Thrombosis and Haemostasis, Table of Contents Thromb Haemost 1966; 15(01/02): 001-011DOI: 10.1055/s-0038-1649407 Originalarbeiten — Original Articles — Travaux Originaux Schattauer GmbH Activation of Purified Prothrombin in Ammonium Sulfate Solutions: Purification of Autoprothrombin C[*] Authors Author Affiliations W. H Seegers 1 Department of Physiology and Pharmacology, Wayne State University, School of Medicine, Detroit, Michigan, USA D. L Heene 1 Department of Physiology and Pharmacology, Wayne State University, School of Medicine, Detroit, Michigan, USA Ewa Marciniak 1 Department of Physiology and Pharmacology, Wayne State University, School of Medicine, Detroit, Michigan, USA Recommend Article Abstract Buy Article(opens in new window) Summary For the generation of autoprothrombin C activity from prothrombin preparations in concentrated ammonium sulfate solution the optimum conditions were found to be near 2 M at pH 7. In addition to thrombin and autoprothrombin C an inhibitor was obtained. Methods were developed to obtain the autoprothrombin C consistently with a specific activity of about 4,700 units per mg protein, and a yield of one mg per liter of bovine plasma. At an intermediate stage of purification the stability of autoprothrombin C was far better than in the final product which lost activity even by simple freezing and thawing. Preservation of activity in 50% glycerol solutions at pH 7.2 was found to be the most convenient procedure. The alterations produced in purified prothrombin with DEAE cellulose chromatography are discussed with respect to possible significance for thrombin and autoprothrombin C. Full Text References References 1 Milstone J. H. Thrombokinase as prime activator of prothrombin: historical perspectives and present status. Fed. Proc 23: 742 1964; 2 Seegers W. H, Cole E. R, Harmison C. R, Marciniak E. Purification and some properties of autoprothrombin C. Canad. J. Biochem. Physiol 41: 1047 1963; 3 Kowarzyk H, Marciniak E. The significance of prothrombin derivatives. Proceedings of the Eight Congress of the European Society of Haematology. Vienna 1961; 402. 4 Marciniak E, Seegers W. H. Autoprothrombin C: A second enzyme from prothrombin. Canad. J. Biochem. Physiol 40: 597 1962; 5 Seegers W. H, Cole E. R, Aoki N, Harmison C. R. Separation of autoprothrombin III from bovine prothrombin preparations. Canad. J. Biochem 42: 229 1964; 6 Seegers W. H, Aoki N, Marciniak E. Autoprothrombin C and the generation of thrombin from prothrombin activation mixtures. New Istanbul Contr. clin. Sci 05: 170 1962; 7 Esnouf M. P, Williams W. J. The isolation and purification of a bovine plasma protein which is a substrate for the coagulant fraction of Russell’s viper venom. Biochem. J 84: 62 1962; 8 Williams W. J, Esnouf M. P. The fractionation of Russell’s viper (Vipera russellii) venom with special reference to the coagulant protein. Biochem. J 84: 52 1962; 9 Seegers W. H. Enzyme theory of blood clotting. Fed. Proc 23: 749 1964; 10 Purcell G. M, Barnhart M. I. Prothrombin activation with cathepsin C. Biochim. biophys. Acta. (Arnst) 78: 800 1963; 11 Milstone J. H, Oulianoff N, Milstone V. K. Outstanding characteristics of thrombokinase isolated from bovine plasma. J. gen. Physiol 47: 315 1963; 12 Seegers W. H. The purification of prothrombin. Record Chem. Progr 13: 143 1952; 13 Seegers W. H, Levine W. G, Shepard R. S. Further studies on the purification of thrombin. Canad. J. Biochem 36: 603 1958; 14 Ware A. G, Seegers W. H. Two stage procedure for the quantitative determination of prothrombin concentration. Amer. J. clin. Path 19: 471 1949; 15 Seegers W. H, Smith H. P. Factors which influence the activity of purified thrombin. Amer. J. Physiol 137: 348 1942; 16 Cole E. R, Marciniak E, Seegers W. H. Procedures for the quantitative determination of autoprothrombin C. Thrombos. Diathes. haemorrh. (Stuttg) 08: 434 1962; 17 Seegers W. H, Landaburu R. H. Purification of prothrombin and thrombin by chromatography on cellulose. Canad. J. Biochem 38: 1405 1960; 18 Mammen E. F, Thomas W. R, Seegers W. H. Activation of purified prothrombin to autoprothrombin I or autoprothrombin II (platelet cofactor II) or autoprothrombin II-A. Thrombos. Diathes. haemorrh. (Stuttg) 05: 218 1960; 19 Marciniak E, Seegers W. H. Prethrombin as a new subunit of prothrombin. Nature. (Lond.) (In press.) 20 Thomas W. R, Seegers W. H. Terminal amino acids of bovine prothrombin and thrombin preparations. Biochim. biophys. Acta (Amst) 42: 556 1960; 21 Riddle J. M, Bernstein M. H, Seegers W. H. Ultrastructure of prothrombin and thrombin molecules. Thrombos. Diathes. haemorrh. (Stuttg) 09: 12 1963; 22 Marciniak E, Cole E. R, Seegers W. H. Functional Alteration in the prothrombin molecule. Nature (Lond) 195: 1305 1962; 23 Seegers W. H, Marciniak E. Autoprothrombin C in irregular blood clotting. Thrombos. Diathes. haemorrh. (Stuttg) 08: 1 1962;
