The Human Prothrombin-Activating Enzyme^[*]

Summary

The formation and action of human prothrombin-activating enzyme is described. The study of the formation of the enzyme leads to the following conclusions :

1. The enzyme is formed from factor V, factor X and phospholipid in the presence of calcium. If one of the reagents is omitted no activity develops.

2. Factor V and factor X need activation by thrombin and for instance Russell Viper Venom, respectively.

3. A linear relationship exists between the inverse of factor Va concentration and the inverse of enzyme concentration.

4. A linear relationship exists between the inverse of factor Xa concentration and the inverse of enzyme concentration.

5. A linear relationship exists between the inverse of phospholipid concentration and the inverse of enzyme concentration at small phospholipid concentration.

6. A linear relationship exists between the phospholipid concentration and the inverse of enzyme concentration at high phospholipid concentration.

The study of the action of the enzyme leads to the conclusion that human prothrombin is substrate and an inhibitor if present in excess.

The observed phenomena are best explained by the hypothesis that factor Va and factor Xa adsorb onto the phospholipid surface. When both factors are adsorbed close together they are active as an enzyme. This activity depends on two active centers, probably one derived from factor Va and one from factor Xa.

^* The investigations were supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO).

• References

• 1 Barton P. G, Jackson G. M, Hanahan D. J. Relationship between factor V and activated factor X in the generation of prothrombinase. Nature (Lond.) 214: 923 1967;
• 2 Biggs R, MacFarlane R. G. Human Blood Coagulation and its Disorders. Blackwell Scientific Publications; Oxford: 1962
• 3 Biggs R, MacFarlane R. G, Denson K. W. E, Ash B. J. Thrombin and the interaction of factors VIII and IX. Brit. J. Haemat. 11: 276 1965;
• 4 Blombäck B, Blombäck M. Purification of human and bovine fibrinogen. Ark. Kemi. 10: 415 1956;
• 5 Blombäck B, Blombäck M. A method for the assay of factor V. Scand. J. clin. Lab. Invest. 15: 639 1963;
• 6 Breckenridge R. T, Ratnoff O. D. The activation of proaccelerin by bovine thrombokinase. Blood 27: 527 1966;
• 7 Cohn E. J, Strong L. E, Hughes Jr. W. L, Mulford D. J, Ashworth J. N, Melin M, Taylor H. L. Preparations and properties of serum and plasma proteins. J. Amer. ehem. Soc. 68: 459 1946;
• 8 Cole E. R, Koppel J. L, Olwin J. H. Phospholipid-protein interactions in the formation of prothrombin activator. Thrombos. Diathes. haemorrh. (Stuttg.) 14: 431 1965;
• 9 Daemen F. J. M, van Arkel G, Hort H. Ch, van der Drift C, van Deenen L. L. M. Activity of Synthetic Phospholipids in Blood Coagulation. Thrombos. Diathes. haemorrh. (Stuttg.) 13: 194 1965;
• 10 Davis B. J. Disc Electrophoresis II Method and Application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404 1964;
• 11 Deggeller K, van Dorp E. M, Vreeken J, Krijnen H. W. Isolation and separation of the “prothrombin complex”. In: Schroer H. Biochemie und Aktivierung des Prothrombins. 41 F. K. Schattauer Verlag; Stuttgart: 1968
• 12 Deggeller K, Thesis K. Amsterdam 1968.
• 13 Denson K. W. E. The use of antibodies in the study of blood coagulation. Blackwell Scientific Publications; Oxford: 1967
• 14 Dixon M, Webb E. C. Enzymes. Longmans, Green and Co Ltd.; London: 1958
• 15 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;
• 16 Folch J. Brain cephalin, a mixture of phosphatides. Separation from it of a phosphatidyl serine, phosphatidyl ethanolamine, and a fraction containing an inositol phosphatide. J. biol. Chem. 146: 35 1942;
