Human Coumarin Prothrombin

Nicole Cesbron; Catherine Boyer; Marie-Claude Guillin; Doris Ménaché

doi:10.1055/s-0038-1649122

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1973; 30(03): 437-450
DOI: 10.1055/s-0038-1649122

Original Article

Schattauer GmbH

Human Coumarin Prothrombin

Chromatographic, Coagulation and Immunologic Studies

Nicole Cesbron

¹Service Central d’ Immunologie et Hématologie, Hôpital Beaujon, 92/Clichy and CHU Xavier Bichat, Paris, France

,

Catherine Boyer

¹Service Central d’ Immunologie et Hématologie, Hôpital Beaujon, 92/Clichy and CHU Xavier Bichat, Paris, France

,

Marie-Claude Guillin

¹Service Central d’ Immunologie et Hématologie, Hôpital Beaujon, 92/Clichy and CHU Xavier Bichat, Paris, France

,

Doris Ménaché

¹Service Central d’ Immunologie et Hématologie, Hôpital Beaujon, 92/Clichy and CHU Xavier Bichat, Paris, France

› Author Affiliations

Abstract

Summary

The abnormal prothrombin produced in patients under coumarin treatment has been isolated from oxalated plasma. After removal of normal prothrombin by adsorption on to small amounts of barium sulfate, coumarin prothrombin proved to be adsorbable on to larger amounts of barium sulfate. After elution into citrate the coumarin prothrombin was precipitated with ethanol and chromatographed on DEAE cellulose. Using a similar linear gradient than for normal prothrombin, the coumarin prothrombin was eluted ahead of normal prothrombin. Immunological studies performed on chromatographed coumarin prothrombin showed that it shared with normal prothrombin the main antigenic determinants and a complete line of identity was obtained when tested against antihuman normal prothrombin antiserum. On crossed Immunoelectrophoresis the electrophoretic mobility of coumarin prothrombin, unlike normal prothrombin, was not modified by the addition of calcium ions. Coagulation studies revealed that coumarin prothrombin could not be activated by physiological activators whereas conversion to thrombin was possible by non physiological activators such as staphylocoagulase and Echis carinatus venom. The amounts of thrombin generated under staphylocoagulase activation per 100 antigen units were similar to those generated by normal prothrombin. The amounts of thrombin obtained under the action of the venom were less than from normal prothrombin. In addition, the activation of coumarin prothrombin proved to be slower than for normal prothrombin.

