• BMB Reports
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• v.35 no.2
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• pp.199-205
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• 2002

An anticoagulant protein was purified from the edible portion of a blood ark shell, Scapharca broughtonii, by ammonium sulfate precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-75, DEAE-Sephacel, and Biogel P-l00. In vitro assays with human plasma, the anticoagulant from 'S. broughtonii, prolonged the activated partial thromboplastin time (APTT) and inhibited the factor LX in the intrinsic pathway of the blood coagulation cascade. But, the fibrin plate assay did not show that the anticoagulant is a fibrinolytic protease. The molecular mass of the purified S. broughtonii anticoagulant was measured to be about 26.0kDa by gel filtration on a Sephadex G-75 column and SDS-PAGE under denaturing conditions. The optimum activity in the APTT assay was exhibited at pH 7.0-7.5 and $40-45^{\circ}C$ in the presence of $Ca^{2+}$.

• Korean Journal of Food Science and Technology
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• v.29 no.4
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• pp.817-822
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• 1997

Inhibitory mechanism of the anticoagulant polysaccharide purified from the fruit body of Coriolus versicolor was investigated in this paper. The anticoagulant polysaccharide (CV-40-Va-1) was proposed to have functions of the inhibition of intrinsic pathway in the blood coagulation pathway together with the interuption of a human platelet aggregation induced by von Willebrand factor (vWF). CV-40-Va-I inhibited other factors of the coagulation cascade such as factor VIII, IX, and as well as thrombin. Especially, CV-40-Va-I inhibited the fibrin formation mediated by thromin, however the polysaccharide did not affect the fibrin formation directly but affected the anticoagulant activity through the activation of antithrombin III. The sulfation of the anticoagulant polysaccharide increased the anticoagulant activity, showing that the sulfate concentration of anticoagulant polysaccharide was important factor in the blood coagulation cascade. Low molecular weight subfraction (MW 1,000) obtained by partial hydrolysis of the CV-40-Va-1 generated potent antiplatelet activity, but showed decreased anticoagulant activity.

• Archives of Pharmacal Research
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• v.26 no.6
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• pp.441-445
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• 2003

New sulfonated amino ribofuranans were synthesized to elucidate the relationship between structure and specific biological activities such as anti-HIV and blood anticoagulant activities. The synthesis was performed by sulfonation of copolymers having various proportion of (1$\rightarrow5)-\alpha$-D-ribofuranosidic unit. The sulfonation with piperidine N-sulfonic acid produced the sulfonated amino ribofuranans in high yield. The anti-HIV activity of sulfonated 3-amino-3-deoxy-(1$\rightarrow5)-\alpha$-D-ribofuranan showed more potent by increasing the degree of sulfonation and the average molecular weights. This activity was almost equal to the activities of sulfonated ribofuranans and ribopyranans reported before in spite of low molecular weight. The blood anticoagulant activities was observed at 36-48 mg/units, more potent than standard dextran sulfonate, 22.7 mg/units. In addition, the blood anticoagulant activities of sulfamide-copolysaccharide consisting various proportion of (1$\rightarrow5)-\alpha$-D-ribofuranan units were potentiated by increasing sulfonated amino-ribofuranan units from 13 to 21 mg/units.

• BMB Reports
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• v.29 no.6
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• pp.500-506
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• 1996

We have isolated a water-extracted novel regulator for blood coagulation from an earthworm, Lumbricus rubellus. As a folk remedy, the earthworm has been known to facilitate blood circulation. After complete heat inactivation of endogenous proteases in the earthworm, an anticoagulant(s) was purified through ammonium sulfate fractionation and three consecutive gel permeation chromatography of Sephacryl S-300, Sephadex G-75, and G-150 by measuring activated partial thromboplastin time (APTT) The anticoagulant was further purified to 2,800 fold with a C4 reversed-phase HPLC This activity was stable under heat ($100^{\circ}C$ for 30 min) and acidic conditions (0.4 N HCl). The effects of this partially purified anticoagulant on thrombin were observed with various substrates such as N${\alpha}$-benzoyl-DL-arginine-p-nitroanilide (BApNA), H-D-phenylalanyl-L-pipecoyl-L-arginine-p-nitroanilide (S-2238), N${\alpha}$-p-tosyl-L-arginine methyl ester (TAME), and fibrinogen as a natural substrate. Only TAME hydrolysis, due to an esterase activity of the enzyme, was inhibited among the chromogenic substrates. In addition, the anticoagulant not only inhibited the conversion of fibrinogen to fibrin but also prolonged the fibrin clot formation monitored with the in vitro coagulation test. Based on these observations, we suggest the significance of measuring the ability of antithrombotic drugs to inhibit the esterase activity of thrombin. In this report, it was also shown that the earthworm indeed contained a water-extractable, heat- and acid-stable anticoagulant which could be used as a novel antithrombotic agent.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.6
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• pp.1204-1210
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• 1998

