Research advances in the study of anticoagulant active molecules / 药学学报

The incidence of thrombosis-induced cardiovascular diseases is increasing worldwide and poses a serious threat to human health. Three factors, slow speed of blood flow, hypercoagulable blood and vascular damage, have been considered to be causes of thrombosis. Antithrombotic drugs have been classified into three categories based on the mechanism of thrombosis, including anticoagulants, platelet inhibitors and fibrinolytics. The coagulation and anticoagulation systems have drawn increasing attention because of the important role they play in the process of thrombosis. Novel compounds with anticoagulant activity are now emerging, alleviating to some extent some of the problems associated with the clinical use of early approved thrombotic drugs, such as high bleeding risk, slow onset of action and narrow therapeutic windows. In this review, we initially describe the mechanisms of coagulation as well as thrombosis. Meanwhile, a wide range of bioactive compounds and potential antithrombotic candidates reported in recent years have been summarized. In addition, the structure-activity relationship of certain compounds has been discussed, expecting to facilitate the development of molecules with anticoagulant biological activity for the treatment of thrombotic diseases.

Predictive Analysis of Quality Markers of Anticoagulant Activity of Kunning Granules Based on UHPLC-Q-TOF-MS/MS and Spectral Efficacy / 中国实验方剂学杂志

ObjectiveTo perform a predictive analysis of the quality marker（Q-Marker） for the anticoagulant activity of Kunning granules. MethodThe chemical components of Kunning granules were analyzed by ultra high performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry（UHPLC-Q-TOF-MS/MS） on a Waters ACQUITY UPLC HSS T3 column（2.1 mm×100 mm， 1.8 μm） with the mobile phase of acetonitrile（A）-25 mmol∙L-1 ammonium acetate aqueous solution（B） for gradient elution （0-5 min， 5%-22%A； 5-10 min， 22%-30%A； 10-15 min， 30%-95%A； 15-20 min， 95%-5%A； 20-30 min， 5%A）， flow rate of 0.2 mL∙min-1， column temperature at 30 ℃， injection volume of 1 μL， electrospray ionization（ESI）， positive and negative ion detection modes. Interaction analysis between the targets of chemical components and the targets of abnormal uterine bleeding（AUB） was performed by network pharmacology， and the key components were screened through network topology analysis. The fingerprints of 10 batches of Kunning granules were established by high performance liquid chromatography（HPLC）， the anticoagulant activity of the granules was determined by blood coagulation method and fibrinogen plate method， and the spectrum-effective relationship was established. The components co-occurring in the topological analysis and spectrum-effective relationship were selected as Q-Markers， and their anticoagulant activities were verified and confirmed. ResultA total of 475 chemical components were identified from Kunning Granule， of which 22 key components such as salvianolic acid B， paeoniflorin， naringin and neohesperidin， were the potential material basis for the treatment of AUB. The spectrum-effective analysis showed that peaks 7（paeoniflorin）， 9（naringin）， 10（neohesperidin） and 11（salvianolic acid B） were the optimal principal components， and in vitro activity test showed that these four components could better characterize their anticoagulant activity. ConclusionSalvianolic acid B， paeoniflorin， neohesperidin and naringin may be Q-Markers for the anticoagulant activity of Kunning granules.

Rapid Enrichment and Activity Research of Antithrombotic Proteins from Pheretima vulgaris / 中国实验方剂学杂志

Objective:A strong antithrombotic protein component， named PvQ， was purified and enriched from total protein of <italic>Pheretima vulgaris</italic>，<italic> </italic>a<italic> </italic>traditional Chinese medicine. Moreover， we evaluated its fibrinolytic and anticoagulant activity， and expected to provide reference for the research on antithrombotic substances of Pheretima. Method:A rapid <italic>in</italic> <italic>vitro</italic> activity-oriented separation combined with the AKTA-Pure protein purification system conducted on <italic>P. vulgaris</italic>. Meanwhile， the fibrinolytic and anticoagulant activities of PvQ were measured by fibrin plate method and fibrinogen-thrombin time （Fibg-TT） method. And the <italic>in vitro</italic> thrombolysis assay was used for evaluating the lysis ability of PvQ to thrombus. Then the stability of PvQ was also analyzed for its anticoagulant activity at different pH and temperature. Result:The PvQ was successfully enriched and its activity was determined to have significant fibrinolytic and anticoagulant activities. And the result of <italic>in vitro</italic> thrombolysis assay revealed that PvQ could hydrolyze more than 80% of thrombus after 5 h of incubation at 37 ℃. In addition， the changes of temperature and pH had significant effects on antithrombotic activity， and this study showed that PvQ was rapidly inactivated at ≥60 ℃ or in acidic conditions （pH<7）. While， the activity of PvQ was unaffected or less affected at ≤50 ℃ and under alkaline conditions. Conclusion:A feasible preparation method of PvQ is established， and it can affect fibrin and fibrinogen at the same time， thus exerting a dual fibrinolytic effect and possessing significant fibrinolytic and anticoagulant activities. It provides a scientific interpretation for the treatment of thrombotic diseases by PvQ and a reference for the development of antithrombotic protein products of Pheretima.