• 17 Gobbi F, Barbieri U, Ascari E. Physical factors in the thromboplastic activity of phospholipids. The modifications of the thromboplastic activity of cephalin following treatment with surface-active substances. Thrombos. Diathes. haemorrh. (Stuttg.) 17: 495 1967;
• 18 Hemker H. C. The pathways of blood coagulation. Folia med. neerl. 10: 102 1967;
• 19 Hemker H. G, Ménaché D, Soidier J. P. Specific activity of prothrombin. Proc. Europ. Fed. Biochem. Soc. London A 62: 48 1964;
• 20 Hemker H. C, Esnouf M. P, Hemker P. W, Swart A. G. W, Macfariane R. G. Formation of prothrombin converting activity. Nature (Lond.) 215: 248 1967;
• 21 Jobin F, Esnouf M. P. Studies on the formation of the Prothrombin - converting Complex. Biochem. J. 102: 666 1967;
• 22 Jovin T, Grambach A, Naughton M. A. An apparatus for preparative temperature regulated Polyacrylamide gel electrophoresis. Anal. Biochem. 9: 351 1964;
• 23 Lineweaver H, Burk D. The determination of enzyme dissociation constants. J. Amer. chem. Soc. 56: 658 1934;
• 24 Lundblad R. L, Davie E. W. The activation of antihemophilic factor (factor VIII) by activated Christmas factor (activated factor IX). Biochemistry 3: 1720 1964;
• 25 Lundblad R. L, Davie E. W. The activation of Stuart factor (factor X) by activated antihemophilic factor (activated factor VIII). Biochemistry 4: 113 1965;
• 26 MacFarlane R. G. The coagulant action of Russell’s Viper Venom; the use of antivenom in defining its reaction with a serum factor. Brit. J. Haemat. 7: 496 1961;
• 27 MacFarlane R. G, Ash B. J. The activation and consumption of factor X in recalcified plasma; the effect of added factor VIII and Russell’s Viper Venom. Brit. J. Haemat. 10: 217 1964;
• 28 Milstone J. H. Thrombokinase of the blood as a trypsin-like enzyme. J. gen. Physiol. 45: 103 1961;
• 29 Ornstein L. Disc Electrophoresis; I Background and Theory Ann. N. Y. Acad. Sci. 121: 321 1964;
• 30 Owren P. A. The prothrombin activating complex and its clinical significans. 3d. Congress Int. Soc. Haemat. Cambridge. Grune & Stratton; New York: 1951
• 31 Papahadjopoillos D, Hougie C, Hanahan D. J. Influence of surface charge of phospholipids on their clot-promoting activity. Proc. Soc. exp. Biol. (N. Y.) 111: 412 1962;
• 32 PapahadjopoiUos D, Hanahan D. J. Observations on the interaction of phospholipids and certain clotting factors in prothrombin activator formation. Biochim. biophys. Acta (Amst.) 90: 436 1964;
• 33 Papahadjopoulos D, Hougie G, Hanahan D. J. Purification and properties of bovine factor V: A change of molecular size during blood coagulation. Biochemistry 3: 264 1964;
• 34 Prentice C. R. M, Ratnoff O. D, Breckenridge R. T. Experiments on the Nature of the pro-thrombin-converting principle: Alteration of proaccelerin by thrombin. Brit. J. Haemat. 13: 898 1967;
• 35 Rapaport S. I, Schiffman S, Patch M. J, Ames S. B. The importance of Activation of Antihemophilic Globulin and Proaccelerin by Traces of Thrombin in the Generation of Intrinsic Prothrombinase Activity. Blood 21: 221 1963;
• 36 Scarborough J. B. Method Least Squares. Numerical Mathematical Analysis. The Johns Hopkins Press; 339 1950
• 37 Seaman A. J, Owren P. A. An asolectin adsorbed substrate for proaccelerin assay. J. clin. Invest. 35: 145 1956;
• 38 Straub W, Duckert F. The Formation of the Extrinsic Prothrombin Activator. Thrombos. Diathes. haemorrh. (Stuttg.) 5: 402 1961;
• 39 Vroman L. Effects of Hydrophobic Surfaces upon Blood Coagulation. Thrombos. Diathes. haemorrh. (Stuttg.) 10: 455 1964;
• 40 Ware A.G, Seegers W. H. Serum Ac-globulin: Formation from plasma Ac-globulin. Amer. J. Physiol. 152: 567 1948;
• 41 Williams W. J. The activity of Lung Microsomes in Blood Coagulation. J. biol. Chem. 239: 933 1964;