Low Anticoagulant Activity of High Sulphated Heparan Sulphates

Summary

Two high sulphated heparin-like polysaccharides (LI, MW 16,000 and L2, MW 11,700) were isolated from rat liver tissues, after DEAE-cellulose chromatography. Heparan sulphates from heart and lung tissues were isolated for comparison and fractionated according to their molecular weight. The anticoagulant activities in vitro were studied using clotting antifactor Xa, antifactor IIa, and APTT assay methods, falling in a narrow range (5-44 IU/mg) although the wide variability m molecular weight and sulphate content. The heparan sulphate nature of fractions L1 and L2 (sulphate/disaccharide ratio 2.05 and 2.48, respectively) has been verified by: a) low iduronic/glucuronic acid ratio; b) nitrous acid degradation followed by gel chromatography; c) heparinase treatment followed by gel chromatography; d) electrophoretic behaviour. Native proteoglycans have been isolated and the glycosidic chains compared with L1 and L2. Their anticoagulant activities in vitro and the fact that antiXa clotting activity was not neutralized by protamine sulphate are in accordance with the results of structural studies.

• References

• 1 Dietrich CP, Sampaio LO, Toledo OM S. Characteristic distribution of sulfated mucopolysaccharides in different tissues and in their respective mitochondria. Biochem Biophys Res Commun 1976; 71: 1-10
• 2 Gallagher JT, Walker A. Molecular distinctions between heparan sulphate and heparin. Biochem J 1985; 230: 665-74
• 3 Johnson EA. Heparan sulphates from porcine intestinal mucosa. Preparation and physicochemical properties. Thromb Res 1984 35. 583-8
• 4 Lindahl U, Pejler G. Heparin-like polysaccharides in intra- and extravascular coagulation reactions. Acta Med Scand Suppl 1987; 715: 139-44
• 5 Hubbard AR, Jennings CA, Barrowcliffe TW. Anticoagulant properties in vitro of heparan sulphates. Thromb Res 1984; 35: 567-76
• 6 Hovingh P, Piepkorn M, Linker A. Biological implications of the structural, antithrombin affinity and anticoagulant activity relationship among vertebrate heparins and heparan sulphates. Biochem J 1986; 237: 573-81
• 7 Nader HB, Takahashi HK, Strauss AH, Dietrich CP. Selective distribution of the heparin in mammals. Conspicuous presence of heparin in lymphoid tissues. Biochim Biophys Acta 1980 627. 40-8
• 8 Kovensky J, Fernandez Cirelli A. Comparative studies of heparan sulfates from rat tissues. An Asoc Quim Argent 1987; 75 (06) 563-8
• 9 Roden L, Baker J, Cifonclli JA, Mathews MB. Isolation and characterization of connective tissue polysaccharides. Methods Enzy-mol 1972; 28: 73-140
• 10 Terho TT, Hartiala K. Method for determination of the sulfate content of glycosaminoglycans. Analyt Biochem 1971; 41: 471-6
• 11 Di Ferrante N, Nichols BL, Donnelly PV, Neri G, Hrgovcic R, Berglund RK. Induced degradation of glycosaminoglycans in Hurler’s and Hunter’s syndromes by plasma infusion. Proc Natl Acad Sci USA 1971; 68: 303-7
• 12 Dische Z. Color reactions of hexosamines. Methods Carbohydr Chem 1962; 1: 507-12
• 13 Chandrasekaran EV, BeMiller JN. Constituent analysis of glycosaminoglycans. Methods Carbohydr Chem 1980; VIII: 89-96
• 14 Fransson LA, Sjoberg I, Havsmark B. Structural studies on heparan sulphates. Eur J Biochem 1980; 106: 59-69
• 15 Hook M, Lindahl U, Iverius PH. Distribution of sulphate and iduronic acid residues in heparin and heparan sulphate. Biochem J 1974; 137: 33-43
• 16 Bianchini P, Nader HB, Takashi HK, Osima B, Strauss AH, Dietrich CP. Fractionation and identification of heparin and other acidic mucopolysaccharides by a new discontinuous electrophoretic method. J Chromatogr 1980; 196: 455-62
• 17 Oldberg A, Hook M, Obrink B, Pertoft H, Rubin K. Structure and metabolism of rat liver heparan sulphate. Biochem J 1977; 164: 75-81
• 18 Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956; 28: 350-6
• 19 Shively JE, Conrad HE. Formation of anhydro sugars in the chemical depolymerization of heparin. Biochemistry 1976; 15: 3932-42
• 20 Denson KW E, Bonnar J. The measurement of heparin: a method based on the potentiation of the antifactor Xa. Thromb Diathes Haemorrh 1973; 30: 471-9
• 21 Eggleton CA, Barrowcliffe TW, Merton RE, Thomas DP. In vitro and in vivo studies of the anti Xa activity of heparin. Thromb Res 1981; 24: 319-28
• 22 Biggs R. Human Blood Coagulation, Haemostasis and Thrombosis. Blackwell Scientific Publications; Oxford: 1972: 643-5
• 23 Ostergaard PB, Nilsson B, Bergqvist D, Hedner U, Pedersen PC. The effect of low molecular weight heparin on experimental thrombosis and haemostasis. The influence of production method. Thromb Res 1987 45. 739-49
• 24 Casu B. Structure and biological activity of heparin. Adv Carbohydr Chem Biochem 1985; 43: 51-135
• 25 Ofosu FA, Modi GJ, Blajchman MA, Buchanan MR, Johnson EA. Increased sulphation improves the anticoagulant activities of heparan sulphate and dermatan sulphate. Biochem J 1987; 248: 889-96
• 26 Van Ryn-McKenna J, Ofosu FA, Hirsh J, Buchanan MR. Antithrombotic and bleeding effects of glycosaminoglycans with different degrees of sulphation. Bi J Haematol 1989; 71: 265-9
• 27 Linker A, Hovingh P. The heparitin sulfates (heparan sulfates). Carbohydr Res 1973; 29: 41-62
• 28 Poblacion CA, Michelacci YM. Structural differences of dermatan sulfates from different origins. Carbohydr Res 1986; 29: 41-62
• 29 Horner AA. Rat heparins. Biochem J 1986; 240: 171-9