• Asian-Australasian Journal of Animal Sciences
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• v.19 no.1
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• pp.137-143
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• 2006

The purpose of this study was to prepare a binder containing porcine blood transglutaminase (TGase), thrombin and fibrinogen. Extracted TGase, thrombin and fibrinogen were used alone or mixed with different proportions of their volume (v/v/v) by nine combinations as follows were 0.5:1:15, 0.5:1:20, 0.5:1:25, 1:1:15, 1:1:20, 1:1:25, 1.5:1:15, 1.5:1:20 and 1.5:1:25, respectively. Five ml of each combination were mixed with 0.6 ml of 0.25 M calcium chloride before experiment. After storage at 4C for 0, 1, 2, 3, 4 and 5 weeks, enzyme activity, total plate count, pH value, and SDS-PAGE of TGase, thrombin and fibrinogen were tested and pH value, clotting time and gel strength of the nine combination binders were determined. The results showed that total plate count of thrombin and pH value of TGase were significantly higher (p<0.05) than in other treatments. SDS-PAGE results showed that purified TGase, thrombin and fibrinogen from porcine blood plasma compared with commercial products (Sigma) had the same band patterns and nine different combination binders had no significant effect. Enzymatic activity of TGase and thrombin decreased as storage time increased. Total plate count of TGase, thrombin and fibrinogen and clotting time of the binder increased as storage time increased. The higher amount of fibrinogen in combinations, the stronger the gel strength.

• Journal of Life Science
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• v.14 no.3
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• pp.441-444
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• 2004

This study was performed to establish a simple and rapid method for measuring thrombin activity based on weight of fibrin clot formed. The new method was based on the weight measurement of fibrin clot after enzymatic reaction of thrombin with fibrinogen. The fibrin formation depended upon the activities of thrombin used, temperature, incubation time, and centrifugation time. The fibrin formation was increased proportionally up to 1.0 unit/ml of thrombin activity, 4.0mg/ml of fibrinogen concentration, and 5 min of incubation time at 37$^{\circ}C$. The fibrin clot formed was stable by centrifugation at 3,000$\times$g for 5min. This simple assay based on weight of fibrin after centrifugation would be useful for identifying natural food anticoagulants by inhibiting thrombin.

• Biomolecules & Therapeutics
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• v.1 no.2
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• pp.166-170
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• 1993

Hirudin is a potent inhibitor of thrombin, which was originally obtained from the medicinal leech (Hirudo medicinalis) Now it is being produced through the recombinant technology on a large scale. Recombinant hirudin has been assayed for the anticoagulant activity by the measurement of clotting time and the inhibition of thrombin actvity using a chromogenic substrate. The assay range of partial thromboplastin time and thrombin time is within $0.2{\sim}1.0 {\mu}g/mι.$ Thrombin time is more sensitive to the measurement of clot. Ex vivo study showed the level of hirudin in rat plasma was highest in 10 min and then it was eliminated slowly. The half-life of r-hirudin was 80~110 min depending on the assay methods. Intraveneous injection of russel viper venom was used for thrombus induction combined with vents cava ligation. Inhibition of venous thrombosis was observed with i.v. hirudin. It was dependent on the concentration of hirudin.

• Journal of Pharmaceutical Investigation
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• v.39 no.5
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• pp.373-379
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• 2009

This study was conducted to develop a pharmacokinetic-pharmacodynamic (PK/PD) model of a direct thrombin inhibitor, argatroban to predict the concentration-effect profiles in rats. Argatroban was i.v. injected to rats at 0. 2, 0.8 and 3.2 mg/kg doses (n = 4-5 per dose), and plasma drug levels were determined by a validated LC/MS/MS assay. The pharmacokinetics of argatroban was linear over the i.v. dose range studied. The thrombin time (TT) and the activated partial thromboplastin time (aPTT) were measured in rat plasma and they were found to linearly increase with increasing the dose. A 2-compartment pharmacokinetic model linked with an indirect response pharmacodynamic model was successfully utilized to evaluate the drug concentration-response relationship.

• Archives of Pharmacal Research
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• v.28 no.6
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• pp.667-674
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• 2005

The leaves of Persimmon (Diospyros kaki L.) has long been used for tea in Korea since it was thought to be effective against hypertension. An anticoagulant fraction was purified through gel filtration G-100, hydrophobic, gel filtration G-150, and FPLC, Phenyl superpose column chromatographies. The purified fraction was homogenous and its Mr was estimated 10,000 Da by gel filtration and SDS-PAGE. The purified fraction was sensitive to treatment of subtilisin B, but not to heat and its activity was not changed after periodate oxidation, indicating that the activity was not due to carbohydrates. It delayed thrombin time (TT), activated partial thromboplastin time (APTT), and prothrombin time (PT) using human plasma. TT was more sensitive than APTT and PT, suggesting that the anticoagulant activity may be caused by a degradation or a defect of fibrin or thrombin. It did not cause the hydrolysis of fibrin after incubation. However, it inhibited thrombin-catalyzed fibrin formation with a competitive inhibition pattern. These results indicate that it may be an antithrombotic agent and that it is bound to fibrinogen binding sites of thrombin.

