Antithrombin activity and disaccharide composition of dermatan sulfate from different bovine tissues

Dermatan sulfate is a glycosaminoglycan that selectively inhibits the action of thrombin through interaction with heparin cofactor II. Unlike heparin it does not interact with other coagulation factors and is able to inhibit thrombin associated with clots. This property has made dermatan sulfate an attractive candidate as an antithrombotic drug. Previous studies have showed that dermatan sulfate derived from porcine/bovine intestinal mucosa/skin or marine invertebrates is capable of stimulating heparin cofactor II-mediated thrombin inhibition in vitro. This biological activity is reported for the first time in this study using dermatan sulfate derived from mammalian tissues other than intestinal mucosa or skin. Ten different bovine tissues including the aorta, diaphragm, eyes, large and small intestine, esophagus, skin, tendon, tongue, and tongue skin were used to prepare dermatan sulfate-enriched fractions by anion exchange chromatography and acetone precipitation. Heparin cofactor II/dermatan sulfate-mediated thrombin inhibition measured in vitro revealed activity comparable to or higher than the commercial standard with 2-fold differences observed between some tissues. Analysis of the extracted dermatan sulfate using fluorophore-assisted carbohydrate electrophoresis revealed significant differences in the relative percentage of all the mono-sulfated disaccharides, in particular the predominant mammalian disaccharide uronic acid-->N-acetyl-D-galactosamine-4-O-sulfate, confirming previous reports regarding variations in sulfation in dermatan sulfate from different tissues. Overall, these findings demonstrate that dermatan sulfate extracted from a range of bovine tissues exhibits in vitro antithrombin activity equivalent to or higher than that observed for porcine intestinal mucosa, identifying additional sources of dermatan sulfate as potential antithrombotic agents.     

Increased sulphation improves the anticoagulant activities of heparan sulphate and dermatan sulphate.

Heparan sulphate and dermatan sulphate have both antithrombotic and anticoagulant properties. These are, however, significantly weaker than those of a comparable amount of standard pig mucosal heparin. Antithrombotic and anticoagulant effects of glycosaminoglycans depend on their ability to catalyse the inhibition of thrombin and/or to inhibit the activation of prothrombin. Since heparan sulphate and dermatan sulphate are less sulphated than unfractionated heparin, we investigated whether the decreased sulphation contributes to the lower antithrombotic and anticoagulant activities compared with standard heparin. To do this, we compared the anticoagulant activities of heparan sulphate and dermatan sulphate with those of their derivatives resulphated in vitro. The ratio of sulphate to carboxylate in these resulphated heparan sulphate and dermatan sulphate derivatives was approximately twice that of the parent compounds and similar to that of standard heparin. Anticoagulant effects were assessed by determining (a) the catalytic effects of each glycosaminoglycan on the inhibition of thrombin added to plasma, and (b) the ability of each glycosaminoglycan to inhibit the activation of 125I-prothrombin in plasma. The least sulphated glycosaminoglycans were least able to catalyse the inhibition of thrombin added to plasma and to inhibit the activation of prothrombin. Furthermore, increasing the degree of sulphation improved the catalytic effects of glycosaminoglycans on the inhibition of thrombin by heparin cofactor II in plasma. The degree of sulphation therefore appears to be an important functional property that contributes significantly to the anticoagulant effects of the two glycosaminoglycans.     

Dermatan sulfate is the predominant antithrombotic glycosaminoglycan in vessel walls: implications for a possible physiological function of heparin cofactor II

The role of different glycosaminoglycan species from the vessel walls as physiological antithrombotic agents remains controversial. To further investigate this aspect we extracted glycosaminoglycans from human thoracic aorta and saphenous vein. The different species were highly purified and their anticoagulant and antithrombotic activities tested by in vitro and in vivo assays. We observed that dermatan sulfate is the major anticoagulant and antithrombotic among the vessel wall glycosaminoglycans while the bulk of heparan sulfate is a poorly sulfated glycosaminoglycan, devoid of anticoagulant and antithrombotic activities. Minor amounts of particular a heparan sulfate (< 5% of the total arterial glycosaminoglycans) with high anticoagulant activity were also observed, as assessed by its retention on an antithrombin-affinity column. Possibly, this anticoagulant heparan sulfate originates from the endothelial cells and may exert a significant physiological role due to its location in the interface between the vessel wall and the blood. In view of these results we discuss a possible balance between the two glycosaminoglycan-dependent anticoagulant pathways present in the vascular wall. One is based on antithrombin activation by the heparan sulfate expressed by the endothelial cells. The other, which may assume special relevance after vascular endothelial injury, is based on heparin cofactor II activation by the dermatan sulfate proteoglycans synthesized by cells from the subendothelial layer.     

