Isolation of Antithrombin Globulin from Human Blood Plasma

George Y. Shinowara; Dimite J. Buckley

doi:10.1055/s-0038-1654485

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1960; 4(01): 017-030
DOI: 10.1055/s-0038-1654485

Originalarbeiten — Original Articles — Travaux Originaux

Schattauer GmbH

Isolation of Antithrombin Globulin from Human Blood Plasma^[*)]

George Y. Shinowara^**)

¹Department of Pathology, Ohio State University College of Medicine, Columbus, Ohio

,

Dimite J. Buckley^***)

¹Department of Pathology, Ohio State University College of Medicine, Columbus, Ohio

› Author Affiliations

Abstract

Summary

Fraction III + IV obtained by a low temperature — low ionic strength fractionation procedure was found to contain 97 percent of the antithrombin activity of whole plasma. The mean antithrombin activity in this fraction from 88 normal plasma specimens was 172.6 ± S. D. 19.8 units per ml. Further purification experiments resulted in fraction III representing 96 per cent of the antithrombin activity in fraction III + IV. There was no evidence for fibrinogen, prothrombin and antihemophilic globulin contamination in fraction III. Over 90 per cent of the plasma gamma globulins was present in this fraction.

The kinetics of the thrombin-antithrombin reaction were investigated. Under the specific conditions employed, the reaction was found to follow a first order course. The heat of activation was determined to be 11,300 calories. A standard antithrombin unit is defined. A quantitative test procedure which is accurate at both high and low concentrations of the circulating anticoagulant is described and its application on fractions, heat defibrinated plasma and serum is discussed.

Full Text

References

References
(1) Astrup T, Darling S. Measurement and properties of antithrombin. Acta physiol, scand. 4: 293 1942;
(2) Douglas A. S, Biggs R. The consumption of some components involved in physiological blood coagulation. Glasg. med. J. 34: 329 1953;
(3) Folin O, Ciocalteu V. On tyrosine and tryptophane determinations in proteins. J. biol. Chem. 73: 627 1927;
(4) Gerendás M. Inactivation of thrombin. Nature (Lond.) 157: 837 1946;
(5) Glazko A. J, Ferguson J. H. Kinetics of thrombin inactivation as influenced by physical conditions, trypsin and serum. J. gen. Physiol. 24: 169 1940; 1941
(6) Grüning W. The lipoid nature of antithrombin. Naturwissenschaften 31: 299 1943;
(7) Lyttleton J. W. The anti arombin activity of human plasma. Biochem. J. 58: 8 1954;
(8) Mihályi R. Observations on thrombin inactivation by human serum. J. gen. Physiol. 37: 139 1953;
(9) Monkhouse F. C, France E. S, Seegers W. H. Studies on the antithrombin and heparin cofactor activities of a fraction adsorbed from plasma by aluminum hydroxide. Circulation Res. 3: 397 1955;
(10) Morawitz P. Beiträge zur Kenntnis der Blutgerinnung. I. Mitteilung. Arch, klin. Med. 79: 215 432 1904;
(11) Quick A. J. The normal antithrombin of the blood and its relation to heparin. Amer. J. Physiol. 123: 712 1938;
(12) Rettger L. J. The coagulation of blood. Amer. J. Physiol. 24: 406 1909;
(13) Schmidt A. Zur Blutlehre. F. C. W. Vogel; Leipzig: 1892
(14) Shinowara G. Y. Enzyme studies on human blood. II. The substrate in the fibrinogen thrombin reaction. Proc. Soc. exp. Biol. (N. Y.) 68: 55 1948;
(15) Shinowara G. Y. Enzyme studies on human blood. V. Estimation of prothrombin by an homologous-isolation method. Amer. J. Physiol. 159: 303 1949;
(16) Shinowara G. Y. Thromboplastic cell component, the lipoprotein of erythrocytes and platelets. J. biol. Chem. 225: 63 1957;
(17) Shinowara G. Y. The isolation and characterization of antithrombin and gamma globulin in human blood plasma. Abstracts of Communications. XXth International Physiological Congress.. 826 Brussels: 1956
(18) Shinowara G. Y. Studies on the purification of thromboplastic plasma component (antihemophilic substance) in human blood. Amer. J. med. Sei. 223: 528 1957;
(19) Shinowara G. Y, Rosenfeld L. Enzyme studies on human blood. VIII. The effect of fibrinogen concentration on the thrombin clotting time. J. Lab. clin. Med. 37: 303 1951;
(20) Shinowara G. Y, Ruth M. E. An evaluation of a plasma fractionation system. Thromb. Diath. haem. 4: 31 1959;
(21) Stewart J. D, Rourke G. M. On the inactivation of thrombin by plasma protein. J. clin. Invest. 19: 695 1940;
(22) Storz H. