical diagnosis.

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We have purified the tissue-type plasminogen activator (t-PA) produced by human melanoma cell line and have investigated its protein-chemical characteristics and fibrinolytic activity. Either one-chain or two-chain t-PA was obtained in the presence or absence of aprotinin during cell culture in purification steps. The N-terminal amino acid sequence of the one-chain t-PA was shown to start with Ser-Tyr-Gln-Val-. The amidolysis of S-2288 by t-PA was found to be greatly altered when the one-chain form was changed to a two-chain form by plasmin or trypsin treatment. When the fibrinolytic activities of t-PA and urokinase (UK) were compared using clot lysis time assay, t-PA and UK having the same unit base were found to have the nearly similar lysis time. On the other hand when fibrin plate assay was used, UK, having a unit of less than 1/10th of that of t-PA, was found to have the same fibrinolytic activity as t-PA.
Furthermore, when the modified Chandler's loop method was used, t-PA was found to be several times more effective than UK in thrombolysis. Whereas UK drastically lowered the amount of fibrinogen and plasminogen in blood, t-PA hardly showed any effect on them.

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Glu-and Lys-plasminogen (plg) were subjected to low pH (pH 2.0). Their conformation and activation rates were measured. The activtaion rates of Glu-plg enhanced after acidified. The increase was irreversible even after a short interval of acidification. The activation rate of acidified Glu-plg did not increase in the presence of fibrin or fibrinogen. The activation rate of acidified Lys-plg rather decreased and was not influenced in the presence of fibrin or fibrinogen. The fluorescence intensity at 340nm of plasminogen decreased after acidification. The negative molar ellipticity, [Θ], of Glu-plg decreased after acidification but that of Lys-plg did not change.
These results indicate that the conformational change corresponded to the increased activation rate of Glu-plg after the acidification. Although the tertiary conformation of Lys-plg changed after the acidification, its activation rate did not change.

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A radioimmunoassay technique for the fragment Eneo-antigens in plasma was developed. Specific antibody to the Eneo-antigen was obtained from anti-E rabbit serum through a fibrinogen-Sepharose column. There was no interference on binding between Eneo-antibody and ¹²⁵I-E even though hundreds mg of fibrinogen were present in a sample to be tested. Therefore, the method was applicable to plasma sample, permitting quantitation of the E-related neoantigens more than 1ng/ml. On the Eneo-antigenicity, electro-blotting study showed that the products of fragments X and Y which were generated in the early stage of fibrinolysis were bearing the antigenicity. Among the chains of fibrin and fibrinogen molecules, β and γ chains were strongly suggested to be the loci of Eneo-antigenic sites.

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We have developed a radioimmunoassay technique to measure the fragment Eneo-antigens in plasma and applied this technique to healthy persons and patients. The Eneo-antigen level of 174 normal subjects was 2.2±1.9ng/ml. Analyzing on ages, the older persons were found to show higher Eneo-antigen levels. In 42 diabetes mellitus, Eneo level was 9.8±3.3ng/ml which was significantly higher than those of healthy. Hemodialyzed patients showed higher Eneo-antigen levels than healthy and, however, there was no significant change during the dialysis.

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Natural substrates of human leukocyte elastase (ELP) in the blood are suspected to be fibrinogen and fibrin, although ELP is immediately complexed with α₂-macroglobulin (α₂M) and other inhibitors. However, the enzymatic activity of α₂M·ELP complex is not yet clear. This study was undertaken to clarify the activity of the complex.
(1) ELP and α₂M were purified respectively, then the α₂M·ELP complex was prepared.
(2) The complex immediately after preparation degraded a specific synthetic substrate of ELP, Suc-Ala-Tyr-Leu-Val-pNA, independently of NaClO₄ concentration in the reaction medium, different from the mode of action of free ELP.
(3) However, the complex could not degraded fibrin or fibrinogen without the addition of NaClO₄.
(4) The amidolytic and fibrinolytic activities of the complex without addition of NaClO₄ were 45% and 2% of those of free ELP, respectively, and those in the presence of 0.2M NaClO₄ were 35% and 45%, respectively.
(5) After incubation at 37°C for over 24hr, the complex degraded fibrin and fibrinogen as well as synthetic substrate without the addition of NaClO₄.
(6) The inhibition for amidolytic activity of the complex by various inhibitors was compared with those of ELP and pancreatic elastase (PE). The activity of the complex was not inhibited by various trypsin inhibitors and plasma inhibitors but was inhibited by Suc-Ala-Tyr-Leu-Val-CH₂Cl and DFP.

