Oversulfated dermatan sulfate and heparinoid in the starfish Lysastrosoma anthosticta: Structures and anticoagulant activity

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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S PROTEIN/VITRONECTIN NEUTRALIZES THE ANTICOAGULANT ACTIVITY OF GLYCOSAMINOGLYCANS IN THE INHIBITION OF THROMBIN BY HEPARIN COFACTOR II

10.1055/s-0038-1643633 ◽

1987 ◽

Author(s):

K T Preissner ◽

P Sie

Keyword(s):

Dermatan Sulfate ◽

Enzyme Inhibitor ◽

Anticoagulant Activity ◽

Coagulation System ◽

Binary Complex ◽

Heparin Cofactor Ii ◽

S Protein ◽

Adhesive Protein ◽

Inhibition Of Thrombin

The complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin against fast inactivation by antithrombin III. The interference of S protein with glycos-aminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in a purified system. In the presence of 0.3 μg/ml heparin, or 0.5 μg/ml pentosan polyphosphate (SP 54), or 2 μg/ml dermatan sulfate, S protein induced a concentration-dependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo-first order rate constants by about 17-fold (heparin), or about 7-fold (SP 54), but only by about 2-fold for dermatan sulfate at a physiological ratio of S protein to heparin cofactor II. Likewise, S protein significantly counteracted the anticoagulant activity of heparin and SP 54 bot not of dermatan sulfate when tested in an inhibition assay using various concentrations of glycosaminoglycans. For heparin, the activity of S protein at the point of 50% inhibition of thrombin was expressed in the range 0.06-0.6 μg/ml (0.01-0.1 U/ml) and for SP 54 in the range 0.3-2 pg/ml. Exposure of the glycos-aminoglycan-binding region of S protein by reduction and carb-oxymethylation of the protein even increased the neutralizing activity of S protein towards heparin and SP 54. S protein not only was found together with thrombin in a binary complex. S protein also became incorporated into a ternary complex with thrombin and heparin cofactor II as judged by crossed immunoelectrophoresis, regardless whether complex formation was initiated by heparin or dermatan sulfate. These findings underline the role of S protein as potent glycosaminoglycan-neutral-izing protein in plasma and as scavenger protein which may bind to enzyme-inhibitor complexes of the coagulation system.

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INFLUENCE OF THE SULFATION PATTERN ON CERTAIN BIOLOGICAL PROPERTIES OF GALACTOSAMINOGLYCANS

10.1055/s-0038-1643251 ◽

1987 ◽

Author(s):

B Casu ◽

L Marchese ◽

A Naggi ◽

G Torri ◽

J Fareed ◽

...

Keyword(s):

Molecular Weight ◽

Dermatan Sulfate ◽

Anticoagulant Activity ◽

Chlorosulfonic Acid ◽

Biological Properties ◽

Sulfated Polysaccharides ◽

Low Molecular Weight ◽

Antithrombotic Activity

In order to investigate the influence of charge distribution and chain length on the biological properties of sulfated polysaccharides, additional sulfate groups were introduced into the galactosaminoglycans, chondriotin sulfate and dermatan sulfate. Using a flexible method (with sulfuric acid and chlorosulfonic acid) for concurrent sulfation and controlled depolymerization, numerous products were obtained and characterized by chemical, enzymatic and nuclear magnetic resonance spectroscopic methods. The biologic actions of these products were profiled in both in vitro and in vivo assays for antithrombotic activity. Despite a weaker in vitro anticoagulant activity, low molecular weight over sulfated galactosaminoglycans produced significant dose-dependent antithrombotic actions in animal models which were similar to the actions observed with oversulfated low molecular weight heparins. These results suggest that a significant antithrombotic activity can be elicited through non-specific interactions of polysulfates with cellular and plasma components, and that clusters of sulfate groups such as the 4-6 disulfate group on D-galactosaminoglycan residues may be important for these interactions. Furthermore, these results, also suggest that supersulfation of glycosaminogly-cans results in products with biologic activity distinct from the native material.