Isolation, fractionation and anticoagulant activity of a sulfated galactan extracted from the green algae Penicillus capitatus / Isolamento, fracionamento e atividade anticoagulante da galactana sulfatada extraída da alga verde Penicillus capitatus

ABSTRACT: Marine algae are natural sources of macromolecules known as sulfated polysaccharides. This class of compounds has attracted the interest of Pharmaceutical Sciences due to its pharmacological anticoagulant, antiplatelet and antithrombotic properties. Therefore, this study evaluated the anticoagulant potential of sulfated polysaccharides extracted from the algae Penicillus capitatus. The extracted sulfated polysaccharides were purified, partially characterized and their anticoagulant activity was evaluated. The extraction process followed by ethanol precipitation resulted in five fractions. Among the analyzed fractions, F44 contained highest concentration of sulfated polysaccharides. After the purified fraction F23, F44 displayed in vitro anticoagulant activity in a time testing for activated partial thromboplastin time and prothrombin time. The preferential mechanism effect was based on interactions between thrombin and factor Xa. Additional studies on structure pharmacological are required to test the viability of the use of sulfated polysaccharides as therapeutic agents.

RESUMO: As algas marinhas são fontes naturais de macromoléculas conhecidas como polissacarídeos sulfatados. Esta classe de compostos atraiu o interesse das Ciências Farmacêuticas devido às suas propriedades farmacológicas como anticoagulante, antiplaquetária e antitrombótica. Portanto, este estudo tem como objetivo avaliar o potencial anticoagulante de polissacarídeos sulfatados extraídos de algas de Penicillus capitatus. Os polissacarídeos sulfatados extraídos foram purificados, parcialmente caracterizados e sua atividade anticoagulante foi avaliada. O processo de extração seguido pela precipitação com etanol resultou em cinco frações. Entre as frações analisadas, F44 foi a maior concentração de polissacarídeos sulfatados. Após a purificação, as frações F23 e F44 mostraram atividade anticoagulante in vitro em um teste de tempo de tromboplastina parcialmente ativada e tempo de protrombina. Seu mecanismo preferencial é baseado nas interações entre trombina e fator Xa. Estudos adicionais sobre a estrutura farmacológica são necessários para testar a viabilidade do uso como agente terapêutico.

Research progress on proteins and peptides from earthworm / 中草药

Earthworm is one of animal drugs in Chinese materia medica. It was commonly used in clinic with rich resources in China. Modern studies showed that proteins and peptides were the main components in earthworm. It has been used for treating cardiovascular and cerebrovascular diseases because of its various activities, such as anticoagulant, anti-stroke, antibacterial, and antifibrotic activities, etc. In this review, 48 proteins and peptides from different species of earthworm reported since 1983 were summarized, including their names, molecular weights, amino acid sequences, isoelectric points, and activities. In addition, its pharmacological effects of earthworm proteins and peptides were summarized. In all, it will provide a scientific basis for the further study and comprehensive utilization of proteins and peptides of earthworm.

