• Journal of Pharmacopuncture
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• v.16 no.2
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• pp.46-54
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• 2013

Objective: This study was undertaken to isolate a fibrin(ogen)olytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate the enzymatic characteristics and hemorrhagic activity of the isolated enzyme as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were determined by using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrin(ogen)olytic enzyme with the molecular weight of 27 kDa (FE-27kDa) isolated from G. b. siniticus venom consisted of two heterogenous disulfide bond-linked polypeptides with the molecular weights of 15 kDa and 18 kDa. When more than $20{\mu}g$ of FE-27kDa was applied on the fibrin plate, fibrinolysis zone was formed as indicating its fibrinolytic activity. The fibrinolytic activity was inhibited completely by phenylmethanesulfonylfluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA) and partially by thiothreitol and cysteine. Metal ions such as $Hg^{2+}$ and $Fe^{2+}$ inhibited the fibrinolytic activity completely, but $Mn^{2+}$ did not. FE-27kDa preferentially hydrolyzed ${\alpha}$-chain of fibrinogen and slowly hydrolyzed ${\beta}$-chain, but did not hydrolyze ${\gamma}$-chain. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into polypeptides with molecular weights of more than 45 kDa. A dosage of more than $10{\mu}g$ of FE-27kDa per mouse was required to induce hemorrhage beneath the skin. Conclusion: FE-27kDa was a serine proteinase consisting of two heterogeneous polypeptides, hydrolyzed fibrin, fibrinogen, and gelatin, and caused hemorrhage beneath the skin of mouse. This study suggests that the potential of FE-27kDa as pharmacopuncture agent should be limited due to low fibrinolytic activity and a possible side effect of hemorrhage.

• Journal of Pharmacopuncture
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• v.17 no.1
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• pp.44-50
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• 2014

Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as $Fe^{2+}$ and $Hg^{2+}$ inhibited the fibrin hydrolysis completely, but $Zn^{2+}$ enhanced it. FE-32kDa hydrolyzed ${\alpha}$-chain but did not hydrolyze ${\beta}$-chain and ${\gamma}$-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of $2{\mu}g$. Conclusions: FE-32kDa is a ${\alpha}$-fibrin(ogen)olytic metalloprotease that requires $Zn^{2+}$ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

• BMB Reports
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• v.34 no.2
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• pp.134-138
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• 2001

By adding sodium chloride (2.5%) into a Bacillus amyloliquefaciens DJ-4 culture broth, two serine-type fibrinolytic proteases with a molecular weight of 29 (subtilisin DJ-4) and 38-kDa were stimulated on the SDS-fibrin zymogram or inhibitor gels. B. amyloliquefaciens DJ-4 showed the highest proteolytic activity (5.52 plasmin NIH unit/ml) on the fibrin plate based on the molar ratio when cells were subjected to the 2.5% NaCl. Using a fibrin plate, the secreted protease from this strain in the presence of 5% NaCl showed that about 49% of the enzyme's activity remained after incubation at $60^{\circ}C$ for 30 min, but as the salt concentration was increased (10% NaCl) the activity nearly disappeared (0.14 plasmin NIH unit/ml). However, through a fibrin zymography assay, three fibrinolytic enzymes (38, 53 and 80-kDa) from the cells in the presence of 10% NaCl were detected. Also, two salt-activated serine-type fibrinolytic professes (29 and 38kDa) showed thermostability from 65 to $70^{\circ}C$ for 30 min. Furthermore, these professes also showed stability, pH 6-11. In particular, 29-kDa (subtilisin DJ-4) was very stable in the pH range of 4-11 at $4^{\circ}C$ for 48 h.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.04a
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• pp.104-104
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• 2003

In this study, seven grain seeds(sorghum maize, buckwheat, soy bean, mung bean, red bean, and barngrass) and germinated seven grain seeds were examined the fibrinolytic activity through fibrin plate assay and SDS-PAGE. The results obtained were as follows : 1. In the fibrin plate assay, the extracts of maize, barngrass, sorghum and buckwheat showed fibrinolytic activity. Especially, Maize of them showed fibrinolytic activity that was almost similar to plasmin, fibrinolytic enzyme used as a positive control. 2. In the SDS-PAGE of seven grain seeds, fibrinolytic activity was remarkably shown in mung bean and red bean. 3. In the fibrin plate assay of germinated grain seeds, buckwheat(5 mm), buckwheat(10 mm) and soy bean(10 mm) showed a level of fibrinolytic activity that was about 0.3 fold than 1.0 unit of plasmin, Also maize(10 mm) of them showed a level of fibrinolytic activity that was about 0.5 fold than 1.0 unit of plasmin. As a result, maize of grain seeds was found that it has a strong fibrinolytic activity.