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Comparative Anticoagulant and Thrombin Generation Inhibitory Profile of Heparin, Sulodexide and Its Components

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029620954913 ◽

2020 ◽

Vol 26 ◽

pp. 107602962095491

Author(s):

Fakiha Siddiqui ◽

Debra Hoppensteadt ◽

Emily Bontekoe ◽

Ambar Farooqui ◽

Walter Jeske ◽

...

Keyword(s):

Thrombin Generation ◽

Dermatan Sulfate ◽

Medical Center ◽

Anticoagulant Activity ◽

Pharmaceutical Ingredients ◽

Thrombin Generation Assay ◽

Venous Diseases ◽

Individual Determination ◽

Normal Human ◽

The Individual

Introduction: Sulodexide represents a mixture of fast-moving heparin (FMH) and dermatan sulfate (DS) and has been used for the management of venous diseases such as DVT and related disorders. The purpose of this study is to compare sulodexide and its components with unfractionated heparin (UFH) to determine its suitability for the indications in which UFH is used. Materials and Method: Active pharmaceutical ingredients (API) versions of sulodexide, FMH and DS were obtained from Alfasigma. API versions of UFH were obtained from Medefil Inc. Normal human citrated plasma was obtained from blood bank of the Loyola University Medical Center. Each of the individual agents were supplemented in plasma at a graded concentration of 0.0-10 µg/mL. Clotting assays (PiCT, aPTT, PT and TT), anti-Xa and anti-IIa and thrombin generation studies were carried out. Results were compiled as mean ± SD of 3 individual determination. Result: In the clot based (PiCT, aPTT and TT), anti-Xa and IIa assays, both the UFH and FMH produced stronger activities in these assays followed by sulodexide. DS did not show any anticoagulant activity. In the thrombin generation assay, FMH and UFH produced comparable inhibition of thrombin generation as measured by various parameters. Sulodexide was slightly weaker in this assay, whereas DS produced relatively weaker effects. Conclusion: In comparison to sulodexide, both UFH and FMH exhibit comparable anticoagulant activity despite differences in their molecular weight. These results suggest that sulodexide can be developed as a parenteral anticoagulant for indications in which UFH is used.

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Highly sulfated dermatan sulfate from the skin of the ray Raja montagui: anticoagulant activity and mechanism of action

Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology ◽

10.1016/j.cbpb.2010.03.010 ◽

2010 ◽

Vol 156 (3) ◽

pp. 206-215 ◽

Cited By ~ 30

Author(s):

Mohamed Ben Mansour ◽

Manel Dhahri ◽

Mohsen Hassine ◽

Nadine Ajzenberg ◽

Laurence Venisse ◽

...

Keyword(s):

Mechanism Of Action ◽

Dermatan Sulfate ◽

Anticoagulant Activity

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Prevention and Therapy of Experimental Venous Thrombosis in Rabbits by Desmin 370

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615198 ◽

1998 ◽

Vol 80 (08) ◽

pp. 338-341 ◽

Cited By ~ 4

Author(s):

Maria Rosaria Rossiello ◽

Miriam Barbanti ◽

Fiorella Calanni ◽

Nicola Semeraro ◽

Mario Colucci

Keyword(s):

Therapeutic Efficacy ◽

Therapeutic Agent ◽

Dermatan Sulfate ◽

Anticoagulant Activity ◽

Low Molecular Weight ◽

Dependent Manner ◽

Jugular Vein Thrombosis ◽

Vein Thrombosis ◽

Drug Inhibition ◽

Dose Dependent

SummaryDesmin 370 (D370), a low molecular weight dermatan sulfate, has been shown to reduce the size of preformed thrombi in rats, via a mechanism largely independent of its anticoagulant activity. In the present study we investigated the therapeutic efficacy of D370 in rabbits with experimental jugular vein thrombosis. Experiments performed to evaluate the antithrombotic dosages in rabbits indicated that D370 prevented the formation of venous thrombi (Wessler model) in a dose-dependent manner with complete inhibition at 20 mg/kg. When injected to rabbits bearing a 30 min aged thrombus, D370 caused a time- and dose-dependent reduction in thrombus weight. Thrombi harvested 2 h after injection of 50 mg/kg of D370 were 71% smaller than thrombi from saline-treated rabbits and 50% smaller than pretreatment thrombi, suggesting a double effect of the drug: inhibition of thrombus accretion and reduction of the existing thrombus. Interestingly, pretreatment with the fibrinolytic inhibitor EACA (1 g/kg), significantly attenuated the therapeutic efficacy of D370, suggesting a possible involvement of the fibrinolytic system. Heparin (50 and 200 U/kg) was less active as therapeutic agent, the maximal decrease in thrombus weight, as compared to untreated rabbits, amounting to 38%. Heparin, moreover, caused a more pronounced prolongation of APTT than comparable antithrombotic dosages of D370. Our present data extend previous results on the therapeutic efficacy of D370 and underscore its potential as an alternative antithrombotic drug.

