Evidence for an Ester Bond between Thrombin and Heparin Cofactor^[*]

Summary

Heparin cofactor, a thrombin inhibitor, is purified from human plasma by affinity chromatography on heparin-agarose. The nature of the binding between thrombin and the inhibitor is studied by treatment of the complex with 6 M guanidinium chloride, hydroxylamine, and dilute alkali. The complex is not dissociated during gel chromatography in 6 M guanidinium chloride. This result supports an earlier proposal that formation of the complex includes the formation of a covalent bond. Treatment of dodecyl sulfate-denatured complex with hydroxylamine results in dissociation of the complex to yield free thrombin and heparin cofactor. Hydroxylamine does not dissociate the complex unless it is denatured. The complex is also dissociated in dilute sodium hydroxide (pH 12) solutions. These results indicate that the covalent bond between thrombin and the inhibitor is a carboxylic ester.

^* Presented at the Vth Congress of the International Society on Thrombosis and Haemostasis, Paris, July, 1975.

• Reference

• 1 Abildgaard V, Fagerhol M. K, Egeberg O. 1970; Comparison of progressive antithrombin activity and the concentrations of three thrombin inhibitors in human plasma. Scandanavian Journal of Clinical Investigation 26: 349.
• 2 Aronson D. L. 1966; Chromatographic differentiation of human prothrombins. Thrombosis et Diathesis haemorrhagica 16: 491.
• 3 Cuatrecasas P. 1970; Protein purification by affinity chromatography. The Journal of Biological Chemistry 245: 3059.
• 4 Davis B. J. 1964; Disc electrophoresis. II. Method and application to human serum proteins. Annals of the New York Academy of Science 121: 404.
• 5 Fish W. F, Mann K. G, Tanford C. 1969; The estimation of polypeptide chain molecular weights by gel filtration in 6M guanidine hydrochloride. The Journal of Biological Chemistry 244: 4989.
• 6 Heimburger N. 1974 Proteinase inhibitors of human plasma – their properties and control functions.. Cold Spring Harbor Symposium on Proteases and Biological Control, Abstracts p. 20.
• 7 Highsmuh R. F, Rosenberg R. D. 1974; The inhibition of human plasmin by human antithrombin-heparin cofactor. The Journal of Biological Chemistry 249: 4335.
• 8 Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680.
• 9 Laskowski Jr. M, Sealock R. W. 1971. Protein proteinase inhibitors – molecular aspects. In: Boyer P. D. (ed.) The Enzymes. Volume III. Academic Press; New York: 376.
• 10 Lundblad R. L. 1971; A rapid method for the purification of bovine thrombin and the inhibition of the purified enzyme with phenylmethylsulfonyl fluoride. Biochemistry 10: 2501.
• 11 Miller-Andersson M, Borg H, Andersson L. O. 1974; Purification of antithrombin III by affinity chromatography. Thrombosis Research 5: 439.
• 12 Nelson C. A. 1971; The binding of detergents to proteins. The Journal of Biological Chemistry 246: 3895.
• 13 Owen W. G, Esmon C. T, Jackson C. M. 1974; The conversion of prothrombin to thrombin. The Journal of Biological Chemistry 249: 594.
• 14 Owen W. G, Jackson C. M. 1973; Activation of prothrombin with Oxyuranus scutellatus scutellatus (taipan snake) venom. Thrombosis Research 3: 705.
• 15 Robinson D. W, Jencks W. P. 1965; The effect of compounds of the urea-guanidinium class on the activity coefficient of acetyltetraglycine ethyl ester and related compounds. Journal of the American Chemical Society 87: 2462.
• 16 Rosenberg R. D, Damus P. S. 1973; The purification and mechanism of action of human antithrombin-heparin cofactor. The Journal of Biological Chemistry 248: 6490.
• 17 Ruhlman A, Kukla D, Schwager P, Bartels K, Huber R. 1973; Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Journal of Molecular Biology 77: 417.
• 18 Yashphe J, Halpern Y. S, Grossowicz N. 1960; A quantitative method for the differential determination of hydroxylamine and beta-aspartyl hydroxamate in mixtures. Analytical Chemistry 32: 518.
• 19 Yin E. T, Eisenkramer L, Butler J. 1975. Heparin interaction with activated factor X and its inhibitor. In: Bradshaw R. A, Wessler S. (eds.) Heparin.. Plenum Publishing Corp.; New York: 239.
• 20 Yin E. T, Wessler S. 1970; Heparin-accelerated inhibition of activated factor X by its natural plasma inhibitor. Biochimica Biophysica Acta 201: 387.
• 21 Yin E. T, Wessler S, Stoll P. J. 1971; Identity of plasma-activated factor X inhibitor with antithrombin III and heparin cofactor. The Journal of Biological Chemistry 246: 3712.