This study was focused on the purification, characterization and promotion mode of an anticoagulant polysaccharide from Hizikia fusiforme. The anticoagulant crude polysaccharide(HF 0) was obtained by using hot water extraction at 100oC for 3 hrs after homogenizing desalted Hizikia fusiforme. The anticoagulant polysaccharide(HF 2 3 1a) was purified from the crude extract(HF 0) through stepwise gradient ethanol precipitation(HF 2), DEAE Toyopearl 650C(HF 2 3), Sephadex G 75(HF 2 3 1), Sepharose CL 6B(HF 2 3 1a) chromatography and HPLC to homogeneity. HF 2 3 1a was estimated at 5.3$\times$105 Da molecular weight and composed of fucose(51.92%), galactose(19.34%), mannose(13.92%), xylose (7.14%), arabinose(3.95%) and rhamnose(3.78%), and comprimised 29.7 % sulfate residue. The sulfated anticoagulant polysaccharide from HF 2 3 1a was proposed to inhibit via the intrinsic pathway and common pathway in the blood coagulation. The HF 2 3 1a exhibited the anticoagulant activity by activating an antithrombin III and the activity depended on the concentration of HF 2 3 1a. Acute toxicity of HF 2 in mice was not detected. Only 14 of 33 control mice(11.4%) that had taken saline survived for 30 min after injecting thrombin(100 NIH unit/ml).

• Journal of the Korean Society of Food Science and Nutrition
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• v.31 no.5
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• pp.917-923
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• 2002

Inhibitory mechanism of the anticoagulant polysaccharide purified from Codium fragile was investigated. The anticoagulant compounds (Cf-30-IV-4-ii, CF-30-IV) prolonged the clotting time at both activated partial thrombo-plastin time (aPTT) and thrombin time (TT). The Inhibition factor assay of instrinsic coagulation pathway in the blood showed that the anticoagulant polysaccharide (CF-30-IV-4-ii) inhibited other factors such as Ⅷ, Ⅸ, Ⅵ and Ⅷ of the coagulation cascade, which did not affect the lupus anticoagulant AB activity. In the thrombin inhibition pattern the CF-30-IV-4-ii did not directly influence the fibrine formation mediated by thrombin but af-fected the anticoagulant activity through the activation of antithrombin III. Base on these result, the anticoaglant polysaccharide (CF-30-IV-4-ii) was considered to inhibit serine pretense involved in the blood coagulation cascade through the enhancing antithrombin III activity. The residual effects of anticoagulant activity and antithrombosis were tested with ICR mice. The anticoagulant polysaccharide (CF-30-W) kept its anticoagulant activitv for 6 hrs with 100% survival at a dose of 150 mg/kg in the antithromboisis test. The anticoagulant effect of CF-30-RF in ex vivo was proportional to the concentration of intravenously injected dose up to 100 mg/kg.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2000.05a
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• pp.90-91
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• 2000