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The Additive Effect of Low Molecular Weight Heparins on Thrombin Inhibition by Dermatan Sulfate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649602 ◽

1993 ◽

Vol 70 (03) ◽

pp. 443-447 ◽

Cited By ~ 6

Author(s):

Benilde Cosmi ◽

Giancarlo Agnelli ◽

Edward Young ◽

Jack Hirsh ◽

Jeffrey Weitz

Keyword(s):

Molecular Weight ◽

Antithrombin Iii ◽

Dermatan Sulfate ◽

Anticoagulant Activity ◽

Additive Effect ◽

Low Molecular Weight ◽

Low Molecular Weight Heparins ◽

Heparin Cofactor Ii ◽

Thrombin Inhibition ◽

Sds Page

SummaryThe aim of this study was to investigate the mechanism by which the anticoagulant activity of dermatan sulfate (DS) is increased by low molecular weight heparin (LMWH). In platelet poor plasma, LMWH enhances the effect of DS on thrombin (IIa) inhibition as determined by thrombin clotting times and with a chromogenic substrate assay. Analysis of the results of the chromogenic assays using either the algebraic fractional or the graphic isobole method suggests that LMWH has an additive effect on the anti-IIa activity of DS. This additive effect was lost when the experiments were repeated in plasma immunodepleted of antithrombin III (ATIII), indicating that the anti-IIa activity of LMWH is ATIII-dependent. To further explore the mechanism of the interaction between LMWH and DS, 125I-labeled IIa was added to plasma in the presence or absence of DS and/or LMWH and the formation of IIa-inhibitor complexes was assessed using SDS-PAGE followed by autoradiography. DS addition selectively increases the formation of heparin cofactor II (HCII)-IIa complexes, whereas LMWH enhances ATIII-IIa complex generation. Compared to plasma containing DS alone, the formation of ATIII-IIa complexes also is increased when the combination of DS and LMWH is added. These findings suggest that the additive effect of LMWH on the anti-IIa activity of DS reflects their different modes of IIa inhibition; DS potentiates IIa inhibition by HCII, while LMWH catalyses ATIII-dependent IIa inactivation. The potential clinical significance of these findings requires further investigation.

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Plasma dermatan sulfate proteoglycan in a patient on chronic hemodialysis

Blood ◽

10.1182/blood.v82.11.3380.3380 ◽

1993 ◽

Vol 82 (11) ◽

pp. 3380-3385 ◽

Cited By ~ 8

Author(s):

MA Delorme ◽

N Saeed ◽

A Sevcik ◽

L Mitchell ◽

L Berry ◽

...

Keyword(s):

