Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.     

Anticoagulant activity of sulfated polysaccharide isolated from fermented brown seaweed Sargassum fulvellum

A sulfated polysaccharide with anticoagulant properties was isolated from the fermented brown seaweed Sargassum fulvellum. Freeze-dried S. fulvellum was fermented in an incubator for 10^th week at 25°C to convert seaweed macromolecules into anticoagulant sulfated polysaccharides (ASP). Anticoagulant activity was determined by an activated partial thromboplastin time (APTT) test using citrated human blood plasma. The 8^th week S. fulvellum crude seaweed extract (SWE) exhibited the highest blood anticoagulant activity. Therefore, 8^th week crude SWE was used for purification of ASP by two steps; DEAE cellulose anion-exchange followed by Sepharose 4B chromatography. The isolated ASP showed a single spot on agarose gel electrophoresis, which confirmed the purification status of our ASP. Polyacrylamide gel electrophoresis (PAGE) analysis showed that the molecular mass of the purified ASP was between 8 and 20 kDa. Polysaccharide and sulfate concentrations of the purified ASP were 180 and 29.70 μg mL⁻¹ respectively. ASP recovery was 1.32% (w/w) from the crude polysaccharide applied to the DEAE column. Purified ASP had a pH of 3.86 and was considered an acidic polysaccharide. Moreover, both ASP and heparin showed a relative clotting factor of 27.47 at the concentrations of 180 and 60 μg mL⁻¹ respectively. Therefore, S. fulvellum ASP can be considered a weaker anticoagulant than heparin. Results of the APTT, PT, and TT clotting assays showed that ASP was able to inhibit both intrinsic and extrinsic blood coagulation pathways. Finally, this study established a feasible and simple experimental protocol to isolate anticoagulant from fermented seaweeds leading to potential further development of anticoagulant agent for the pharmaceutical industry.     

Chemical characteristic and anticoagulant activity of the sulfated polysaccharide isolated from Monostroma latissimum (Chlorophyta)

A polysaccharide was isolated from marine green algae Monostroma latissimum, and its chemical characteristic and anticoagulant activity were investigated. The results demonstrated that the polysaccharide was high rhamnose-containing sulfated polysaccharide, and was mainly composed of 1,2-linked l-rhamnose residues with sulfate groups substituted at positions C-3 and/or C-4. The sulfated polysaccharide exhibited high anticoagulant activities by assays of the activated partial thromboplastin time (APTT) and thrombin time (TT). The anticoagulant property of the sulfated polysaccharide was mainly attributed to powerful potentiation thrombin by heparin cofactor II.     

Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO3Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.     

Heparinoid-active two sulfated polysaccharides isolated from marine green algae Monostroma nitidum

Two sulfated polysaccharides WF1 and WF3 were isolated from marine green algae Monostroma nitidum, and their structural characteristics were determined. Anticoagulant activities of WF1 and WF3 were evaluated by assays of the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), antithrombin and anticoagulation factor Xa activities. The results showed that WF1 and WF3 had similar high contents of rhamnose, whereas their sulfate contents, sulfation positions, molecular sizes and linkage patterns of rhamnose residues were different. The bioassay results demonstrated that WF1 and WF3 had high anticoagulant activities, and were potent thrombin inhibitors mediated by heparin cofactor II, especially WF3. They also hastened thrombin and coagulation factor Xa inhibition by potentiating antithrombin III, but at a lower effectiveness. The differences of anticoagulant activities between WF1 and WF3 were directly due to their structural features discrepancy.     