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It is known that endotoxin activates the complement system via the alternative pathway and induces many pathophysiological changes. We compared the effect of OKY, a specific thromboxane synthetase inhibitor, with that of FUT-175 (FUT), a protease inhibitor, on endoxin-induced decrease in platelet count and complement activation. OKY 10mg/kg and FUT 0.3mg/kg but not aspirin 1mg/kg, inhibited the decrease in platelet count induced by injection of endotoxin in rabbits. PGI₂ 10μg/kg also inhibited the endotoxin-induced platelet decrease. OKY markedly inhibited the endotoxin-induced increase in plasma TXB₂ level and enhanced 6-keto-PGF_1α level, but FUT did not. Endotoxin released lactate dehydrogenase (LDH) which is a marker enzyme of platelt lysis from rabbit plateets in vitro. FUT 10^-5M and PGI₂ 3×10^-7M, but not OKY 10^-4M, inhibited the LDH release from platelets. FUT 10^-6-10^-5M dose-dependently inhibited the decrease in total hemolytic activity of complement system (CH 50). On the other hand PGI₂ inhibited the binding of complement (C3 and C5) to guinea-pig platelets induced by endotoxin. These results suggest that OKY inhibited the decrease in platelet count induced by endotoxin may be due to formation of PGI₂ which inhibits the binding of complement to platelet, while FUT inhibited this via inhibition of complement activation.

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Previously reported boosting effect of ultrasonic vibration on fibrinolytic activity of urokinase has been investigated experimentally in 40 Wistar rats.
Five and 24 hours after the injection of sodium laurate into a femoral artery to produce arteial occlusion, urokinase, 12.000iμ/kg each, given intravenously of followed directly by ultrasonic waves administration in bath in 12 rats, urokinase only was used 12 rats, ultrasonic vibration only were employed in 8 rats, and physiological saline solution was injected in 8 rats. The changes in the legs with laurate administration were observed for 12 days.
In rats with ultrasonic vibration only and also in those with saline solution, fingers developed gangrene after 24 hours of laurate injection, as were lost after 4 days. The leg was mummited after 12 days. In animals with urokinase and ultrasonic vibration, neither nocrotic changes, nor mummifications were seen. In rats with urokinase only, necroses in fingers were incomplete and mummifications were limited in fingers.
It was suggested that ultrasonic wave administration might reinforce fiibrinolytic effect of urokinase in vivo.

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Heat-treatment of lyophilized factor VIII concentrate at 60°C resulted in a slight decrease in the factor VIII procoagulant activity (VIIIC), whereas a great decrease in the VIII C activity was detected at more than 70°C. A complete inactivation of viruses such as Entero, Orthomyxo, Paramyxo, Toga, Herpes and Adeno viruses in lyophilized factor VIII concentrate was attained by heating at 60-65°C for 5-30 hours. Optimal heating condition as to inactivation of hepatitis virus, where temperature and incubation time were at 65°C and for 96 hours, respectively, was chosen on the basis of the experiments using parvo virus. Effectiveness of heat-treatment (65°C, 96 hours in lyophilized state) in the inactivation of non-A, non-B agent appeared to be confirmed in chimpanzee test.

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Changes in blood coagulation and fibrinolysis in the tube of the hydrogel with long polyethyleneoxide chains (PEO) were evaluated and compared with other synthetic materials. Whole blood clotting time was 45 minutes in the PEO tube, 5 minutes in glass, 15 minutes in plasticized polyvinylchloride, 23 minutes in silicone rubber. Also, by studying scanning electron micrographs, fibrin formation of the surface of the PEO tube was not found until 50 minutes after injection of whole blood into the tube. Excellent antithrombogenicity of the PEO was supposed due to inhibition of the contact activation in blood coagulation by the effect of volume restriction repulsion of the PEO.

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The releasing ability of vascularplasminogen activator (VA) from vessel wall is considered to be a key to the development of thrombosis and its prognosis.
Applying the venous occlusion (VO) test by Nilson and Pandolfi as an index of the decrease in VA or difficulty in VA release, the author was clinically established VO test method measuring the euglobulin clot lysis time (ELT) before and after venous occlusion for 10 minutes under intermediate pressure between diastolic and systolic pressures. Clinically, the VO test was used in collagen disease. The following results were obtained.
1. In normal adult, ELT measured before and after venous occlusion, there was no difference in terms of age or sex.
2. Coagulation and fibrinolytic factors measured before and after the VO test were observed within the normal range.
3. In active SLE cases within 5 years after the onset, it was recognized that VA at rest and in venous occlusion were increased. In both active and inactive SLE cases at more than 10 years from the onset, VA at rest and VA in venous occlusion were decreased. With SLE cases in the comparatively early stage, there was increased VA release due to vasculitis, while in SLE cases passed about 10 years exhaustion and unreleasing of VA were noticed.
4. In progressive systemic sclerosis, the fibrinolytic activity was increased. In the Behçet cases, however, fibrinolytic activity was decreased and VA release was exhausted. In rheumatoid arthritis cases, findings suggested impaired VA release.
5. It could be established that the author's clinically and fundamental analysis on VO test could well serve as an important index for the fibrinolytic activity of thrombogenesis and the key to the development and prognosis of thrombosis.

