• Archives of Pharmacal Research
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• v.29 no.5
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• pp.418-423
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• 2006

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-lle-Asp-Lys-Val-Arg. The protein was activated by $Cu^{2+}\;and\;Zn^{2+}$, and the optimal conditions were found to be at pH $3\sim6\;and\;40\sim70^{\circ}C$. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.

• Toxicological Research
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• v.14 no.3
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• pp.333-339
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• 1998

Sambutoxin, a newly purified mycotoxin in Koea, caused hemorrhage in the stomach and intestine of rats. To elucidate the mechanism of hemorrhage, effects of sambutoxin on rabbit platelet aggregation were investigated. First of all, the effects of sambutoxin on the platelet aggregation response and ATP release from platelet by various appregating factors were investigated. And then the role of $Ca^{2+}$ on the platelet aggregation was investigated by flow cytometer. Finally, morphological effect of sambutoxin on platelet ultrastructure was examined by transmission electron microscope. Sambutoxin inhibited aggregation induced by ADP, collagen, thrombin, and arachidonic acid and decreased platelet activating factor-induced disaggregation time in a dose dependent manner. Sambutoxin also decreased thrombin and arachidonic acid-induced ATP release, but increased all factors induced $Ca^{2+}$ release. Sambutoxin showed severe ultrastructural changes of platelet such as appearance of disorganization debri of cellular organelle in intercellular space. Our results indicate that sambutoxin inhibitis rabbit platelet aggregation, and it may be party due to the decrease of ATP release. However, it is not clear whether the antiaggregating effect of sambutoxin is related to $Ca^{2+}$ increase.

• The Korean Journal of Physiology
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• v.29 no.2
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• pp.217-223
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• 1995

Adenosine 5'-tetraphosphate (ATPP) was identified and quantified in extracts of rabbit platelets by elution of extracts containing authentic adenosine 5'-tetraphosphate and comparison of retention time with nucleotide standards using high-performance liquid chromatography technique. The amount of adenosine 5'-tetraphosphate was $0.62\;nmoles/10^{9}$ cells which was 62-fold lower than that of ATP but only 10-fold lower than that of ADP. During platelet aggregation induced by thrombin, adenosine 5'-tetraphosphate was released to a relatively high extent. The degradation rates and halflives of adenosine 5'-tetraphosphate were measured during incubation of platelets in whole blood, erythrocyte suspension and plasma, respectively. The results suggest that plasma contributes more than blood cells to the catabolism of adenosine 5'-tetraphosphate. The pattern of degradation indicates that ATPP may be degraded mainly to AMP by soluble enzymes in plasma and very slowly to ADP and/or AMP by ectoenzymes on blood cells such as erythrocyte. The nature of the enzymes responsible fer the degradation of adenosine 5'-tetraphosphate is yet to be identified.

• Korean Journal of Veterinary Research
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• v.36 no.4
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• pp.887-894
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• 1996

Cyclopiazonic acid(CPA) known as stimulating the release of intracellular calcium, aflatoxin $B_1(AFB_1)$ causing gastrointestinal hemorrhage frequently were used as model toxic mycotoxins in these studies. First of all, the effects of various mycotoxins on the platelet aggregation response were determined. The effects of mycotoxins on the ATP release from platelet by aggregating factors were investigated. The results and conclusions obtained from these studies are : 1) CPA promoted ADP, collagen, thrombin, A.A. and PAF-induced rabbit platelet aggregation. $AFB_1$ inhibited collagen, A.A. and PAF-induced rabbit platelet aggregation only. 2) CPA increased both aggregation and disaggregation time, whereas $AFB_1$ decreased in a dose dependent manner. 3) CPA increased ADP, thrombin, A.A. and PAF-induced ATP release. $AFB_1$ increased A.A.-induced ATP release and decreased PAF-induced release in a dose dependent manner. In conclusion, CPA promoted platelet aggregation by the increase of ATP. Antiaggregating effects of AFB1 may be due to decreases of ATP. These data provide the basis for the future study of roles of ATP release in platelet aggregation.

• Journal of the Korean Society of Food Science and Nutrition
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• v.35 no.2
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• pp.132-138
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• 2006

Direct thrombin inhibitor, which is effective to prevent or cure the thrombosis, has been investigated in worldwide. In this study, we tried to screen antithrombosis agent from edible or medicinal plant. A strong antithrombin activity was identified from methanol or $95\%$ ethanol extract of buckwheat seeds. The solvent fractionation of buckwheat extracts using hexane, ethylacetate, butanol revealed that the butanol fraction has a prominent antithrombin activity. Thrombin time (blood-clot formation time) exceeded to over $2,000\%$ by addition of the butanol fraction at concentration of $312.5{\mu}g/mL$, whereas thrombin time extended to $336\%$ by addition of aspirin at concentration of $1,500{\mu}g/mL$. The butanol fraction showed anthrone-positive and ninhydrine-negative reaction. The active components were heat-liable, acid-unstable non-proteinous macromolecules (>30 KD). In vivo analysis using ICR male mouse showed that the buckwheat extract was superior than the aspirin in pulmonary thrombosis, KCN-induced coma and death. Our results suggest that the buckwheat is a potential as an antithrombosis agent and medicinal food.

• Natural Product Sciences
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• v.6 no.2
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• pp.53-55
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• 2000

In the course of continous work for the discovery of anti-thrombotic constituents from tubers of Gastrodia elata, citric acid 1,5-dimethyl ester was isolated from ethylacetate soluble fraction prepared from the methanol extract. The structure of the compound was determined by the spectroscopic data. The anti-thrombotic effect of this compound was observed with prolonging the bleeding time in thrombin-induced thrombosis model of mice.