Optimized expression of heparin sulfotransferases and their application in sulfation of animal derived heparin / 生物工程学报

Heparin is a very important anticoagulant drug. Currently, heparin is mainly extracted from porcine mucosa. However, animal-derived heparin shows low anticoagulant activity due to the low proportion of the anticoagulant active unit, the GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S pentasaccharide. In this study we proposed an enzymatic strategy to sulfate the animal-sourced heparin to increase the proportion of anticoagulant pentasaccharide and the anticoagulant activity. First, three sulfotransferases HS2ST, HS6ST, and HS3ST were expressed tentatively in Escherichia coli and Pichia pastoris. After measuring the sulfotransferase activity, we confirmed P. pastoris GS115 is the better host for sulfotransferases production. Then, the maltose binding protein (MBP) and thioredoxin (TrxA) were fused separately to the N-terminal of sulfotransferases to increase enzyme solubility. As a result, the yields of HS2ST and HS6ST were increased to (839±14) U/L and (792±23) U/L, respectively. Subsequent sulfation of the animal-sourced heparin with the recombinant HS2ST, HS6ST and HS3ST increased the anticoagulant activity from (76±2) IU/mg to (189±17) IU/mg.

Research progress on chemical ingredients of Aleppo Avens and their pharmacological effects / 中草药

Aleppo Avens, a traditional Chinese medicine, has been commonly used for the treatment of headache and dizziness, infantile convulsion, hypertension, lumbar and leg pain, irregular menstruation, and so on. However, the underlying mechanisms of the functions remain unclear. Up to the present, terpenoids (monoterpene, sesquiterpene and three terpenoids), tannins and phenylpropyl esters were found as the main chemical composition in Aleppo Avens. Accumulating studies showed that Aleppo Avens possess the pharmacological profile of anticoagulant, antihypertensive, anti-inflammatory, anticancer, and anti-ischemia. The present paper reviewed the researches of active ingredients and pharmacological effects of Aleppo Avens in recent 40 years, and provided the pharmacological basis for the clinical application and further rational development of Aleppo Avens.

Preparation of anticoagulant peptide from casein and study on thrombolysis and anticoagulation in vitro / 中草药

Objective: To study the preparation and separation method for anticoagulant peptide and the effect of anticoagulation and thrombolysis in vitro. Methods: The casein was hydrolyzed to prepare anticoagulant peptide using the mixed four enzymes such as papain, pineapple proteinase, neutral protease, and alkali protease. The anticoagulant peptide was extracted using immobilized thrombin. The effect of haemolysis and anticoagulation in vitro was investigated through the New Zealand rabbits experiments. Results: The conditions of preparation anticoagulant peptide were as follows: quality of casein was 15%, papain proteinase, pineapple proteinase, neutral protease, and alcalase dosage were 1500, 2400, 1000, and 1250 U/(g casein), respectively, temperature was 50℃, pH value was 7.0, and hydrolysis time was 4 h. The conditions for the extraction of anticoagulant peptide were as follows: the initial concentration was 6 ATU (Anti Thrombin Unit)/mL, temperature was 30℃, pH value was 5.0, and time was 30 min. Anti-extraction temperature was 30℃, pH value was 7.6, and time was 40 min. The purified anticoagulant peptide was analyzed via high performance size exclusion chromatography. The molecular weight of purified anticoagulant peptide was equal to N-Hippuryl-His-Leu hydrate and the main components were three peptides. The time of anticoagulation was more than 72 h and the time of hemolysis was 24 h in vitro. Conclusion: The main components of anticoagulant peptides are three peptides. The effect of hemolysis and anticoagulation in vitro is good.

A Research Progress and the Prospective of Hirudo Nipponica / 世界科学技术-中医药现代化

Hirudo nipponica Whitman,a Chinese medicine which has a strong effect on platelet aggregation and antithrombin activity,is involved in the studies.The current situation is adverse to the cultivation,development and utilization of H.nipponica.Prior to summarizing the current research status and the unresolved problems and the prospective of H.nipponica,domestic and foreign literatures over the H.nipponica was discussed and judged,aiming at providing a reference for the comprehensive development and utilization of H.nipponica.

In vitro anticoagulant activity of different processed products of Whitmania pigra by water extraction and bionic extraction / 中国中药杂志

In order to determine the scientificalness of traditionally processed Whitmania pigra, water extraction method and bionic extraction method were used respectively to extract the anticoagulating active components in W. pigra hanging dry products, talcum powder fried products and wine immersing-baked products. Activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and antithrombin activity were selected as the activity indexes to evaluate the anticoagulant activities of different processed W. pigra. Then the contents of protein in different processed W. pigra were measured by Coomassie brilliant blue method to preliminarily explain the reason of anticoagulant activity changes. When water extraction method was used, the results of APTT, PT, TT and antithrombin activity showed that the anticoagulant activities of W. pigra were decreased both in talcum powder fried products and wine immersing-baked products, and the activity order was as follows: hanging dried products> wine immersing-baked products>talcum powder fried products. This order was same as the protein content order. While when bionic extraction was used, APTT was shortened in talcum powder fried products, but all the other results indicated the anticoagulant activities of W. pigra processed products were increased, and the activity order was as follows: wine immersing-baked products>talcum powder fried products>hanging dry products. As compared with water extraction, the bionic extraction was more similar to the absorption process of W. pigra in human digestive system after oral administration and was more scientific. Therefore, the traditional processing method can not only modify the taste and smell, but also enhance the anticoagulant activity of W. pigra.