Molecular Weight ◽

Antithrombin Iii ◽

Dermatan Sulfate ◽

Anticoagulant Activity ◽

Psoas Muscle ◽

Chronic Hemodialysis ◽

Gel Chromatography ◽

Thrombin Time ◽

Heparin Cofactor Ii ◽

Dermatan Sulfate Proteoglycan

Abstract A 68-year-old man on chronic hemodialysis for 6 years, presented with a spontaneous psoas muscle hemorrhage. Investigations showed intermittently elevated activated partial-thromboplastin time and thrombin time. Preliminary investigations suggested a heparin-like inhibitor in the patient's plasma, but no anti-Xa activity could be detected. Investigation of the ability of patient plasma to inhibit exogenous thrombin showed that most thrombin was inhibited by heparin cofactor II, in contrast to normal plasma in which most thrombin was inhibited by antithrombin III. Treatment of plasma with glycosaminoglycan-degrading enzymes suggested the presence of dermatan sulfate (DS) in patient plasma. This was confirmed in a heparin cofactor II-dependent antithrombin assay for DS that showed anticoagulant equivalent to 2.2 +/- 0.3 micrograms/mL (mean +/- SD) of porcine mucosal DS. Of this activity, approximately 90% was sensitive to enzymes that degrade DS. The glycosaminoglycan containing fraction of plasma was isolated and subjected to gel chromatography. Anticoagulant activity eluted from Sephadex G-100 (Pharmacia, Montreal, Quebec, Canada) as two peaks with Kav of 0.10 and 0.45. After treatment with base, the Kav of the higher molecular weight species was increased to 0.55. This activity was completely sensitive to enzymes that degrade DS. Thus, the active DS was present as a proteoglycan. The lower molecular weight material was not sensitive to enzymes that degrade DS or heparan sulfate and it was active in the heparin cofactor II- dependent antithrombin assay but not in an antithrombin III-dependent antithrombin assay. This activity was not degraded by heating. Subsequently, measurement of DS activity was performed in plasmas obtained from eight other patients on hemodialysis before administration of heparin that showed that all patients had DS activity present that varied from 0.05 to 0.4 microgram/mL. No enzyme-resistant activity could be shown in these patients. In summary, a circulating anticoagulant with properties of DS is present in patients requiring hemodialysis.

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Modulation of Heparin Cofactor II Function by S Protein (Vitronectin) and Formation of a Ternary S Protein-Thrombin-Heparin Cofactor II Complex

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646979 ◽

1988 ◽

Vol 60 (03) ◽

pp. 399-406 ◽

Cited By ~ 15

Author(s):

Klaus T Preissner ◽

Pierre Sié

Keyword(s):

Dermatan Sulfate ◽

Enzyme Inhibitor ◽

Anticoagulant Activity ◽

Factor Xa ◽

Binding Domain ◽

Coagulation System ◽

Pentosan Polysulfate ◽

Heparin Cofactor Ii ◽

S Protein ◽

Inhibition Of Thrombin

SummaryThe complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin as well as factor Xa against fast inactivation by antithrombin III. The interference of S protein with glycosaminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in these studies. S protein significantly counteracted the anticoagulant activity of heparin and pentosan polysulfate but not of dermatan sulfate. In the presence of 0.3 μg/ml heparin, 0.5 μg/ml pentosan polysulfate, or 2 μg/ml dermatan sulfate, S protein induced a concentrationdependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo first-order rate constants by about 17-fold (heparin), or about 7-fold (pentosan polysulfate), whereas no neutralization of dermatan sulfate was demonstrable at a physiological ratio of S protein to heparin cofactor II. Exposure of the glycosaminoglycan-binding region of S protein by reduction and carboxymethylation of the protein increased the neutralizing activity of S protein towards heparin and pentosan polysulfate. The results of these functional experiments correlated well with the demonstration of direct binding of S protein to both polysaccharides but not to dermatan sulfate. While reduced/carboxymethylated S protein remained also ineffective in neutralizing other dermatan sulfate compounds with varying degree of sulfation, a synthetic highly basic tridecapeptide, representing a portion of the glycosaminoglycan-binding domain of S protein, counteracted their anticoagulant activity. Independent on the polysaccharide used, S protein was found incorporated within a ternary complex with thrombin and heparin cofactor II during the inhibition reaction as judged by crossed immunoelectrophoresis, ultracentrifugation as well as ELISA analysis, emphazising the function of S protein as scavenger protein for enzyme-inhibitor complexes of the coagulation system. These findings demonstrate the role of S protein as effective neutralising plasma protein of the anticoagulant activity of various glycosaminoglycans also with respect to heparin cofactor II. Although the glycosaminoglycan-binding domain of S protein readily neutralized different dermatan sulfate compounds, physiological modulation of heparin cofactor-II-dependent inhibition of thrombin by native S protein appears to be restricted to the vascular compartments, where other glycosaminoglycans than dermatan sulfate appear to be operative.

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