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It has been well known that heparin inhibits thrombin action through antithrombin-III (AT-III). However, the inhibition by heparin on thrombin-induced platelet aggregation in human washed platelet suspension (WPS), which seems to be lack of AT-III, was reported by Eika. In our experiment, we confirmed his observation and studied the mechanism of this action of heparin using canine WPS.
Results are summarized as follows:
1. Heparin inhibited thrombin-induced platelet aggregation in the absence of AT-III.
2. It is assumed that inhibitory action of heparin is not caused by direct interaction of heparin with thrombin or platelet membrane, but possibly due to the indirect action of heparin bearing electro-negative charge as a barrier to the binding of thrombin to the platelet membrane.

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Changes in blood coagulation and fibrinolysis due to hepatic ischemia by using mongrel dogs were investigated.
Under the heparinized hydrophilic catheter-bypass between splenic and femoral vein, the hepatoduodenal ligament was ligated to interrupt hepatic blood inflow in 5 dogs, and temporary interruption of hepatic blood inflow for 15, 30, 60, 90 minutes was performed in each of 5 dogs.
In this experimental study, changes of systemic blood coagulation and fibrinolysis, and histological changes of liver were investigated periodically.
Continuous interruption of hepatic blood inflow decreased blood coagulability gradually. Temporary interruption of hepatic blood inflow for more than 60 minutes caused remarkable decrease of blood coagulability after declamping, and histologically fibrin deposits were found in sinusoid and portal vein of Glisson's area, indicating that apparent DIC occured in the liver at more than 60 minutes after clamping. One hour after declamping of 90 minutes interruption of hepatic blood inflow, most of fibrin deposits disappeared, but degeneration of endothelial cell of sinusoid and hepatocyte indicating the disturbance of hepatic microcirculaion by which caused acute liver failure and systemic DIC were observed, thereby finally led to death of shock.

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Eicosapentaenoic acid (EPA) is known by its anti-atherogenic and anti-thrombotic properties. This protective character of the fatty acid is usually explained through the shift of prostanoid production toward anti-thrombotic direction including depression of platelet aggregability. Since 1981 we have been reporting the effect of EPA on hemorheology. Because there is a possibility that viscosity of fatty acids themselves may be reflected in membrane fluidity, hence RBC filterability, we measured viscosities of triglycerides and ethylesters of EPA, and other fatty acids. It was found that triglycerides and ethylester of EPA are the less viscous oils than triglycerides and ethylesters containing other fatty acids measured in the experiment, respectively. It is important to prove directly that an increase in EPA content in RBC membrane could increase membrane fluidity.

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Neuraminidase in platelets was puified by colominic acid starch gel affinity chromatography.
Platelets in 0.01% triton X-100 of 1/5 vol. ACD-saline were sonicated by 50W for 2min at 5°C and fractioned with saturated ammonium sulfate. Neuraminidase in the fraction was purified by affinity chromatography. The highest concentration of neuraminidase was obtained with 20ml of 100mM acetate buffer, pH 5.0. The specific activity of the neuraminidase was 9.030u/mg when the fraction was ultrafiltrated, though other trace amount of enzymes such as galactosidase, glucosidase and mannosidase were found.
The study on neuraminidase in platets revealed the following. By using SDS-PAGE, two bands wends found indicating moleculecular weights of 82000 and 69000. The activity of neuraminidase in the platelets was approximately 10^-3 units/10⁸ p latelets. The optimum pH's for enzyme activity in neuraminidase were 4.2 when colominic acid substrate was used and 3.0 or less when NANA-4MU was used. The values of Km in NANA-lactose were 2.0×10^-5M, 4.0×10^-4M and 1.6×10^-5M for fetuin, colominic acid and NANA-4MU substrates respectively.
The study also indicated that neuraminidase appeared to adhere firmly to the platelet membrane.

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Previously we reported that nitroglycerine (NTG) enhances the PGI₂ generation via activation of phospholipase A2 of arachidonic acid cascade in the cultured human vascular endothelial cells. This paper reports the effects of NTG on the PGI₂ generation with reference to cyclic nucleotide metabolism. In the presence of MIX, intracellular cAMP or cGMP level was increased and PGI₂ generation of the endothelial cells was decreased. Addition of NTG had no effect on the level of intracellular cAMP in this experimental system, while cGMP was increased 1.21 folds by NTG. Addition of dibutyryl CAMP or 8-bromo cGMP increased intracellular cAMP or cGMP and diminished PGI₂ generation. Addition of exogenous PGI₂ caused the increase of intracellular cAMP level without affecting cGMP level. Through these experiments, it is speculated that enhancement of PGI₂ generation is regulated by increased CAMP due to increased PGI2, that is negative feedback mechanism of PGI2 generation, and that increase of cGMP level by NTG may also regulate PGI2 generation.

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[in Japanese]

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[in Japanese]

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