Experimental study on anticoagulant and antiplatelet aggregation activity of a chemically sulfated marine polysaccharide YCP

A polysaccharide YCP was prepared from a marine filamentous fungus Keissleriella sp. YS4108, which exhibited as a molecular weight (Mw) of 2.4x10(3) kDa and its three sulfated derivatives (YCP-SL, YCP-SM and YCP-SH) were synthesized, the degree of substitution (DS) of which were determined to be 0.13, 0.99 and 1.3, with the average molecular weight 0.64x10(3), 0.57x10(3) and 0.45x10(3) kDa, respectively. Anticoagulant activity and antiplatelet aggregation activity of these sulfated derivates were evaluated by activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and platelet aggregation assay. The results showed that YCP sulfates significantly prolonged APTT, TT and PT. The derivates showed no effects on thrombin in the presence or in the absence of antithrombin III (AT III) or heparin cofactor II (HC II), while the derivates effectively inhibited factor Xa in the presence of AT III. At the same time, YCP-SH also possessed potent antiplatelet aggregation activity in vitro compared with aspirin. YCP sulfates specifically interfered with different stages of the coagulation cascade, and the anticoagulant activity improved with the increasing DS and decreased Mw.     

Simple separation of anticoagulant sulfated galactan from marine red algae

In this study, hot water extracts of 22 red algal species were evaluated for their potential anticoagulant activities. The extracts from eight species (Grateloupia elliptica, Sinkoraena lancifolia, Halymenia dilatata, Grateloupia lanceolata, Lomentaria catenata, Martensia denticulata, Schizymenia dubyi, Chondrus crispus) showed potent activated partial thromboplastin time (APTT). Of these eight algae, the crude polysaccharide fraction (CpoF) from the hot water extracts of L. catenata and S. dubyi showed the highest APTT activity. Lomentaria catenata and S. dubyi were selected and an enzymatic digestion process which could effectively separate a crude polysaccharide fraction with higher yields from raw algae materials was applied. The 10 enzymes tested included five carbohydrases and five proteases. The Ultraflo and Celluclast digests of L. catenata and the AMG digest of S. dubyi exhibited the most potent anticoagulant activity. Furthermore, in both species, the active compounds were mainly concentrated in the >30 kDa fraction through ultrafiltration and showed strong APTT (>1000) and thrombin time (TT) >1000 activity. The active compounds were shown to be sulfated galactans with a greater than 80% galactose content and an 0.22 ∼ 0.31 sulfate to total sugar ratio.     

Synthesis and anticoagulant activity of the quaternary ammonium chitosan sulfates

Quaternary ammonium chitosan sulfates with diverse degrees of substitution (DS) ascribed to sulfate groups between 0.52 and 1.55 were synthesized by reacting quaternary ammonium chitosan with an uncommon sulfating agent (N(SO₃Na)₃) that was prepared from sodium bisulfite (NaHSO₃) through reaction with sodium nitrite (NaNO₂) in the aqueous system homogeneous. The structures of the derivatives were characterized by FTIR, ¹H NMR and ¹³C NMR. The factors affecting DS of quaternary ammonium chitosan sulfates which included the molar ratio of NaNO₂ to quaternary ammonium chitosan, sulfated temperature, sulfated time and pH of sulfated reaction solution were investigated in detail. Its anticoagulation activity in vitro was determined by an activated partial thromboplastin time (APTT) assay, a thrombin time (TT) assay and a prothrombin time (PT) assay. Results of anticoagulation assays showed quaternary ammonium chitosan sulfates significantly prolonged APTT and TT, but not PT, and demonstrated that the introduction of sulfate groups into the quaternary ammonium chitosan structure improved its anticoagulant activity obviously. The study showed its anticoagulant properties strongly depended on its DS, concentration and molecular weight.     

Chemical characterization of cell-wall polysaccharides from tank-cultivated and wild plants ofDelesseria sanguinea (Hudson) Lamouroux (Ceramiales,delesseriaceae): culture patterns and potent anticoagulant activity

Tank cultivation ofDelesseria sanguinea was investigated in order to manipulate conditions for vegetative growth and to provide biomass for the analysis of cell wall polysaccharides. Seasonality is subject to short-day photoperiodic control. Night-break or long-day conditions prevented fertility in tetrasporophytes and gametophytes and triggered outgrowth of new blades. Long-day illuminations allowed a 1% daily growth rate. Seawater temperature below 13 °C was necessary for inducing formation of new blades. Both wild and cultivated ofD. sanguinea plants contained a non gelling sulfated heteropolysaccharide composed of a galactosyl backbone branched with xylosyl residues. The hot water extract at neutral pH displayed the highest anticoagulant activity (5 μg ml^-1 polysaccharide concentration in APTT clotting assay). No obvious differences were found in polysaccharide chemical composition and properties between gametophytes and sporophytes or between cultivated and wild plants.     