Structural composition and anticoagulant activity of dermatan sulfate from the skin of the electric eel, Electrophorus electricus (L.)

We determined the disaccharide composition of dermatan sulfate (DS) purified from the skin of the electric eel Electrophorus electricus. DS obtained from the electric eel was composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. The anticoagulant, antithrombotic and bleeding effects of electric eel skin DS were compared to those of porcine DS and also to those described previously for DS purified from skin of eel, Anguilla japonica. DS from electric eel is a potent anticoagulant due to a high heparin co-factor II (HC II) activity. The electric eel DS has a higher potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the porcine DS. Interestingly, it was recently demonstrated that DS obtained from skin of the eel Anguilla japonica, which possesses a disaccharide composition very similar to that of electric eel skin DS described here, did not show anticoagulant activity. Thus, the anticoagulant activity of electric eel skin DS is not merely a consequence of its charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to different arrangements of the disulfated disaccharide domain for binding to HC II within their polysaccharide chains and that it may be more efficiently arranged along the carbohydrate chain in electric eel skin DS than in the two other types of DS.     

Inhibition of factor X and factor V activation by dermatan sulfate and a pentasaccharide with high affinity for antithrombin III in human plasma

There is evidence that by catalyzing thrombin inhibition, several glycosaminoglycans can inhibit the thrombin-mediated amplification reactions of coagulation and thereby delay prothrombin activation. The two amplification reactions can apparently be catalysed by endogenously generated factor Xa and thrombin. This study provides evidence which suggests that on a molar basis, an agent which can only catalyse thrombin inhibition is approximately 10 times more effective than an agent which can only catalyse factor Xa inhibition in their ability to inhibit intrinsic prothrombin activation. We determined the concentrations of each of heparin, dermatan sulfate and a pentasaccharide with high affinity for antithrombin III, to delay intrinsic prothrombin activation for at least 15s. Heparin catalyses both thrombin and factor Xa inhibition; dermatan sulfate catalyses only thrombin inhibition, while the pentasaccharide only catalyses factor Xa inhibition. Efficient prothrombin activation, which coincided with both factor X activation and factor V proteolysis, was first observed 45s after CaC12 was added to contact-activated plasma. Heparin (approximately 0.1 microM) prolonged by at least 30 s the time required for the activation of the three clotting factors to begin. The minimum concentrations of the pentasaccharide and dermatan sulfate to delay the activation of prothrombin, factors X and V were approximately 50 microM and approximately 5 microM, respectively. Thus, each anticoagulant could inhibit intrinsic prothrombin activation only when it inhibited activation of both factors X and V. A combination of approximately 5 microM pentasaccharide and approximately 0.05 microM dermatan sulfate similarly delayed the activation of all three clotting factors. Thus, while catalysis of thrombin inhibition is a more effective pathway than catalysis of factor Xa inhibition for delaying prothrombin activation, the simultaneous catalysis of thrombin and factor Xa inhibition can synergistically improve the ability of a sulfated polysaccharide to delay prothrombin activation.     

Osmotic stress regulates the anticoagulant efficiency of dermatan sulfate.

