Antithrombin III Binding to Surface Immobilized Heparin and Its Relation to F Xa Inhibition

Summary

The mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depoly-merized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 HTid 11 pic.omoles/cmf, respectively Thus the estimated density of biologically active high and low ailmity heparm respectively would be 40 and 90 ng/cm² The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. Tt was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromized AT binding sequences.

Keywords

Antithrombin III - Factor Xa - Heparin - Immobilized heparin - Thromboresistant surface

• References

• 1 Lärm O, Larsson R, Olsson R. A new non-thrombogenic surface prepared by selective binding of heparin via a modified reducing terminal residue. Biomat Med Dev Art Org 1983; 11: 161-163
• 2 Pasche B, Kodama K, Larm O, Olsson P, Swedenborg J. Thrombin inactivation on surfaces with covalently bonded heparin. Thromb Res 1986; 44: 739-748
• 3 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505
• 4 Marcum JS, Rosenberg RD. Heparin-like molecules with anticoagulant activity are synthesized by cultured endothelial cells. Biochem Biophys Res Com 1985; 126: 365-372
• 5 Marcum JA, McKenny JB, Rosenberg RD. Acceleration of thrombin-antithrombin complex formation in rate hindquarters via heparin-like molecules bound to the endothelium. J Clin Invest 1984; 74: 341-350
• 6 Thunberg L, Lindahl U, Tengblad A, Laurent TC, Jackson CM. On molecular-weight-dependence of the anticoagulant activity of heparin. Biochem J 1979; 181: 241-243
• 7 Nordenman B, Nordling K, Björk I. A differential effect of low-affinity heparin on the inhibition of thrombin and Factor Xa antithrombin. Thromb Res 1980; 17: 595-600
• 8 Holmer E, Kurachi K, Söderström G. The molecular-weight dependence of rate-enhancing effect of heparin on the inhibition of thrombin. Factor Xa, Factor IXa, Factor XIa, Factor Xlla and kallikrein by antithrombin. Biochem J 1981; 193: 395-400
• 9 Höök M, Björk I, Hopwood J, Lindahl U. Anticoagulant activity of heparin: separation of high-activity and low-activity heparin species by affinity chromatography on immobilized heparin. FEBS Lett 1976; 66: 90-93
• 10 Bitter T, Muir HM. A modified uronic acid carbazole reaction. Anal Biochem 1962; 4: 330-334
• 11 Abildgaard U, Lie M, Odegaard OR. Antithrombin (heparin cofactor) assays with “new” chromogenic substrate. Thromb Res 1977; 11: 549-553
• 12 Laurent TC, Tengblad A, Thunberg L, Höök M, Lindahl U. The molecular-weight-dependence of the anticoagulant activity of heparin. Biochem J 1978; 175: 691-701
• 13 Rollason G, Sefton MV. Factor Xa inactivation by a heparinized hydrogel. Thromb Res 1986; 44: 517-525
• 14 Hennink H JS, Kripmeijer A SK, Engberg G HM, Dost L, Feijen J. Is the use of low molecular weight heparin in blood compatible surface coatings advantagenous over that of nonfractionated heparin?. Proceedings Polymers in Medicine, 7/1-7/11. 1986. Levenhorst; Holland:
• 15 Bauer KA, Goodman TL, Kass BL, Rosenberg RD. Elevated factor Xa activity in the blood of asymptomatic patients with congenital antithrombin deficiency. J Clin Invest 1985; 76: 826-836
• 16 Fuchs HE, Salvatore VP. Regulation of Factor Xa in vitro in human and mouse plasma and in vivo in mouse. J Clin Invest 1983; 72: 2041-2049
• 17 Bindslev L, Gouda J, Inacio J, Kodama K, Lagergren H, Larm O, Nilsson E, Olsson P. Extracorporeal elimination of carbon dioxide using a surface-heparinized veno-venous by-pass system. Trans ASAIO 1986; Vol 32 (01) 530-533
• 18 Bindslev L, Eklund J, Norlander O, Swedenborg J, Olsson P, Inacio J, Nilsson E, Larm O, Gouda J, Malmberg A, Scholander E. Treatment of acute respiratory failure by extracorporeal carbon dioxide elimination performed with a surface heparinized artificial lung. Anaesthesiology 1987; 67 (01) 117-120
• 19 Arnander C, Dryjski M, Larsson R, Olsson P, Swedenborg J. Thrombin uptake and inhibition on endothelium and surfaces with a stable heparin coating. A comparative in vitro study. J Biomed Mat Res 1986; 20: 235-246