玉米芯木聚糖硫酸酯抗凝血活性及其机制的研究

采用活化部分凝血活酶时间(APTT)、凝血酶时间(TT)和凝血酶原时间(PT)检测了玉米芯木聚糖硫酸酯(wisX-SB)的抗凝活性,结果表明wisX-SB能明显延长APTT和TT,而不影响PT,提示wisX-SB是通过内源性和/或共同途径发挥抗凝血作用的。采用发色底物法及纤维蛋白原转化实验分别考察了wisX-SB对凝血酶及纤维蛋白原的作用,结果提示wisX-SB的抗凝机制包括:直接抑制纤维蛋白原的转化;通过增强抗凝血酶III(AT-III)的活性,抑制凝血酶活性,从而达到抗凝目的。较低浓度时以前者为主,较高浓度下两者皆起作用。     

Improvement of artemisinin production by chitosan in hairy root cultures of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 μg mg⁻¹ dry wt over 6 days by adding 150 mg chitosan l⁻¹. The increase was dose-dependent. Similar treatment of hairy roots with methyl jasmonate (0.2 mM) or yeast extract (2 mg ml⁻¹) increased artemisinin production to 1.5 and 0.9 μg mg⁻¹ dry wt, respectively.     

Identification of anticoagulant activities in the salivary glands of the soft tick,Ornithodoros savignyi

Salivary gland extracts of the sand tampan, Ornithodoros savignyi, prolonged the activated partial thromboplastin time (APTT) and prothrombin time (PT) significantly in a concentration-dependent manner. There was also a pronounced inhibition of human activated factor Xa (fXa) by salivary gland extracts. The salivary gland extracts inhibited chromogenic assays specific for both fXa and thrombin. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the salivary gland proteins followed by elution of specific areas or bands from a polyvinylidene difluoride (PVDF)-membrane, showed that various anticoagulant factors are present when screened by means of the APTT assay. The most active component was associated with a band of M _r of 14 kDa. Partial purification of this component was achieved using isoelectric focusing (IEF) and size-exclusion highperformance liquid chromatography (HPLC).     

Influence of functional groups on the in vitro anticoagulant activity of chitosan sulfate

A new method for the chemical modification of chitosan sulfate was used to prepare N-propanoyl-, N-hexanoyl- and N,O-quaternary substituted chitosan sulfate. Structural analysis by elemental analysis, FTIR, 13C NMR, and 1H NMR spectroscopy, and gel-permeation chromatography showed that these methods could conveniently be used for the introduction of functional groups. The influences of the acyl or quaternary groups on the anticoagulant activity of the polysaccharides were studied with respect to activated partial thromboplastin time (APTT) thrombin time (TT), and prothrombin time (PT). The propanoyl and hexanoyl groups increased the APTT activity, and the propanoyl groups also increased the TT anticoagulant activity slightly, while the N,O-quaternary chitosan sulfate showed only a slight TT coagulant activity.     

Sulfated polysaccharides from marine green algae Ulva conglobata and their anticoagulant activity

Sulfated polysaccharides from the green algae Ulva conglobata were isolated and prepared by extraction in hot water, precipitation with ethanol and purification by ion-exchange and size-exclusion column chromatography. The characterizations of the sulfated polysaccharides were defined, and containing 23.04–35.20% sulfate ester groups, 10.82–14.91% uronic acid and 3.82–4.51% protein. Gas chromatography analysis shows that the sulfated polysaccharides from Ulva conglobata are mainly consisted of rhamnose with variable contents of glucose and fucose, trace amounts of xylose, glactose and mannose. The anticoagulant properties of the sulfated polysaccharides were compared with those of heparin by studying the activated partial thromboplastin time using normal human plasma. The sulfated polysaccharide from Ulva conglobata collected in Qingdao, China is the most potent among the sulfated polysaccharides tested. The mechanism of anticoagulant activity mediated by the sulfated polysaccharides is due to the direct inhibition of thrombin and the potentiation of heparin cofactor II.     