Glycosaminoglycans (GAGs) in pericellular and interstitial spaces help to maintain local water homeostasis and blood coagulation balance. This study explored whether dehydrating microenvironment conditions influence dermatan sulfate's (DS) anticoagulant activity. Water transfer during antithrombin activation by dermatan sulfate was measured using osmotic stress techniques. Anticoagulant activity was determined from the change in the rate of coagulation factor Xa (fXa) inhibition. Osmotic stress accelerated reaction rates, indicating water transfer from reactants to bulk. The net volume transferred, measured using osmotic probes similar in size to the reacting proteins, was approximately 2500 mol of water per mole of fXa inhibited. The reaction efficiency, V(sat)/K 1/2 (rate at saturation/concentration resulting in half-maximal rates), determined in titrations with monosulfated dermatan sulfate and disulfated dermatan sulfate (DDS), were 4x10(4) and 2x10(5) M-1 s-1 under osmotic stress and in the presence of calcium, corresponding to 34- and 81-fold increases over efficiency measured under standard conditions. These results indicate that dermatan sulfate can contribute significantly to antithrombin activation, and that in dehydrating environments and depending of ionic conditions, its anticoagulant efficiency can exceed that of heparan sulfate (HS).     

Heparin cofactor IIOslo. Mutation of Arg-189 to His decreases the affinity for dermatan sulfate

Heparin and dermatan sulfate increase the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the proteinase bind. A variant form of HCII that binds heparin but not dermatan sulfate has been described recently in two heterozygous individuals (Andersson, T.R., Larsen, M.L., and Abildgaard, U. (1987) Thromb. Res. 47, 243-248). We have now purified the variant HCII (designated HCIIOslo) from the plasma of ne of these individuals. HCIIOslo or normal HCII (11 nM) was incubated with thrombin (9 nM) for 1 min in the presence of heparin or dermatan sulfate. Fifty percent inhibition of thrombin occurred at 26 micrograms/ml dermatan sulfate with normal HCII and greater than 1600 micrograms/ml dermatan sulfate with HCIIOslo. In contrast, inhibition of thrombin occurred at a similar concentration of heparin (1.0-1.5 micrograms/ml) with both inhibitors. To identify the mutation in HCIIOslo, DNA fragments encoding the N-terminal 220 amino acid residues of HCII were amplified from leukocyte DNA by the Taq DNA polymerase chain reaction and both alleles were cloned. A point mutation (G----A) resulting in substitution of His for Arg-189 was found in one allele. The same mutation was constructed in the cDNA of native HCII by oligonucleotide-directed mutagenesis and expressed in Escherichia coli. The recombinant HCIIHis-189 reacted with thrombin in the presence of heparin but not dermatan sulfate, confirming that this mutation is responsible for the functional abnormality in HCIIOslo.     

Suppression of experimental autoimmune encephalomyelitis by dermatan sulfate

The effect of dermatan sulfate (DS) on the treatment of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was examined. DS, a sulfated glycosaminoglycan, has been reported to exhibit anticoagulant and fibrinolytic activities. DS treatment (50 mg/kg/day) facilitates recovery from the clinical manifestations of EAE. In this study, the fibrinolytic activity was higher in DS-treated rats than in saline-treated rats. Although the degree of perivascular mononuclear cell infiltration in the spinal cord was not suppressed in DS-treated rats compared to that in saline-treated rats, perivascular fibrin deposition was markedly suppressed in DS-treated rats. These findings suggest that DS would act as an effective therapeutic agent for EAE by preventing fibrin deposition.     

Purification and characterization of dermatan sulfate from the skin of the eel,Anguilla japonica

Glycosaminoglycans were isolated from the eel skin (Anguilla japonica) by actinase and endonuclease digestions, followed by a beta-elimination reaction and DEAE-Sephacel chromatography. Dermatan sulfate was the major glycosaminoglycan in the eel skin with 88% of the total uronic acid. The content of the IdoA2Salpha1-->4GalNAc4S sequence in eel skin, which shows anticoagulant activity through binding to heparin cofactor II, was two times higher than that of dermatan sulfate from porcine skin. The anti-IIa activity of eel skin dermatan sulfate was determined to be 2.4 units/mg, whereas dermatan sulfate from porcine skin shows 23.2 units/mg. The average molecular weight of dermatan sulfate was determined by gel chromatography on a TSKgel G3000SWXL column as 14 kDa. Based on 1H NMR spectroscopy, the presence of 3-sulfated and/or 2,3-sulfated IdoA residues was suggested. The reason why highly sulfated dermatan sulfate does not show anticoagulant activity is discussed. In addition to dermatan sulfate, the eel skin contained a small amount of keratan sulfate, which was identified by keratanase treatment.     

