In-vitro anticoagulant activity of fucoidan derivatives from brown seaweed Laminaria japonica

The in vitro anticoagulant activity of 41 water extracts of various seaweeds from Baja California Sur, Mexico was evaluated. In this study, nine extracts exhibited anticoagulant activity in the prothrombin time assay and 29 extracts were positive in the activated partial thromboplastin time assay. The water extract obtained at 25 °C from the brown seaweed Eisenia arborea was the most active in both assays, increasing the normal blood clotting-time over 300 s at 100 g mL-1. The fractionation of this extract by anion exchange chromatography yielded 3 fractions. Fraction 2 eluted with 1.0 M sodium chloride increased the clotting-time over 300 s in the activated partial-thromboplastin time assay at 5 g mL-1, being more active than sodium heparin. Chemical and spectroscopic analysis of fraction 2 showed it to be a sulfated heterofucan composed of 56.2 % ± 0.1% of total sugars and 45 % of sulfates.The neutral sugar constituents of the active heterofucan was determined to be 47.6 % fucose, 35.5 % xylose and 16.9 % rhamnose, with substitutions of sulfate groups at C-4 (axial), and minor substitutions at C-2 and-or C-3. Monitoreo de anticoagulantes en algas marinas de México y caracterización parcial de polisacáridos sufatados activos de Eisenia arborea Se evaluó la actividad anticoagulante in vitro de 41 extractos acuosos de diversas algas de Baja California Sur. Nueve extractos exhibieron actividad anticoagulante en el ensayo de tiempo de protrombina y 29 extractos fueron activos en el ensayo de tiempo de tromboplastina parcial activada. El extracto acuoso de Eisenia arborea obtenido a 25 °C fue el más activo en ambos ensayos, incrementando el tiempo normal de coagulación a más de 300 s, a una concentración de 100 g mL-1. El fraccionamiento de este extracto por cromatografía de intercambio iónico resultó en 3 fracciones. La fracción 2 eluída con cloruro de sodio 1.0 M incrementó el tiempo de coagulación a más de 300 s en el ensayo de tiempo de la tromboplastina parcial activada a una concentración de 5 g mL-1; resultando más activa que el control positivo (heparina de sodio). Los análisis químicos y espectroscópicos mostraron que la fracción 2 era un heterofucano sulfatado, compuesto por 56.2 % ± 0.1 % de azúcares totales y 45% de sulfatos. Los azúcares neutros constituyentes del heterofucano activo fueron determinados como 47.6 % fucosa, 35.5 % xylosa y 16.9 % ramnosa, con sustituciones de los grupos sulfato en C-4 (axial) y sustituciones menores en C-2 y/o C-3.

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Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614856 ◽

1999 ◽

Vol 82 (11) ◽

pp. 1462-1468 ◽

Cited By ~ 22

Author(s):

José Fernández ◽

Jari Petäjä ◽

John Griffin

Keyword(s):

Molecular Weight ◽

Unfractionated Heparin ◽

Protein C ◽

Dermatan Sulfate ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Factor V ◽

Factor Va ◽

Anticoagulant Action

SummaryUnfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.

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Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646437 ◽

1991 ◽

Vol 66 (04) ◽

pp. 453-458 ◽

Cited By ~ 15

Author(s):

John T Brandt

Keyword(s):

Specific Activity ◽

Anticoagulant Activity ◽

Factor Xa ◽

Clinical Importance ◽

Potential Method ◽

Quantitative Expression ◽

Increased Risk ◽

Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

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The Anticoagulant Properties of a Modified Form of Protein S

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647048 ◽

1988 ◽

Vol 60 (02) ◽

pp. 298-304 ◽

Cited By ~ 17

Author(s):

C A Mitchell ◽

S M Kelemen ◽

H H Salem

Keyword(s):

Monoclonal Antibody ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Factor Xa ◽

Protein S ◽

Human Neutrophil Elastase ◽

Factor X ◽

Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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Effects of Two Different Doses of Hirudin on APTT, Determined with Eight Different Reagents

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653754 ◽

1995 ◽

Vol 73 (02) ◽

pp. 219-222 ◽

Cited By ~ 4

Author(s):

Manuel Monreal ◽

Luis Monreal ◽

Rafael Ruiz de Gopegui ◽

Yvonne Espada ◽

Ana Maria Angles ◽

...

Keyword(s):

Ex Vivo ◽

Anticoagulant Activity ◽

Subcutaneous Administration ◽

In Vitro Study ◽

Ex Vivo Model ◽

Suitable Candidate ◽

Significant Prolongation ◽

High Doses ◽

Different Doses

SummaryThe APTT has been considered the most suitable candidate to monitor the anticoagulant activity of hirudin. However, its use is hampered by problems of standardization, which make the results heavily dependent on the responsiveness of the reagent used. Our aim was to investigate if this different responsiveness of different reagents when added in vitro is to be confirmed in an ex vivo study.Two different doses of r-hirudin (CGP 39393), 0.3 mg/kg and 1 mg/kg, were administered subcutaneously to 20 New Zealand male rabbits, and the differences in prolongation of APTT 2 and 12 h later were compared, using 8 widely used commercial reagents. All groups exhibited a significant prolongation of APTT 2 h after sc administration of hirudin, both at low and high doses. But this prolongation persisted 12 h later only when the PTTa reagent (Boehringer Mannheim) was used. In general, hirudin prolonged the APTT most with the silica- based reagents.In a further study, we compared the same APTT reagents in an in vitro study in which normal pooled plasma was mixed with increasing amount of hirudin. We failed to confirm a higher sensitivity for silica- containing reagents. Thus, we conclude that subcutaneous administration of hirudin prolongs the APTT most with the silica-based reagents, but this effect is exclusive for the ex vivo model.