Sulfation pattern of the fucose branch is important for the anticoagulant and antithrombotic activities of fucosylated chondroitin sulfates

Background

The aim is to compare the structures, anticoagulant and antithrombotic activities of two fucosylated chondroitin sulfates isolated from sea cucumbers Isostichopus badionotus (fCS-Ib) and Pearsonothuria graeffei (fCS-Pg), which were known to have different sulfation patterns on the fucose branches.

Methods

The structures of fCSs were identified using 2D NMR. Anticoagulant activities were measured by activated partial thromboplastin time (APTT) and thrombin time (TT), and inhibition of factors IIa, Xa and XIIa was assessed in vitro. Antithrombotic activity was determined ex vivo by measuring the length and weight of the thrombus generated.

Results

The two fCSs had identical chondroitin sulfate E backbones and similar fucose branches, but different sulfation patterns of the fucose branches. The fucose branch in fCS-Ib was mainly 2,4-O-sulfated whereas that in fCS-Pg was mainly 3,4-O-sulfated. In vitro assay indicated that fCS-Pg possessed much lower potency than fCS-Ib in prolonging APTT/TT and in inhibition of thrombin. However, they both exhibited similar inhibitory effects on factor X activation by intrinsic tenase complex, and on thrombus generation. Furthermore, both fCSs significantly activated factor XII, which has been proved to be associated with adverse clinical events associated with heparin contaminated by oversulfated chondroitin sulfate.

Conclusion

The 2,4-O-sulfated fucose branch is the key structural factor of fCSs for prolonged APTT/TT and inhibition of thrombin, whereas the inhibitory effect of fCSs on factor X, XII activation and thrombus generation was attributed to the overall structure of fCS polysaccharide.

General importance

Both fCSs have well defined structures and can be readily quality-controlled, and therefore may be potential alternatives for heparin as anticoagulant and antithrombotic drugs.     

Thermal depolymerization of alginate in the solid state

A new method of introduction carboxyl groups to chitosan sulfate by the acylation reaction between hydroxyethyl chitosan sulfates and butane dioic anhydride in homogeneous solution was used to obtain carboxybutyrylated hydroxyethyl chitosan sulfates. The structures of the derivatives were characterized by element analysis, FT-IR, ¹³C-NMR, and gel permeation chromatography. The content and position of the carboxyl groups could be controlled favorably. Their anticoagulant activity was determined for human plasma with respect to activated partial thromboplastin time (APTT), thrombin time (TT), and prothombin time (PT). The introducing of carboxyl groups to amino groups greatly prolonged the APTT and TT. The best result occurred when the degree of substitution of the carboxyl groups was about 0.4/unit that prolonged APTT and TT with about 5 and 1.5 times compared to that of the uncarboxylated hydroxyethyl chitosan sulfates; another conclusion is that introducing of carboxyl groups into N,O-position gave better results than that just into N-positions. Low S% chitosan sulfate and 6-O-desulfated chitosan sulfate showed little anticoagulant activity but their N,O-carboxybutyrylated derivatives (0.6/unit ds) showed increased APTT or TT, while their N-carboxybutyrylated derivatives (0.6/unit ds) gave no improvement. Generally, the introducing of carboxyl groups could not increase PT in spite of the position introduced.     