Experimental studies on the anticoagulant and antithrombotic effects of sodium and calcium pentosan polysulphate.

In the present study we have compared the antithrombotic and anticoagulant properties of sodium and calcium derivatives of pentosan polysulphate (Na-PPS, Ca-PPS). The antithrombotic effect of these agents have been investigated in an experimental thrombosis model in which rat mesenteric venules diameter of 20-30 microm were injured by well defined Argon laser lesions. Furthermore, the in vivo and in vitro anticoagulant activities (aPTT, Heptest) of these agents have been studied. Thrombus formation was significantly inhibited after s.c. injection of Na-PPS and Ca-PPS in doses above 10 mg/kg. The duration of the antithrombotic effect lasted 8 h for Na-PPS and 12 h for Ca-PPS. After oral administration of Na-PPS an antithrombotic effect was not observed. Oral application of Ca-PPS in doses higher than 20 mg/kg significantly inhibited thrombus formation. Na-PPS and Ca-PPS markedly prolonged clotting time in aPTT and Heptest in concentrations ranging from 0.01 to 0.2 mg/ml rat PTT. Two h after s.c. administration of these agents in a dose 10 mg/kg, the aPTT increased 3-fold and Heptest 2.5-fold compared to controls. After oral application of 50 mg/kg Na-PPS and Ca-PPS no effect on coagulation test could be measured.     

Anticoagulant activity of fucosylated chondroitin sulfate isolated from Cucumaria syracusana

Fucosylated chondroitin sulfate (FCScs) isolated from sea cucumber Cucumaria syracusana was characterized by Fourier Transform InfraRed spectroscopy (FT-IR), Nuclear Magnetic Resonance (NMR) spectroscopy and high performance size exclusion chromatograph, a multi-angle laser light scattering detector, a viscometer and a differential refractive index (dRI) detector (HPSEC-MALLS-dRI). The anticoagulant activities of FCScs were studied by the classical clotting time assays and the purified systems containing thrombin and antithrombin or heparin cofactor II. The effect on thrombin generation was investigated using calibrated automated thrombography (CAT). The results obtained showed that the FCS with high sulfate content 31 % and relatively low average molecular weight of 36.3 kDa was isolated from C. syracusana in amount of ∼ 35.6 mg/g dry body wall. Structural analysis of this polysaccharide revealed the presence backbone structure of chondroitin sulfate chain branched by two types of fucose 2,4-O-di and 3,4-O-disulfated residues in respective ratios of 57.5 and 42.5 %. The FCScs exhibited a high anticoagulant activity mediated essentially by heparin cofactor II (HCII) and to lesser extent by antithrombin (AT) with IC₅₀ values of 0.05 μg/mL and 0.09 μg/mL, respectively. Furthermore, the results of CAT assay showed that the velocity index decreases 3-times at 50 μg/mL in comparison with normal plasma. The overall results showed high anticoagulant activity attributed to the high sulfate content and abundance of disulfated fucose branches of FCScs which made it a promising candidate of anticoagulation drug.     

Evaluation of the reticuloendothelial system function by the vascular clearance of chondroitin sulfate iron colloid

Vascular clearance of chondroitin sulfate iron colloid (CSFe) was evaluated as a test for the reticuloendothelial system (RES) in rabbits. Injected CS59Fe was taken up by the RES in the liver (49%) and bone marrow (41%) after 60 min, suggesting its application for the RES function test. The clearance rate (K-value) of CSFe from the blood was calculated by measuring serum Fe concentrations after releasing iron from CSFe at certain intervals after injection. Depending upon different injected doses, K-values were varied and the phagocytic velocity, computed by multiplying K-values by corresponding injected doses, reached a plateau at high doses, indicating the phagocytosis of CSFe by the RES takes a saturation process. Double-reciprocal plotting of the dose and the phagocytic velocity showed a linear relationship, which provided the data on the maximum phagocytic velocity (Vmax), 0.122 mg/kg/min, and the CSFe concentration producing 1/2 Vmax (Kp), 0.426 mg/kg. Thus, this CSFe clearance test can be used for the evaluation of the RES function.     