Full Text

References

References
1 Aronson D. L, and Menache D. 1966; Chromatographic analysis of the activation of human prothrombin with human thrombokinase. Biochemistry 5: 2635.
2 Astrup T, and Darling S. 1943; Measurement and properties of antithrombin. Acta Physio-logica Scandinavica 4: 293.
3 Ganrot P. O, and Niléhn J. E. 1968; Plasma prothrombin during treatment with dicoumarol. II. Demonstration of an abnormal prothrombin fraction. The Scandinavian Journal of Clinical and Laboratory Investigation 22: 23.
4 Hemker H. C, Veltkamp J. J, Hensen A, and Loeliger E. A. 1963; Nature of prothrombin biosynthesis : preprothrombinaemia in vitamin K-deficiency. Nature 200: 589.
5 Hemker H. C, Veltkamp J. J, and Loeliger E. A. 1968; Kinetic aspects of the interaction of blood clotting enzymes. III. Demonstration of an inhibitor of prothrombin conversion in vitamin K deficiency. Thrombosis et Diathesis Haemorrhagica 19: 346.
6 Josso F, Lavergne J. M, Gouault M, Prou-Wartelle O, and Soulier J. P. 1968; Différents états moléculaires du facteur II (prothrombine). Leur étude a l’aide de la staphylocoagulase et d’anticorps antifacteur II. I. Le facteur II chez les sujets traités par les antagonistes de la vitamine K. Thrombosis et Diathesis Haemorrhagica 20: 88.
7 Josso F, Lavergne J. M, and Soulier J. P. 1970; Les dysprothrombinémies constitutionnelles et acquises. Nouvelle Revue Française d’Hématologie 10: 633.
8 Josso F, Lavergne J. M, Weilland C, and Soulier J.P. 1967; Etude immunologique de la prothrombine et de la thrombine humaines. Thrombosis et Diathesis Haemorrhagica 18: 312.
9 Laurell C. B. 1965; Antigen-antibody crossed electrophoresis. Analytical Biochemistry 10: 358.
10 Laurell C. B. 1966; Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analytical Biochemistry 15: 45.
11 Losito R. 1965; Investigations into the presence of a competitive inhibitor (preprothrombin) in the plasma of chicks. Acta Chemica Scandinavica 19: 2229.
12 Malhotra O. P. 1972; a Atypical prothrombin in purified preparations from dicoumarol-treated steers. Life Sciences 11: 901.
13 Malhotra O. P. 1972; b Atypical prothrombins induced by dicoumarol. Nature New Biology 239: 59.
14 Menache D. 1971. Hémostase. In: Hartmann L. (ed.) Techniques Modernes de Laboratoire et Explorations Fonctionnelles. L’Expansion Scientifique Française; Paris: 683.
15 Nelsestuen G. L, and Suttie J. W. 1972; a The purification and properties of an abnormal prothrombin protein produced by dicoumarol-treated cows. The Journal of Biological Chemistry 247: 8176.
16 Nelsestuen G. L, and Suttie J. W. 1972; b Mode of action of vitamin K. Calcium binding properties of bovine prothrombin. Biochemistry 11: 4961.
17 Niléhn J. E, and Ganrot P. O. 1968; Plasma prothrombin during treatment with dicoumarol. I. Immunochemical determination of its concentration in plasma. The Scandinavian Journal of Clinical and Laboratory Investigation 22: 17.
18 Ouchterlony Ö. 1962. Diffusion in gel methods for immunological analysis. In: Kallos P, and Waksman B. H. (ed.) Progress in Allergy. Karger; Basel: vol. 6 30.
19 Ranhotra G. S, and Johnson B. C. 1970; Vitamin K and prothrombin formation. Life Sciences 9: 79.
20 Shah D. V, and Suttie J. W. 1971; Mechanism of action of vitamin K: evidence for the conversion of a precursor protein to prothrombin in the rat. Proceedings of National Academy of Sciences, USA 68: 1653.
21 Soulier J. P, and Larrieu M. J. 1952; Etude analytique des temps de Quick allongés. Dosage de prothrombine, de proconvertine et de proaccélérine. Le Sang 23: 549.
22 Soulier J. P, and Larrieu M. J. 1953; Nouvelle méthode de diagnostic de l’hémophilie. Dosage des facteurs antihémophiliques A et B. Le Sang 24: 3.
23 Soulier J. P, and Prou-Wartelle O. 1966; Etude comparative des taux de cofacteur de la staphylocoagulase (CR.F.) et des taux de facteur II (Prothrombine) dans diverses conditions. Nouvelle Revue Française d’Hématologie 6: 623.
24 Soulier J. P, and Prou-Wartelle O. 1970; Dosage de l’activité “prothrombine-coagulase”. Nouvelle Revue Française d’Hématologie 10: 41.
25 Stenflo J. 1970; Dicoumarol-induced prothrombin in bovine plasma. Acta Chemica Scandinavica 24: 3762.
26 Stenflo J. 1972; Vitamin K and the biosynthesis of prothrombin. II. Structural comparison of normal and dicoumarol-induced bovine prothrombin. The Journal of Biological Chemistry 247: 8167.
27 Stenflo J, and Ganrot P. O. 1972; Vitamin K and the biosynthesis of prothrombin. I. Identification and purification of a dicoumarol-induced abnormal prothrombin from bovine plasma. The Journal of Biological Chemistry 247: 8160.
28 Stenflo J, and Ganrot P. O. 1973; Binding of Ca²⁺ to normal and dicoumarol-induced prothrombin. Biochemical and Biophysical Research Communications 50: 98.
29 Ware A. G, and Seegers W. H. 1949; Two stage procedure for the quantitative determination of prothrombin concentration. American Journal of Clinical Pathology 19: 741.
30 Weilland C, and Soulier J. P. 1959; Purification de la prothrombine par la bentonite. I. Préparation de la prothrombine purifiée. Pathologie-Biologie 7: 2531.