Quantitative Determination of Total Antithrombin III in Plasma

Summary

A relatively simple method has been developed to evaluate the total amount of antithrombin III in plasma. Since purified and stable bovine thrombin was readily prepared in our laboratory, it was selected to be used in our assay system. Purified α₂-macroglobulin was found to inactivate thrombin following a pseudo first-order rate of reaction. However, purified antithrombin III inactivated thrombin following a bimolecular type of reaction. The second-order rate constant for antithrombin III and thrombin interaction was found to be 94 1/M/sec. under these conditions. A least-square straight line was drawn between 15 and 90 minute intervals and the intercept yielded the total amount of antithrombin III expressed NIH thrombin units. This assay system and the graphical analysis yielded the total amount of antithrombin III in plasma quantitatively. The results are reproducible.

• References

• 1 Abildgaard U. 1967; Purification of two progessive antithrombins of human plasma. Scandinavian Journal of Clinical and Laboratory Investigations 19: 190.
• 2 Abildgaard TJ. 1969; Binding of thrombin to antithrombin III. Scandinavian Journal of Clinical and Laboratory Investigations 24: 23.
• 3 Abildgaabd U, Gravem K, and Godal H.C. 1970; Assay of progressive antithrombin in plasma. Thrombosis et Diathesis Haemorrhagica 24: 224.
• 4 Astrup T, and Darling S. 1942; Measurement and properties of antithrombin. Acta Physiologica Scandinavica 4: 293.
• 5 Bertho A, and Grassmann W. 1938. Laboratory Methods of Biochemistry. MacMillan and Co. Ltd.; London: 36.
• 6 Biggs R, Denson K.W.E, Akman N, Borrett R, and Hadden M. 1970; Antithrombin III, antifactor X_a and heparin. Brithish Journal of Haematology 19: 283.
• 7 Dombrose F.A, Seegers W.H, and Sedensky J. A. 1971; Antithrombin: Inhibition of thrombin and autopro thrombin C (F-X_a) as a mutual depletion system. Thrombosis et Diathesis Haemorrhagica 26: 103.
• 8 Esnouf M. P, and Williams W. J. 162 The isolation and purification of a bovine-plasma protein which is a substrate of the coagulation fraction of Russell’s-Viper Venom. Biochemical Journal 84: 62.
• 9 Heimburger N, Haupt H, and Schwick H. G. 1971 Proteinase inhibitor of human plasma. In: Fritz H, and Tschesche H. (ed.) Proceedings of the International Research Conference on Proteinase Inhibitors. Munich, November 4-6, 1970, p. 1.
• 10 Heimburger N, and Trobisch H. 1971; Blood Coagulation and Fibrinolysis. Angewandte Chemie International Edition 10: 85.
• 11 Monkhouse F. C, France E. S, and Seegers W. H. 1955; Studies on the antithrombin and heparin cofactor activities of a fraction absorbed from plasma by aluminium hydroxide. Circulation Research 3: 397.
• 12 Quick A.J. 1951. The physiology and pathology of hemostasis. Lea and Febiger; Philadelphia: 168.
• 13 Seeger W.H. 1968; Antithrombin as a proteinase inhibitor. Annals of the New York Academy of Sciences 146: 593.
• 14 Seegers W. H, Cole E. R, Harmison C. R, and Monkhouse F. C. 1964; Neuralization of autopro thrombin C activity with antithrombin. Canadian Journal of Biochemistry 42: 359.
• 15 Seegers N. H, and Marciniak E. 1962; Inhibition of autoprothrombin C activity with plasma. Nature 93: 1188.
• 16 Seegers W. H, and Smith H. P. 1943; Antithrombin activity of plasma: quantitative interrelationships. Proceeding of the Society for Experimental Biology 52: 159.
• 17 Yin E. T, Wessler S, and Stole P.J. 1971; a Rabbit plasma inhibitor of the activated species of blood coagulation factor X. Purification some properties. Journal of Biological Chemistry 246: 3694.
• 18 Yin E.T, Wessler S, and Stoll P.J. 1971; b Biological properties of the naturally occurring plasma inhibitor to activated factor X. Journal of Biological Chemistry 246: 3703.
• 19 Yin E.T, Wessler S, and Stoll P. J. 1971; c Identity of plasma-activated factor X inhibitor with antithrombin III and heparin cofactor. Journal of Biological Chemistry 246: 3712.
• 20 Yue R.H, Starr T, and Gertler M.M. 1972; The binding of calcium ions to bovine factor X. Federation Proceedings 31: 241 Abstract.