Antithrombotic and profibrinolytic activities of eckol and dieckol

In order to develop new anticoagulant agents, two single compounds (eckol and dieckol) were isolated from Eisenia bicyclis and examined their anticoagulant activities by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT) as well as cell-based thrombin and activated factor X (FXa) generation activities. And the effects of eckol and dieckol on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells (HUVECs). Data showed that eckol and dieckol prolonged aPTT and PT significantly and inhibited thrombin and FXa activities. They also inhibited the generation of thrombin or FXa in HUVECs. In accordance with these anticoagulant activities, eckol or dieckol showed anticoagulant effect in vivo. Furthermore, eckol and dieckol inhibited TNF-α induced PAI-1 production and the ratio between PAI-1 and t-PA was found to be significantly decreased by eckol and dieckol. Surprisingly, these anticoagulant and profibrinolytic effects of dieckol were better than those of eckol indicating that hydroxyl group in eckol positively regulated anticoagulant function of eckol. Therefore, these results suggest that eckol or dieckol possesses antithrombotic activities and provides a possibility to develop as an agent for the anticoagulation.     

Molecular size of dermatan sulfate oligosaccharides required to bind and activate heparin cofactor II

Heparin cofactor II (HCII) inhibits thrombin rapidly in human plasma in the presence of heparin or dermatan sulfate. To determine the minimum structure of dermatan sulfate required to activate HCII, the glycosaminoglycan was partially degraded by sequential treatment with periodate, [3H]borohydride, and sulfuric acid. Labeled oligosaccharide fragments were separated by gel filtration chromatography. Purified fragments were then applied to a column of HCII bound to concanavalin A-Sepharose, and bound oligosaccharides were eluted with a gradient of sodium chloride. Di-, tetra-, and hexasaccharide fragments did not bind to HCII, while 15% of the octasaccharides and up to 45% of larger fragments bound. Octasaccharides that bound to the HCII column had a greater negative charge than the run-through material based on anion-exchange chromatography, suggesting that they contained a greater number of sulfate groups per molecule. Fragments of dermatan sulfate containing a minimum of 12-14 sugar residues accelerated inhibition of thrombin by HCII. Fragments of this length that bound to the column of immobilized HCII had molar specific activities greater than those of the fragments that did not bind. These studies suggest that HCII is activated by dermatan sulfate fragments greater than or equal to 12 residues in length that contain a specific octasaccharide sequence required for binding to the inhibitor.     

Anticoagulant activities of curcumin and its derivative

Curcumin, a polyphenol responsible for the yellow color of the curry spice turmeric, possesses antiinflammatory, antiproliferative and antiangiogenic activities. However, anticoagulant activities of curcumin have not been studied. Here, the anticoagulant properties of curcumin and its derivative (bisdemethoxycurcumin, BDMC) were determined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT) as well as cell-based thrombin and activated factor X (FXa) generation activities. Data showed that curcumin and BDMC prolonged aPTT and PT significantly and inhibited thrombin and FXa activities. They inhibited the generation of thrombin or FXa. In accordance with these anticoagulant activities, curcumin and BDMC showed anticoagulant effect in vivo. Surprisingly, these anticoagulant effects of curcumin were better than those of BDMC indicating that methoxy group in curcumin positively regulated anticoagulant function of curcumin. Therefore, these results suggest that curcumin and BDMC possess antithrombotic activities and daily consumption of the curry spice turmeric might help maintain anticoagulant status.     