Potential Use of Polysaccharides from the Brown Alga Undaria pinnatifida as Anticoagulants

Undaria pinnatifida (U. pinnatifida) is a highly invasive species and has caused concern all over the world because it has invaded coastal environments, has the potential to displace native species, significantly alters habitat for associated fauna, and disturbs navigation. Any attempt to eradicate it would be futile, owing to the elusive, microscopic gametophyte, and because the alga thrives in sites rich in anthropic activities. Venice Lagoon is the largest Mediterranean transitional environment and the spot of the highest introduction of non-indigenous species, including U. pinnatifida, which is removed as a waste. We demonstrated that polysaccharide extracts from U. pinnatifida have an anticoagulant effect on human blood in vitro and are not cytotoxic. The results obtained by PT (normal values 70-120%) and APTT (normal values 28-40s) assays were significantly prolonged by the polysaccharide extracts of U. pinnatifida, therefore algal extracts are ideal candidates as antithrombotic agents.

Physical and chemical properties of acetylated low molecular weight heparin and its antineoplastic effect in vitro / 中国药理学通报

Aims To synthesize acetylated low molecu-lar weight heparin( ALMWH) and to detect its physico-chemical properties and antineoplastic activity. Meth-ods LMWH was prepared by degradation of UFH with sodium periodate oxidation and sodium borohydride re-duction, then the LMWH was acetylated by acetic an-hydride where N, N′, -dicyclohexylcarbodiimide ( DCC ) and 4-dimethylaminopyridine ( DMAP ) were used as catalysts. X-ray diffraction(XRD)and Differ-ential Scanning Calorimetry ( DSC ) of ALMWH were obtained. The antiproliferative activity and anti-inva-sive activity were determined on MDA-MB-231 and MCE-7 human breast cancer cells. Results XRD a-nalysis showed that the LMWH degraded from UFH and ALMWH synthesized by acetylation of LMWH be-longed to amorphous structure, however, their DSC curves were significantly different. Compared with LM-WH, ALMWH had more powerful capacity for binding water and lowering anticoagulant activity, more signifi-cantly ALMWH exhibited stranger anti-proliferative and anti-invasive activity than LMWH, especially when it was used in low concentrations. Conclusion The syn-thesized ALMWH possesses a low anticoagulant activi-ty, certain anti-proliferative, anti-invasive and anti-metastatic activity. This study provides a basic method for screening of antineoplastic drug with low toxicity.

Assay of anticoagulation activity of leeches by using biological standardization method / 中国药学杂志

OBJECTIVE: To assay the anticoagulant activity of leeches by using biological standardization method with the aim to explore the quality control method which can reflect the biological activity. METHODS: Heparin was used as the reference substance, and APTT value was used as the index of anticoagulant activity evaluation. Four species of leeches were determined, including Hirudo nipponia Whitman, Poecilobdella manillensis Lesson, Poecilobdella javanica Wahlberg, and Whitmania pigra Whitman. The determination results were calculated with standard curve and bioassay statistics. RESULTS: The concentration-response curves of APTT of the four species of leeches were similar to that of heparin, and their variation ranges were parallel. CONCLUSION: APTT values can reflect the comprehensive anticoagulant activity of different species of leeches, which may have more clinical significance. Biological standardization is a good supplement to the current quality control methods, also a proper technology for the quality control of TCM.