• Journal of Life Science
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• v.20 no.6
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• pp.838-844
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• 2010

A fibrinolytic enzyme of Streptomyces corcohrussi from soil sediment was purified by chromatography using DEAE-Sephadex A-50 and Sephadex G-50. The analysis of SDS-polyacrylamide gel suggested that the purified enzyme is a homogeneous protein and the molecular mass is approximately 34 kDa. The purified enzyme showed activity of 0.8 U/ml in a plasminogen-rich fibrin plate, while its activity in a plasminogen-free fibrin plate was only 0.36 U/ml. These results suggested that the purified enzyme acts as a plasminogen activator. The fibrinolytic activity of the enzyme under the supplementation of protease inhibitors, $\varepsilon$-ACA, t-AMCHA and mercuric chloride in the enzyme reaction was less than 24%, indicating that it could be modulated by the plasmin and/or fibrinogen inhibitors involved in the fibrinogen-to-fibrin converting process. As time passed, $Zn^{2+}$, a heavy metal ion, inhibited the activity to 34.1%. The optimum temperature of the purified enzyme was approximately $50^{\circ}C$ and over 92% of the enzyme activity was maintained between pH 5.0 and 8.0. Therefore, our results provide a potential fibrinolytic enzyme as a noble thrombolytic agent from S. corcohrussi.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.987-993
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• 2005

A bacterium, producing a fibrinolytic enzyme, was screened from a decaying rice plant. The bacterium was identified as Bacillus subtilis by morphological, biochemical, and physiological properties and named Bacillus subtilis BK-17. The fibrinolytic enzyme (BK) was purified from supernatant of Bacillus subtilis BK-17 culture broth. The molecular weight was 31 kDa as determined by SDS-PAGE. The effect of temperature, pH, and plasminogen on the activity of the bacillokinase (BK) was analysed and the activity was compared with urokinase. The optimal temperature and pH were $50^{circ}C$ and pH 7, pH 8, respectively. The BK activity was inhibited to $45\%$, $35\%$, and $23\%$ with 1mM EDTA, $Zn^{2+}$, and $Ca^{2+}$, respectively. However, $Mg^{2+}$, $Mn^{2+}$, and $Co^{2+}$ ions did not have any significant effect on the enzyme activity The BK showed the artivity in the both plates, plasminogen-free fibrin plate and plasminogen-rich fibrin plate. The result indicates that the BK can directly act the fibrin. In comparison of fibrinolytic activity with urokinase on the fibrin plate, the BK shows about 20 folds higher activity than that of the urokinase.

• Restorative Dentistry and Endodontics
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• v.47 no.2
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• pp.17.1-17.8
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• 2022

Endodontic microsurgery is a predictable treatment option when orthograde treatment or retreatment is unsuccessful or unfeasible. However, when there is a gross compromise of periapical bone, achievement of bone regeneration after the surgical procedure may be hampered. In such cases, the application of guided tissue regeneration principles, with adjunctive use of leukocyte platelet-rich fibrin to fill the bone defect as a bone substitute and as a membrane to cover the site, provides a cost-effective solution with the benefits of accelerated physiological healing and reduced post-surgical pain and discomfort. This case report presents 2 cases of endodontic microsurgery of the upper lateral incisors with loss of buccal cortical plate, where platelet-rich fibrin was successfully applied.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.434-438
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• 2005

In the previous study, we isolated a myulchikinase (MK), which has fibrinolytic activity and cytotoxicity to the tumor cell line, from myl- chi-jeot-gal. In this study, the effect of NaCl concentration, metallic ions, pH, temperature, and plasminogen on the activity of MK was analysed. The MK activity was maintained at least $80\%$ activity up to $30\%$ NaCl, which indicates that the enzyme may be halotolerant. The optimal pH and temperature were 8 and $40^{\circ}C$, respectively. The fibrinolytic activity of MK was completely inhibited with 0.5 mM $Hg^{2+}$ and inhibited to $50^{\circ}C$ with 1 mM $Cu^{2+}\;and\;Zn^{2+}$. The MK showed strong activity in plasminogen- rich fibrin plate but not in plasminogen-free fibrin plate. The result indicates that the MK may be a plasminogen activator type fibrinolytic enzyme.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.357-359
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• 2006

Crude extract prepared from jujube (Zizyphus mauritiana) possseses fibrinolytic activity hydrolyzing fibrin. A clear transparent region is observed where fibrin is hydrolyzed, and its diameter increased as the added jujube extract increased. The fibrinolytic activity of jujube extract seems to be heat and acid stable since its activity was retained after heat treatment at $100^{\circ}C$ for 10 min or an acid treatment by incubating at pH 2.0, 3.0, and 4.0 for 3 hours. But the jujube extract dialyzed with a cellulose membrane with molecular weight cutoff of 12,000 and 2,000 lost its activity, which suggests the fibrinolytic activity might be compounds of low molecular weight.

• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.589-593
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• 1998

Extracts from 65 species of mushrooms in mount Chiak were screened for their fibrinolytic activities. Extracts from Armillariella mellea, Calocybe sp., Lepista nuda and Trichaptum abietinum showed to have almost twice of activity of plasmin 1.5 U/ml, 193%, 213%, 198%, and 193% respectively. Collybia maculata showed 98% of activity, and Coprinus comatus and Lepiota sp. 56% and 58% activities, respectively. Other two, Agrocybe sp. and Stropharia rugosoannulata were less than 10% of activities.