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

The dermatan sulfate (DS) isolated from the ray skin Raja montagui was identified and characterized. Its average molecular weight (Mw) and sulfate content were 39 kDa and 25% w/w, respectively. This DS prolonged thrombin time and activated partial thromboplastin time and inhibited the thrombin generation in a concentration-dependent manner whereas it had no effect on the anti-Xa assay and on platelet function. Data from the anti-IIa assay allowed the assessment of the specific anticoagulant activity which was 40 units/mg. The kinetics of the thrombin inhibition by heparin cofactor II (HCII) has been studied as a function of DS concentration according to a kinetic model in which the polysaccharide binds quickly to the inhibitor and forms a complex more reactive than the free inhibitor towards thrombin. This DS accelerated thrombin inhibition exclusively by HCII. The dissociation constant of the DS-HCII complex, K(DSHCII), and the rate constant of the thrombin inhibition by this complex, k, were (2.93+/-0.25)x10(-6)M and (2.2+/-0.35)x10(9)M(-1)min(-1), respectively. Our findings indicated that the major polysaccharide in the skin of the ray Raja montagui was a DS endowed with a high anticoagulant effect mediated by HCII and which may constitute an anticoagulant drug of interest in anticoagulant therapy.

[1]  G. Calderón‐Domínguez,et al.  Detection, isolation, and characterization of exopolysaccharide produced by a strain of Phormidium 94a isolated from an arid zone of Mexico , 2004, Biotechnology and Bioengineering.

[2]  F. R. Melo,et al.  Structural composition and differential anticoagulant activities of dermatan sulfates from the skin of four species of rays, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and Potamotrygon motoro. , 2004, Biochimie.

[3]  M. Petitou,et al.  Structure-activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity. , 1983, Biochemical and biophysical research communications.

[4]  F. Maccari,et al.  Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All. , 2005, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[5]  P. E. Bock,et al.  The Preferred Pathway of Glycosaminoglycan-accelerated Inactivation of Thrombin by Heparin Cofactor II* , 2004, Journal of Biological Chemistry.

[6]  F. Chaubet,et al.  Polysaccharides from the skin of the ray Raja radula. Partial characterization and anticoagulant activity. , 2009, Thrombosis research.

[7]  T. Tsegenidis,et al.  Isolation and characterisation of a small dermatan sulphate proteoglycan from ray skin (Raja clavata) , 1999 .

[8]  D. Tollefsen,et al.  Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity. , 1990, The Journal of biological chemistry.

[9]  A. Nurden,et al.  Cross‐linking of α and γ‐thrombin to distinct binding sites on human platelets , 1988 .

[10]  D. Tollefsen,et al.  Activation of heparin cofactor II by dermatan sulfate. , 1983, The Journal of biological chemistry.

[11]  T BITTER,et al.  A modified uronic acid carbazole reaction. , 1962, Analytical biochemistry.

[12]  A. Fischer,et al.  Mechanism of thrombin inhibition by heparin cofactor II in the presence of dermatan sulphates, native or oversulphated, and a heparin-like dextran derivative. , 1997, Biomaterials.

[13]  A. Bezeaud,et al.  Interaction of human α‐thrombin and γ‐thrombin with antithrombin III, protein C and thrombomodulin , 1985 .

[14]  A. Manouras,et al.  Isolation and high-performance liquid chromatographic analysis of ray (Raja clavata) skin glycosaminoglycans , 1991 .

[15]  Jennifer W Mitchell,et al.  Aspartic Acid Residues 72 and 75 and Tyrosine-sulfate 73 of Heparin Cofactor II Promote Intramolecular Interactions during Glycosaminoglycan Binding and Thrombin Inhibition* , 2002, The Journal of Biological Chemistry.

[16]  A. Fischer,et al.  Thrombin inhibition by antithrombin in the presence of oversulfated dermatan sulfates. , 2006, Carbohydrate research.

[17]  J. Shively,et al.  Formation of anhydrosugars in the chemical depolymerization of heparin. , 1976, Biochemistry.

[18]  C. Branford-White,et al.  Structural features and anti-HIV-1 activity of novel polysaccharides from red algae Grateloupia longifolia and Grateloupia filicina. , 2007, International journal of biological macromolecules.

[19]  F. Chaubet,et al.  Mechanism of thrombin inhibition by heparin cofactor II and antithrombin in the presence of the ray (Raja radula) skin dermatan sulfate. , 2009, Thrombosis research.

