Removal from Bovine Prothrombin of the Substrate for Russell’s Viper Venom^[*]

Summary

Bovine prothrombin was prepared by adsorption on barium sulfate. After elution, it was passed through thick filter-cakes of Standard Super-Cel, which removed some venom substrate (factor X). Almost all the remaining venom substrate was removed by repeated passage through columns of DEAE-cellulose. Finally, the ratio of venom substrate to prothrombin was considerably less than 1/1,000 that of plasma. The prothrombin was also poor in factor V. It yielded very little thrombin upon incubation with Russell’s viper venom, factor V, phospholipid and calcium chloride. However, inclusion of bovine plasma at a final dilution of 1/10,000 caused the mixture to produce thrombin rapidly. This system offers promise for the assay of venom substrate in plasma.

Thrombokinase derived from bovine plasma was able, at 0.000071 mg/ml, to substitute for both the venom and its substrate in thrombin-producing systems. However, with this small amount of thrombokinase, phospholipid was indispensable. The system was sensitive to 0.00001 mg phospholipid/ml.

With 1,000 times as much thrombokinase, prothrombin was activated without addition of accessory factors, in the presence of oxalate. Removal of venom substrate did not affect this response of prothrombin. Nor did removal of venom substrate from the prothrombin prevent its activation by crystallized trypsin in the presence of oxalate.

^* This investigation was supported by Research Grant HE-03906 from the National Heart Institute, United States Public Health Service.

• References

• 1 Milstone J. H. Chromatography of blood-clotting factors and serum proteins on columns of diatomaceous earth. J. gen. Physiol 38: 743 1955;
• 2 Hougie C. Effect of Russell’s viper venom (Stypven) on Stuart clotting defect. Proc. Soc. exp. Biol. (N. Y) 93: 570 1956;
• 3 Telfer T. P, Denson K. W, Wright D. R. A ‘new’ coagulation defect. Brit. J. Haemat 02: 308 1956;
• 4 Macfarlane R. G. 2. Perspectives. Thrombos. Diathes. haemorrh. (Stuttg) 07 suppl. 1, 17 (1961-1962).
• 5 Macfarlane R. G. 17. Purification of factor X, its activation by Russell’s viper venom, and also by physiologic factors. Thrombos. Diathes. haemorrh. (Stuttg) 07 suppl. 1, 222 (1961—62).
• 6 Esnouf M. P, Williams W. J. 16. Purification, properties and activation of factor X. Thrombos. Diathes. haemorrh. (Stuttg) 07 suppl. 1, 213 (1961-62).
• 7 Milstone J. H. Thrombokinase as prime activator of prothrombin: historical perspectives and present status. Fed. Proc 23: 742 1964;
• 8 Ferguson J. H. Thromboplastic enzymes. Fed. Proc 23: 762 1964;
• 9 Milstone J. H. Preparation of thrombokinase from bovine plasma. J. gen. Physiol 42: 665 1959;
• 10 Milstone J. H. Fractionation of plasma globulin for prothrombin, thrombokinase, and accessory thromboplastin. J. gen. Physiol 35: 67 1951;
• 11 Esnouf M. P, Jobin F. The isolation of factor V from bovine plasma. Biochem. J 102: 660 1967;
• 12 Milstone J. H. Effect of blood thrombokinase, as influenced by soybean trypin inhibitor, ultracentrifugation, and accessory factors. J. gen. Physiol 38: 757 1955;
• 13 Milstone J. H, Oulianoff N, Milstone V. K. Outstanding characteristics of thrombokinase isolated from bovine plasma. J. gen. Physiol 47: 315 1963;
• 14 Kerwin D. M, Milstone J. H. Removal of thrombokinase from commercial thrombin. Thrombos. Diathes. haemorrh. (Stuttg) 17: 247 1967;
• 15 Sherry S, Troll W. The action of thrombin on synthetic substrates. J. biol. Chem 208: 95 1954;
• 16 Lowry O. H, Rosebrough N. J, Farr A. L, Randall R. J. Protein measurement with the Folin phenol reagent. J. biol. Chem 193: 265 1951;
• 17 Seegers W. H. Prothrombin, pp.412, 435. Harvard Univ. Press; Cambridge, Mass: 1962
• 18 Milstone J. H. Protein thrombokinase and lipoid thromboplastin as distinct factors with complementary functions. Yale J. Biol. Med 25: 19 1952;
• 19 Eagle H, Harris T. N. Studies in blood coagulation. V. The coagulation of blood by proteolytic enzymes (trypsin, papain). J. gen. Physiol 20: 543 1937;
• 20 Alexander B, Pechet L. Proteolytic and physiologic activation of some clotting components: pathologic significance. Proc. Eighth Congr. Europ. Soc. Haemat 02: 404 1961;
• 21 Milstone J. H. Chromatography of blood thrombokinase on “DEAE”-cellulose. Nature (Lond.) 187: 1127 1960;
• 22 Seegers W. H, Landaburu R. H. Purification of prothrombin and thrombin by chromatography on cellulose. Canad. J. Biochem 38: 1405 1960;
• 23 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;
• 24 Deutsch E, Lechner K. On the separation of prothrombin and factor X. New Istanbul Contr. clin. Sei. Suppl 01: 26 1965;
• 25 Lechner K, Deutsch E. Activation of factor X. Thrombos. Diathes. haemorrh. (Stuttg) 13: 314 1965;