• Korean Journal of Pharmacognosy
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• v.35 no.1 s.136
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• pp.52-61
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• 2004

Inhibitory activities of 264 methanol extracts, which were prepared from different parts of 210 kinds of wild and medicinal plants, against human thrombin were evaluated. Based on the anti-coagulation activity determined by thrombin time and activated partial thromboplastin time, the 14 extracts were screened. The fibrinolytic activity, heat stability and inhibition of other proteolytic digestive enzymes, such as pepsin, papain, trypsin and chymotrypsin, of the 14 extracts were further determined, and Ginko biloba (herba), Ephedra sinica (radix), Reynoutria elliptica (herba), Amomum tsao-ko Crevost (fructus), and Magnolia officinalis Rehd. et Wils (bark) were finally selected as possible plant sources for anti-thrombosis agent. These results suggested that medicinal and wild plants could be the potential source of thrombin inhibitor.

• BMB Reports
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• v.29 no.3
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• pp.225-229
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• 1996

Effects on thrombin by an amphipathic cation, dequalinium, which has been recognized as an anticarcinoma agent were investigated with small chromogenic substrates such as Na-benzoyl-DL-argininep-nitroanilide (BApNA), H-D-phenylalanyl-L-pipecoyl-L-arginine-p-nitroanilide (S-2238), and Na-p-tosyl-L-arginine methyl ester (TAME). Among them, only TAME hydrolysis due to an esterase activity of the enzyme was significantly activated to 81% at 20 ${\mu}M$ dequalinium in the absence of NaCl. This stimulation became even higher in the presence of 0.2 M NaCl to 3.5-fold at 60 ${\mu}M$ dequalinium. This specific activation of thrombin was well correlated with the results of in vitro coagulation tests measuring the activated partial thromboplastin time (APTT) and the prothrombin time (PT) It is pertinent. therefore, to suggest that the esterase activity should be examined in addition to the effects on 5-2238 hydrolysis when especially any regulators not directed to an active site of thrombin need to be studied. We also expect that dequalinium could be a useful tool for studying structure-function relationship of thrombin and blood coagulation.

• Archives of Plastic Surgery
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• v.36 no.1
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• pp.19-23
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• 2009

Purpose: Platelet transplantation is a novel therapeutic strategy for acceleration of wound healing. When applying platelets, efficacy of adding thrombin to stimulate growth factor release from platelets has already been proved. However, no quantitative data of the thrombin treatment has been reported. The purpose of this study was to determine the optimal thrombin concentration to maximize growth factor release of platelets. In particular, this study was designed to quantify levels of platelet derived growth factor(PDGF)-BB, which is a major growth factor contained in the platelets, in vitro. Methods: Fresh platelets were obtained from a blood bank. They were suspended in DMEM/F - 12 and incubated with thrombin of various concentrations. The concentrations of thrombin tested were 0, 6.25, 12.5, 25, 50, 100, 200, and 400 IU/ml. After 30 minutes, 1, 3, 5, and 7 days, the levels of PDGF - BB were measured using enzyme linked immunosorbent assay. Platelets from four donors were included in this study. Each sample was tested in triplicate and the mean value was used as a data for each sample. Results: The addition of thrombin increased the level of PDGF - BB. Increases in storage time of platelets resulted in decreased levels of PDGF - BB. Higher levels of PDGF were detected in consort with increased thrombin concentrations. However, there was no significant difference between samples of 200 and 400 IU/ml concentrations. Conclusion: The results indicate that adding thrombin accelerates the release of growth factors from platelets and the optimal thrombin concentration to maximize this function is 200 IU/ml.

• Journal of Life Science
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• v.14 no.3
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• pp.509-513
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• 2004

Inhibitory activities of 58 methanol extracts, which were prepared from different parts of 55 kinds of edible and medicinal plants, against human thrombin were evaluated. Analysis of the anti-coagulation activity determined by thrombin time enabled us to select 8 herb extracts as possible sources of anti-thrombosis agent. Further analysis of anti-thrombosis activity determined by activated partial thromboplastin time and fibrinolytic activity, and heat stability and inhibition against other proteolytic digestive enzymes provided new information that Pimpinella brachycarpa, Lysimachia clethroides, and Salix gracilistyla could be used as a potential anti-thrombosis agent. Our results suggest that edible and medicinal plants could be the potential source of thrombin inhibitor.

• Korean Journal of Veterinary Service
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• v.18 no.2
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• pp.182-188
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• 1995

Hemodynamic values were assessed in cows with naturally mastatis. hemodynamic tests included WBC, RBC, PCV, Hemoglobin, Monocyte, Eosinophil, Neutrophil, Lymphocyte, and prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen, platelet, antithrombin-III, and plasminogen activities. Significant changes were observed in the mean values of most analytses : WBC, monocytes, eosinophil, neutrophil were increased and Iymphocyte were decreased. prothrombin time was increased： activated partial thromboplastin time, thrombin time. increased : activated partial thromboplastin time, thrombin time, fibrinogen concentration, plasminogen activity and platelet concentration were decreased : and RBC, PCV, hemoglobin and antithrombin-III activity were unchanged, compared with normal mean values. Thesse data indicated activation of hemodynamic mechanisms, initiated either directly by bacteria produced endotoxin of secondaly inflammatory mediators produced in response to caused bacteria and naturally acquired mastitis was very similar to the experimental endotoxin-induced mastitis.