[20]  H. Coenraad Hemker,et al.  Calibrated Automated Thrombin Generation Measurement in Clotting Plasma , 2003, Pathophysiology of Haemostasis and Thrombosis.

[21]  T. Warkentin.  Pathophysiology of Haemostasis and Thrombosis HIT : Lessons Learned , 2006 .

[22]  A. Valente,et al.  Highly Sulfated Dermatan Sulfates from Ascidians , 1998, The Journal of Biological Chemistry.

[23]  Frédéric Chaubet,et al.  Characterization of a novel dermatan sulfate with high antithrombin activity from ray skin (Raja radula). , 2009, Thrombosis research.

[24]  M. Kyogashima,et al.  Antithrombotic activity of avian crown dermatan sulfate. , 1999, Thrombosis research.

[25]  T. Koide,et al.  Binding of heparin or dermatan sulfate to thrombin is essential for the sulfated polysaccharide‐accelerated inhibition of thrombin by heparin cofactor II , 1987, FEBS letters.

[26]  R. G. Price,et al.  A note on the determination of the ester sulphate content of sulphated polysaccharides. , 1962, The Biochemical journal.

[27]  F. Maccari,et al.  Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes phylippinarum: evidence for the presence of a high content of antithrombin III binding site. , 2004, Glycobiology.

[28]  P. Liaw,et al.  Comparison of Heparin- and Dermatan Sulfate-mediated Catalysis of Thrombin Inactivation by Heparin Cofactor II* , 1999, The Journal of Biological Chemistry.

[29]  S. Anagnostides,et al.  Purification and characterisation of a minor low-sulphated dermatan sulphate-proteoglycan from ray skin. , 1999, Biochimie.

[30]  Shinobu Sakai,et al.  Purification and characterization of dermatan sulfate from the skin of the eel, Anguilla japonica. , 2003, Carbohydrate research.

[31]  A. Fischer,et al.  Mechanism of thrombin inhibition by antithrombin and heparin cofactor II in the presence of heparin. , 1997, Biomaterials.

[32]  CO Passos,et al.  Comparative biochemistry of human skin: glycosaminoglycans from different body sites in normal subjects and in patients with localized scleroderma , 2003, Journal of the European Academy of Dermatology and Venereology : JEADV.

[33]  F. Smith,et al.  Colorimetric Method for Determination of Sugars and Related Substances , 1956 .

[34]  T. Yamagata,et al.  Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. , 1968, The Journal of biological chemistry.

[35]  D. Heinegård,et al.  [17] Isolation and characterization of proteoglycans , 1987 .

[36]  F. R. Melo,et al.  Structural composition and anticoagulant activity of dermatan sulfate from the skin of the electric eel, Electrophorus electricus (L.). , 2007, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[37]  R Mendelsohn,et al.  FTIR microscopic imaging of collagen and proteoglycan in bovine cartilage. , 2001, Biopolymers.

[38]  K. Nordling,et al.  Extension and structural variability of the antithrombin-binding sequence in heparin. , 1984, The Journal of biological chemistry.

[39]  P. Mourão,et al.  Dermatan sulfate is the predominant antithrombotic glycosaminoglycan in vessel walls: implications for a possible physiological function of heparin cofactor II. , 2005, Biochimica et biophysica acta.

[40]  M. Dautzenberg,et al.  Influence of the oversulfation method and the degree of sulfation on the anticoagulant properties of dermatan sulfate derivatives. , 1990, Thrombosis research.

[41]  F. Maquart,et al.  Cellulose acetate electrophoresis of glycosaminoglycans. , 2001, Methods in molecular biology.

[42]  M. Pavão,et al.  Isolation and characterization of a heparin with low antithrombin activity from the body of Styela plicata (Chordata-Tunicata). Distinct effects on venous and arterial models of thrombosis. , 2007, Thrombosis research.

[43]  B. Mulloy,et al.  A Unique Dermatan Sulfate-like Glycosaminoglycan from Ascidian: , 1995, The Journal of Biological Chemistry.

[44]  W. Garnjanagoonchorn,et al.  Determination of chondroitin sulfate from different sources of cartilage , 2007 .

[45]  T. Maruyama,et al.  Preparation and anticoagulant activity of fully O-sulphonated glycosaminoglycans. , 1999, International journal of biological macromolecules.