Influencing Factors in Determination of Anticoagulant Activity of Whitmania Pigra Whitman by Thrombin Titration / 中国药师

Objective: To analyze the influencing factors in thrombin titration for the determination of anticoagulant activity of Whitmania Pigra Whitman. Methods: The white porcelain plates were used as the titration carriers instead of tubes in the titration ( called white porcelain method for short) . The effect of different carriers, interval time of titration and thrombin concentration on the results of anticoagulant activity test was studied. Results:Under the same conditions, the anticoagulant activity was more accurate and stable using white porcelain method. Using white porcelain method with 20 u·ml-1 or 10 u·ml-1 as the thrombin concentration and titrating 5μl each time, once every minute, the thrombin consumption volume was linear with the sample concentration within the range of 0. 125-0. 333 g·ml-1(r20 =0. 961 and r10 =0. 992), and the anticoagulant activity respectively was (33. 08 ± 2. 64) and (31. 24 ±1.32) u·g-1(RSD20 =8.0% and RSD10 =4.2%). As for a certain sample concentration (0.333 g·ml-1), the theoretical error of determination was not more than 10% and 5%. Conclusion:The improved white porcelain method is more suitable for determining anticoagulant activity of Whitmania Pigra Whitman with more stable results and accurate end point states than tube method. Under the conditions of 10 u·ml-1 thrombin concentration, titrating 5μl each time, once every minute, the linearity, accuracy and precision are all promising.

Study on Anticoagulant Activity in Vivo of Active Component F2-2 from Eupolyphaga seu Steleophaga / 世界科学技术-中医药现代化

Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), plasma fibrino-gen (FIB), platelet aggregation rate and blood clots-fibrinolytic dynamic figure were taken as indexes in the evalua-tion of anticoagulant activity in vivo of active component F2-2 from Eupolyphaga seu Steleophaga. After 5 days of hypodermic injection of adrenaline, the rat model of acute blood stasis was established. Indexes were determined af-ter the model rats were treated with an intragastric administration of F2-2 for 9 days. The results showed that com-pared with the model group, PT/APTT was prolonged, FIB content was decreased, platelet aggregation rate and the largest of blood coagulation were declined after 9 days of intragastric administration in the model group. However, there was no difference on TT. It was concluded that the anticoagulant component F2-2 separated from E. seu Steleophaga showed favorable anticoagulant activity in vivo. However, its mechanism remained unknown and request-ed further researches.

Optimization of wet ultrafine grinding technology for Hirudo extraction / 中草药

Objective: To study the optimal extraction for Hirudo by wet ultrafine grinding technology. Methods: On the basis of single-factor experiments, the response surface analysis was made for exploring the optimal extraction conditions by wet ultrafine grinding technology for Hirudo, with solid-liquid ratio, soaking time, extraction temperature, and grinding time as independent variable and the anticoagulant activity as the response value. According to Box-Behnken principles, the response surface analysis method with four factors and three levels was used. Results: The optimum extraction condition for Hirudo by wet ultrafine grinding technology was as follows: solid-liquid ratio 1:12, soaking time 4.9 h, extraction temperature 6.5°C, grinding time 12 min; The anticoagulant activity could reach 20.13 U/g. Conclusion: The extraction by wet ultrafine grinding technology for Chinese materia medica Hirudo is stable and feasible, and it could be further promoted.

Factors affecting the production of lactulose by Lactobacillus acidophilus NRRL 4495 β-galactosidase and its biological activity

Aim: Production of lactulose and other oligosaccharides by Lactobacillus acidophilus NRRL 4495 β-galactosidase and their biological activity. Methodology and Results: The transgalactosylation activity of Lactobacillus acidophilus NRRL 4495 β-galactosidase was investigated under different conditions for synthesis of lactulose and oligosaccharides. The synthesis was optimized with respect to pH; time; enzyme concentration and substrates ratio (lactose: fructose). Maximum production for lactulose was found to be 25 g/L at pH 6.6 with 40: 20% (w/v) lactose to fructose, respectively and enzyme concentration 4 IU/mL after 7 h. With respect to the other oligosaccharides the maximum yield (19 .68 g/L) was obtained under the same conditions but with enzyme concentration 2 IU/mL and after 10 h. As a new pharmaceutical application the produced lactulose and oligosaccharide and their sulfated derivative were found to have fibrinolytic activity, but they failed to act as anticoagulant. Conclusion significance and impact of study: the research leads to increase the production of lactulose and other oligosaccharides with a significant yield and discovered a new pharmaceutical application for all the products.