Anticoagulant activities of piperlonguminine in vitro and in vivo

Piperlonguminine (PL), an important component of Piper longum fruits, is known to exhibit anti-hyperlipidemic, antiplatelet and anti-melanogenic activities. Here, the anticoagulant activities of PL were examined by monitoring activatedpartial-thromboplastin-time (aPTT), prothrombin-time (PT), and the activities of thrombin and activated factor X (FXa). The effects of PL on the expressions of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were also tested in tumor necrosis factor-α (TNF-α) activated HUVECs. The results showed that PL prolonged aPTT and PT significantly and inhibited the activities of thrombin and FXa. PL inhibited the generation of thrombin and FXa in HUVECs. In accordance with these anticoagulant activities, PL prolonged in vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significantly decreased by PL. Collectively, our results suggest that PL possesses antithrombotic activities and that the current study could provide bases for the development of new anticoagulant agents. [BMB Reports 2013; 46(10): 484-489]     

Anticoagulant and antithrombin activities of oversulfated fucans.

Three species of oversulfated fucans having different sulfate contents (the ratio of sulfate/total sugar residues, 1.38-1.98) were prepared by chemical sulfation of a fucan sulfate (sulfate/sugar ratio, 1.28) isolated from the brown seaweed Ecklonia kurome. The anticoagulant activities of the oversulfated fucans were compared with that of a parent fucan with respect to activated partial thromboplastin time (APTT) and thrombin time (TT) in plasma. The respective activities (for APTT and TT) of the oversulfated fucans increased to 110-119% and 108-140% of the original values with increase in their sulfate content. The anticoagulant activity with respect to APTT (173 units/mg) of an oversulfated fucan (sulfate/sugar ratio, 1.98) was higher than that (167 units/mg) of heparin used as a standard. The heparin cofactor II-mediated antithrombin activity of the oversulfated fucans also increased significantly with increase in sulfate content. The maximum activity was higher than those of the parent fucan and heparin. However, the increment of the anticoagulant and the antithrombin effects gradually decreased with increase in the sulfate content of the fucans. These results indicate that the effects of the fucan sulfate are dependent on its sulfate content until a plateau is reached.     

Anticoagulant and antithrombotic activities of a chemically sulfated galactoglucomannan obtained from the lichen Cladonia ibitipocae

A galactoglucomannan (GGM), isolated from the lichen Cladonia ibitipocae, consisted of a (1-->6)-linked main chain of alpha-mannopyranose units, substituted by alpha- and beta-D-galacto (alpha- and beta-D-Galp)-, beta-D-gluco (beta-D-Glcp)- and alpha-D-mannopyranosyl (alpha-D-Manp) groups, and was sulfated giving a sulfated polysaccharide (GGM-SO4) with 42.2% sulfate corresponding to a degree of substitution of 1.29. NMR studies indicated that after sulfation, the OH-6 groups of galactopyranosyl and mannopyranosyl units were preferentially substituted. GGM-SO4 was investigated in terms of its in vitro anticoagulant and in vivo antithrombotic properties. Those of the former were evaluated by its activated partial thromboplastin (APTT) and thrombin time (TT), using pooled normal human plasma, and compared with that of 140 USP units mg(-1) for a porcine intestinal mucosa heparin. Anticoagulant activity was detected in GGM-SO4, but not in GGM. The in vivo antithrombotic properties of GGM-SO4 were evaluated using a stasis thrombosis model in Wistar rats, intravenous administration of 2 mg kg(-1) body weight totally inhibiting thrombus formation. It caused dose-dependent increases in tail transection bleeding time. The results obtained showed that this sulfated polysaccharides is a promising anticoagulant and antithrombotic agent.     

Anticoagulant activities of oleanolic acid via inhibition of tissue factor expressions

Oleanolic acid (OA), a triterpenoid known for its anti-inflammatory and anti-cancer properties, is commonly present in several medicinal plants but its anticoagulant activities have not been studied. Here, the anticoagulant properties of OA were determined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrin polymerization as well as cell-based thrombin and activated factor X (FXa) generation activities. Data showed OA prolonged aPTT and PT significantly and inhibited thrombin catalyzed fibrin polymerization. In addition, OA inhibited the activities of thrombin and FXa and inhibited the generation of thrombin or FXa in human endothelial cells. OA also inhibited TNF-α-induced tissue factor expression on human endothelial cells. In accordance with these anticoagulant activities, OA showed an anticoagulant effect in vivo. These results indicate that OA possesses antithrombotic activities and suggest that daily consumption of a herb containing OA may be preventing thrombosis in pathological states.