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Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653872 ◽

1995 ◽

Vol 73 (05) ◽

pp. 805-811 ◽

Cited By ~ 12

Author(s):

Yasuo Takahashi ◽

Yoshitaka Hosaka ◽

Hiromi Niina ◽

Katsuaki Nagasawa ◽

Masaaki Naotsuka ◽

...

Keyword(s):

Human Urine ◽

Specific Activity ◽

Anticoagulant Activity ◽

Rabbit Lung ◽

Soluble Thrombomodulin ◽

Molecular Weights ◽

Dose Dependent Fashion ◽

Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

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Toxicity of Heparinoids, with Special Reference to the Precipitation of Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655587 ◽

1964 ◽

Vol 12 (01) ◽

pp. 232-261 ◽

Cited By ~ 2

Author(s):

S Sasaki ◽

T Takemoto ◽

S Oka

Keyword(s):

Molecular Weight ◽

Dextran Sulfate ◽

Anticoagulant Activity ◽

Molecular Structures ◽

Severe Reaction ◽

Clinical Use ◽

Molecular Weights ◽

Close Relationship

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.

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Apoptosis-like cell death upon kinetoplastid induction by compounds isolated from the brown algae Dictyota spiralis

Parasites & Vectors ◽

10.1186/s13071-021-04693-7 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Olfa Chiboub ◽

Ines Sifaoui ◽

Manef Abderrabba ◽

Mondher Mejri ◽

José J. Fernández ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Mitochondrial Membrane ◽

Brown Seaweed ◽

Cell Permeability ◽

Oxygen Species ◽

Potential Drug ◽

Concentration Cell ◽

Drug Candidates

Abstract Background The in vitro activity of the brown seaweed Dictyota spiralis against both Leishmania amazonensis and Trypanosoma cruzi was evaluated in a previous study. Processing by bio-guided fractionation resulted in the isolation of three active compounds, classified as diterpenes. In the present study, we performed several assays to detect clinical features associated to cell death in L. amazonensis and T. cruzi with the aim to elucidate the mechanism of action of these compounds on parasitic cells. Methods The aims of the experiments were to detect and evaluate specific events involved in apoptosis-like cell death in the kinetoplastid, including DNA condensation, accumulation of reactive oxygen species and changes in ATP concentration, cell permeability and mitochondrial membrane potential, respectively, in treated cells. Results The results demonstrated that the three isolated diterpenes could inhibit the tested parasites by inducing an apoptosis-like cell death. Conclusions These results encourage further investigation on the isolated compounds as potential drug candidates against both L. amazonensis and T. cruzi. Graphic abstract

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In Vitro and In Vivo Anti-Inflammatory Effects of Sulfated Polysaccharides Isolated from the Edible Brown Seaweed, Sargassum fulvellum

Marine Drugs ◽

10.3390/md19050277 ◽

2021 ◽

Vol 19 (5) ◽

pp. 277

Author(s):

Lei Wang ◽

Hye-Won Yang ◽

Ginnae Ahn ◽

Xiaoting Fu ◽

Jiachao Xu ◽

...

Keyword(s):

Nitric Oxide ◽

Brown Seaweed ◽

Sulfated Polysaccharides ◽

Raw 264.7 ◽

Raw 264.7 Macrophages ◽

Sargassum Fulvellum ◽

Pharmaceutical Industries ◽

Anti Inflammatory

In the present study, the in vitro and in vivo anti-inflammatory effects of the sulfated polysaccharides isolated from Sargassum fulvellum (SFPS) were evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and zebrafish. The results indicated that SFPS improved the viability of LPS-stimulated RAW 264.7 macrophages from 80.02 to 86.80, 90.09, and 94.62% at the concentration of 25, 50, and 100 µg/mL, respectively. Also, SFPS remarkably and concentration-dependently decreased the production levels of inflammatory molecules including nitric oxide (NO), tumor necrosis factor-alpha, prostaglandin E2, interleukin-1 beta, and interleukin-6 in LPS-treated RAW 264.7 macrophages. In addition, SFPS significantly inhibited the expression levels of cyclooxygenase-2 and inducible nitric oxide synthase in LPS-treated RAW 264.7 macrophages. Furthermore, the in vivo test results indicated that SFPS improved the survival rate of LPS-treated zebrafish from 53.33 to 56.67, 60.00, and 70.00% at the concentration of 25, 50, and 100 µg/mL, respectively. In addition, SFPS effectively reduced cell death, reactive oxygen species, and NO levels in LPS-stimulated zebrafish. Taken together, these results suggested that SFPS possesses strong in vitro and in vivo anti-inflammatory activities, and could be used as an ingredient to develop anti-inflammatory agents in the functional food and pharmaceutical industries.

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