Biological activities of the sulfated polysaccharide from the vascular plant Halodule wrightii

A sulfated polysaccharide (SPSG) was successfully isolated from seagrass Halodule wrightii Asch., Cymodoceaceae, and its antioxidant and anticoagulant activities were investigated. The data presented here showed that the SPSG is a 11 kDa sulfated heterogalactan with a sulfatation degree of 20.63 percent and it also contains glucose and xylose. SPSG antioxidant activities were evaluated using several in vitro assays and the anticoagulant activity was evaluated by aPTT and PT tests. These assays suggested that the SPSG possessed remarkable antioxidant properties in different in vitro assays and an outstanding anticoagulant activity 2.5-fold higher than that of heparin Clexane® in the aPTT test. This data represents the first reported on the sulfated polysaccharide biological activities from seagrass. These results indicate that SPSG can be considered in the future as a drug utilized in treating diseases from these systems.

Anticoagulant activity of marine bivalve Donax incarnates Lin, 1758 Collected from Thazhanguda, Southeast coast of India

Objective: Molluscs are highly delicious seafood and they are also very good source for biomedically imported products. Among the molluscs some have pronounced pharmacological activities or other properties which are useful in biomedical area. Methods: In the present study GAGs was isolated from the bivalve such as Donax incarnates. Results: The isolated GAGs were quantified in crude samples and they were estimated as 6.84 gm/kg crude GAGs in Donax incarnates. The bivalve showed the anticoagulant activity of the crude samples 124.53 USP units/mg in Donax incarnates. FTIR analysis reveals the presence of anticoagulant substance signals at different ranges. Conclusions: The determined in this research show that gastropod Donax incarnates tissue is value medicinal due to high quality of anticoagulant compounds.

Anticoagulant properties of a crude sulfated polysaccharide from the red marine alga Halymenia floresia (Clemente) C. Agardh - doi: 10.4025/actascibiolsci.v33i3.6402 / Anticoagulant properties of a crude sulfated polysaccharide from the red marine alga Halymenia floresia (Clemente) C. Agardh - doi: 10.4025/actascibiolsci.v33i3.6402

Alternative sources of anticoagulants have arisen as a result of the increasing demand for safer anticoagulant clinical therapy, and the sulfated polysaccharides of seaweeds have gained attention in biomedicine. In this study, crude sulfated polysaccharide fractions (denominated Hf1, Hf2 and Hf3) were obtained from the red marine alga Halymenia floresia and the anticoagulant properties of a soluble crude polysaccharide fraction (Hf2s) were assayed. The three differential extractions yielded 38.6%. The polysaccharides are composed mainly of galactose, with small amounts of xylose and glucose. The anticoagulant properties of Hf2s containing 53.8% sulfate and 3% protein was also compared to those of heparin (193.0 IU mg-1) by assays of activated partial thromboplastin time (APTT) and thrombin time (TT) using normal human plasma. Hf2s showed a higher anticoagulant activity (68.4 IU mg-1) than those of Hf1s and Hf3s, whose activities were 37.6 and 36.6 IU mg-1, respectively. The compound was less active than heparin, but its anticoagulant mechanism suggested that it is dependent on cofactor heparin II to inhibit thrombin activity, but not on cofactors VIII and IX. Therefore, the polysaccharide from H. floresia interfered on coagulation cascade.

Alternative sources of anticoagulants have arisen as a result of the increasing demand for safer anticoagulant clinical therapy, and the sulfated polysaccharides of seaweeds have gained attention in biomedicine. In this study, crude sulfated polysaccharide fractions (denominated Hf1, Hf2 and Hf3) were obtained from the red marine alga Halymenia floresia and the anticoagulant properties of a soluble crude polysaccharide fraction (Hf2s) were assayed. The three differential extractions yielded 38.6%. The polysaccharides are composed mainly of galactose, with small amounts of xylose and glucose. The anticoagulant properties of Hf2s containing 53.8% sulfate and 3% protein was also compared to those of heparin (193.0 IU mg-1) by assays of activated partial thromboplastin time (APTT) and thrombin time (TT) using normal human plasma. Hf2s showed a higher anticoagulant activity (68.4 IU mg-1) than those of Hf1s and Hf3s, whose activities were 37.6 and 36.6 IU mg-1, respectively. The compound was less active than heparin, but its anticoagulant mechanism suggested that it is dependent on cofactor heparin II to inhibit thrombin activity, but not on cofactors VIII and IX. Therefore, the polysaccharide from H. floresia interfered on coagulation cascade.