Anticoagulant activity of fucoidans from brown algae

The anticoagulant activity of polysaccharide fucoidans from 11 species of brown algae was studied. The anticoagulant activity was measured by the activated partial thromboplastin time (APTT), prothrombin time, and thrombin time. Inhibitory action of these fucoidans significantly varied from one species to another. Fucoidans from Laminaria saccharina and Fucus distichus exhibited high anticoagulant activity, while fucoidans from Cladosiphon okamuranus and Analipus japonicus were almost inactive. Other fucoidans exhibited intermediate inhibitory activity. The inhibitory effect of fucoidans on thrombin and factor Xa was investigated in the presence or in the absence of natural thrombin inhibitor, antithrombin III (AT III). In contrast to the best-studied anticoagulant, heparin, most of these fucoidans inhibited thrombin in the absence of AT III. In the presence of AT III the inhibitory effect of fucoidans considerably increased. In contrast to heparin, fucoidans weakly influenced factor Xa activity in the presence of AT III and their inhibitory effect was not observed in the absence of AT III. There was no correlation between the anticoagulant activities of this series of fucoidans and their anti-inflammatory action, studied earlier. It is suggested that these two types of fucoidan activities depend on different structural features of fucoidans. Results of this study demonstrate a possibility of preparation of fucoidans with high anti-inflammatory activity but low anticoagulant activity. Anticoagulant activity of the fucoidans did not exhibit direct dependence on the content of fucose, the other neutral sugars and sulfates; no dependence was also found between the anticoagulant activity and the structure of the backbone of their molecules.     

Preparation and anticoagulant activity of N-succinyl chitosan sulfates

In order to develop a promising substitute for heparin, N-succinyl chitosan (NSC) was chemically modified by sulfating agent N(SO(3)Na)(3), which were synthesized with sodium bisulfite and sodium nitrite in aqueous solution. The N-succinyl chitosan sulfates (NSCS) products were characterized by infrared spectroscopy (FT-IR) and (13)C NMR. The degree of substitution (DS) of NSCS depended on the ratio of sulfating agent to N-succinyl chitosan, reaction temperature, reaction time and pH of sulfation agent. N-succinyl chitosan sulfates with DS of 1.97 were obtained under optimal conditions. The in vitro coagulation assay of NSCS was determined by activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) assays. The results showed that NSCS obviously prolonged APTT. The anticoagulant activity strongly depended on DS, molecular weight (M(w)) and concentration of NSCS. The anticoagulant activity of NSCS promoted with the increase of DS and concentration, and NSCS exhibited the best anticoagulant activity with the M(w) of 1.37×10(4).     

Screening for anticoagulant activity in marine algae from the Northwest Mexican Pacific coast

Disorders in blood coagulation can lead to an increased risk of bleeding (hemorrhage) or clotting (thrombosis). These illnesses have increased over the last decades and no useful new substances have been discovered to remediate them. In search of new compounds from marine natural resources, macroalgae from the Northwest Mexican Pacific coast were investigated in order to detect anticoagulant activity. Egregia menziesii, Ulva neumatoidea, Porphyra perforata, Silvetia compressa, and Codium fragile were collected from Ensenada coasts. Collected materials were cleaned, dried, milled, and stored until use. Proximate chemical composition and sulfate content were determined in dried powder. Hot and cold aqueous extracts were obtained from the dried algae in order to isolate polysaccharides and similar compounds. Methanol-soluble compounds were separated by means of Soxhlet extraction. Organic and aqueous extracts were screened for anticoagulant activity in both intrinsic and extrinsic pathways of clot formation. Clotting activity was studied by standardized plasma coagulation tests (activated partial thromboplastin time (aPTT) and prothrombin time (PT)). Heparin, a sulfated glycosaminoglycan widely used in anticoagulant therapy, was used as reference. Effects were defined either as aPTT index (Sample aPTT/Control aPTT ratio) or PT index (Sample PT/Control PT ratio). Some of the fractions showed anticoagulant activity over intrinsic pathways, whereas they were found to be coagulants on the extrinsic pathway. The highest aPTT index was 1.8 for U. nematoidea (1 μg mL⁻¹). Hot aqueous extracts from E. menziesii (1 μg mL⁻¹) showed the highest potency, with an aPTT index of 1.4. Sulfate content and anticoagulant activity were not correlated.     

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.