The physiological systems that control blood fluidity are both complex and elegant. Blood must remain fluid within the vasculature and yet clot quickly when exposed to nonendothelial surfaces at sites of vascular injury. There are two principle mechanisms to control a delicate balance in higher organisms (Davie & Ratnoff, 1964). Present evidence suggests that the intrinsic pathway play an important role in the growth and maintenance of fibrin formation in the coagulation cascade while a second overlapping mechanism, called the extrinsic pathway, is critical in the initiation of fibrin formation. Coagulation factors is in two mechanisms, and in order to clot blood, they are activated by a cooperation with $Ca^{2+}$, phospholipid and vitamin K etc. For example, the human placental anticoagulant protein (PAP of PAP- I), which is a $Ca^{2+}$ -dependent phospholipid binding protein (Funakoshi et al., 1987) inhibited the activity of factor Xa, so that it prolonged fibrin formation. We wondered whether any other protein was involved in regulation of the coagulant system as an anticoagulant protein from natural organisms. Natural agents would have not harmful side-effects in comparision with chemically synthesized materials such as warfarin, aspirin, phenindione, etc.. But anticoagulant agents from natural, especially marine organisms have hardly been researched except for polysaccharides from marine algae. (omitted)

• BMB Reports
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• v.40 no.5
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• pp.832-838
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• 2007

A novel inhibitory protein against blood coagulation factor Va (FVa) was purified from muscle protein of granulated ark (Tegillarca granosa, order Arcoida, marine bivalvia) by consecutive FPLC method using anion exchange and gel permeation chromatography. In the results of ESI-QTOF tandem mass analysis and database research, it was revealed that the purified T. granosa anticoagulant protein (TGAP) has 7.7 kDa of molecular mass and its partial sequence, HTHLQRAPHPNALGYHGK, has a high identity (64%) with serine/threonine kinase derived from Rhodopirellula baltica (order Planctomycetales, marine bacteria). TGAP could potently prolong thrombin time (TT), corresponding to inhibition of thrombin (FIIa) formation. Specific factor inhibitory assay showed that TGAP inhibits FVa among the major components of prothrombinase complex. In vitro assay for direct-binding affinity using surface plasmon resonance (SPR) spectrometer indicated that TGAP could be directly bound with FVa. In addition, the binding affinity of FVa to FII was decreased by addition of TGAP in dose-dependant manner ($IC_{50}$ value = 77.9 nM). These results illustrated that TGAP might interact with a heavy chain of FVa ($FVa_H$) bound to FII in prothrombin complex. The present study elucidated that non-cytotoxic T. granosa anticoagulant protein (TGAP) bound to FVa can prolong blood coagulation time by inhibiting conversion of FII to FIIa in blood coagulation cascade. In addition, TGAP did not significantly (P < 0.05) show fibrinolytic activity and cytotoxicity on venous endothelial cell line (ECV 304).

• Food Science and Biotechnology
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• v.17 no.4
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• pp.870-872
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• 2008

Barley $\beta$-glucan was subjected to chemical modification and the anticoagulant activity of the derivative was investigated. The barley $\beta$-glucan was successfully sulfated, showing the degree of substitution calculated by elemental analysis of 0.40. In addition, the Fourier transform infrared (FT-IR) spectra of the derivative confirmed the sulfation, which generated two new absorption bands at 1,250/cm (S=O) and 810/cm (C-O-S) compared to the native. Specially, the anticoagulant activity of barley $\beta$-glucan was created by sulfation, which increased in a concentration-dependent manner. This result demonstrated that the incorporation of sulfate groups into the $\beta$-glucan structure added a blood clotting prevention effect.

• Korean Journal of Food Science and Technology
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• v.36 no.6
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• pp.1008-1013
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• 2004

Effects of extraction conditions and molecular fractionation on anticoagulant activities of major brown seaweeds in Korea were investigated. Hot water extracts of C. costata, U. pinnatifida (Sporophyte), L. japonica, K. crassifolia, E. stolonifera, E. bicyclis, S. horneri, and E. kurome increaced activated partial thromboplastin time (APTT) over 190 seconds, which may be related to intrinsic pathway of blood coagulation. Hot water extract of E. Kurome (EKJ) was further fractionated by ethanol precipitation. EKJ-eim, ethanol-insoluble material of EKJ, showed higher anticoagulant activity than EKJ. EKJ-eim was further fractioned with ultrafiltration. EKJ-eim 1, (over 100 kDa) fraction showed higher APTT activity than EKJ-eim. A EKJ-eim 1 was sulfated polysaccharide consisting of fucose, xylose, mannose, galactose, glucose and, sulfate at molar ratio of 1 : 0.05 : 0.10 : 0.15 : 0.17 : 1.46. The anticoagulant activity increased as sulfate content and molecular weight increased.