• Tuberculosis and Respiratory Diseases
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• v.44 no.5
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• pp.1114-1124
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• 1997

Background : Endothelin(ET) is a very potent vasoconstrictive peptide produced by endothelial cells of pulmonary artery. The endothelin level was increased in plasma of primary pulmonary hypertension and acute pulmonary thromboembolism and it was suggested that the endothelin might do a critical role in the cardiopulmonary dysfunction in these two conditions. But the exact mechanism of increase of ET has not been known. In these two conditions, platelet activation and thrombosis are the main pathophysiologic findings. So there is a possibility that the platelet might stimulate endothelin secretion from endothelial cells. Therefore, we performed this study to evaluate the role of platelet and its mediators on endothelin production in bovine pulmonary artery endothelial(BPAE) cells. Method : Bovine pulmonary artery endothelial cells, ATCC certified cell line 209, were cultured and treated with human platelets($10^6{\sim}10^8/ml$), thrombin (0.1~10u/ml), TGF-${\beta}1$(1~100uM), serotonin(1~100uM), and endotoxin(1ug/ml) in a final volume of 500ul for 18 hours. Levels of ir(immunoreactive)-ET in each conditioned medium were measured by a radioimmunoassay specific for ET. Result : The increase of ir-ET levels was platelet number and time dependent over 18 hours. When washed human platelets were added($10^8/ml$), the ir-ET levels were significantly higher than that of control(p<0.05) at 8 and 18 hours after culture. Subthreshold concentration of platelets($10^7/ml$) coincubated with endotoxin(1ug/ml) or subthreshold dose of thrombin(0.1u/ml) stimulated ir-ET secretion from BPAE cells significantly(p<0.05) compared with control. Thrombin(1ug/ml, 10ug/ml) and TGF-${\beta}1$(100pM, 1000pM) significantly increased ir-ET secretion from BP AE cells(p<0.05) compared with control, but serotoin(1~100uM) and endotoxin(1ug/ml) did not stimulate the ir-ET secretion. Conclusions : Platelets stimulate endothelin secretion from bovine pulmonary artery endothelial cells. The mechanism of increase of endothelin secretion seems to be a stimulation by platelet itself or by mediators, such as TGF-${\beta}1$, secreted from activated platelets. And, in this study, the priming effect of platelets on endothelin secretion from BPAE cells could be another possibility.

• Archives of Pharmacal Research
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• v.26 no.3
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• pp.224-231
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• 2003

The anti-thrombotic effects of LB30057, a direct thrombin inhibitor, were evaluated with in vivo rat and dog thrombosis models. In rats, 1 mg/kg of LB30057 inhibited half of the clot formations in the inferior vena cava at 5 minutes after intravenous application. When measured at 2 hours after oral application, 100 mg/kg prevented approximately half of the clot formations in the inferior vena cava and 50 mg/kg prolonged the mean occlusion time from $15.6{\pm}1.3$ minutes to $47.2{\pm}8.3$ minutes in the carotid artery. In dogs, the formation of thrombus in the jugular vein was reduced to half at a dose range of 20-30 mg/kg at 6 hours after oral application. In addition, the LB30057 dosage required to reduce venous clot formation by approximately 80-90% in dogs was only about 10% of that required for the same reduction in rats. This is probably due to the variation in its time-dependent blood concentration profiles in each species; for example, the plasma half-life of LB71350 in dogs was longer than that in rats ($153.0{\pm}3.0$ vs. $129.7{\pm}12.7$ min at 30 mg/kg, i.v., respectively). AUG, $T_{max},{\;}G_{max}$, and BA in dogs were 59, 8.9, 9.17, and 13.3 times higher than those in rats at oral 30 mg/kg, respectively. Taken together, these results suggest that LB30057 administered orally is effective in the prevention of arterial and venous thrombosis in rats and dogs. It therefore represents a good lead compound for investigations to discover a new, orally available, therapeutic agent for treating thrombotic diseases.

• Journal of Biomedical Engineering Research
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• v.16 no.3
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• pp.367-375
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• 1995

Ampicillin sodium (AMP-Na) was loaded Into fibrin glue (FG) in two different ways and was tried to achieve sustained release from FG. One was loading of AMP-Na in a simple mixing and the other was loading of bovine serum albumin (BSA) microspheres which contained ANP-Na. In case of simple mixing, the release control of AWP-Na from FG was tried by variation of FBNG concentration, but failed. However, the loading of BSA mlcrosphere containing ANP-Na into FG showed sustained re- lease of AMP-Na, especially when microsphere was crosslinked with glutaraldehyde (tO.9 : 33hr). The maximum adhesive strength of FG showed at concentration of FBWG and thrombin, 5.0 % and 25-50 NIHU/ml, respectively. The concentration of Factor Xlll (0-500 U/1g of FBNG) did not affect the adhesive strength of FG. The optimal incubation time was 60 min. The AMP-Na or BSA microsphere which was loaded into FG had no significant effect on the adhesive strength of FG.

• Korean Journal of Pharmacognosy
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• v.35 no.4 s.139
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• pp.346-349
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• 2004

Black colored rice has long been consumed in Oriental countries and is considered to be a healthy food. Extracts from rice with three different colors (white, black, and green) were prepared by using 80% MeOH and subjected to biological screening activities. The levels of antioxidative activity of black colored rice cultivars such as C3GHi and Heugjinjubyeo in radical scavenging activity were higher than those of the related white and green rice ones. When the effect of the extracts on thrombin time inhibition was analyzed, the extract from Heugjinjubyeo showed a dose-dependent suppressive activity. In addition, all the extracts tested did not inhibit the cyclooxygenase activity. Therefore the health benefits of the colored rice are attributed in part to their unique phytochemical composition such as high level of anthocyanin pigments with other minor strong antioxidant components for potential use in neutraceutical or functional food formulations.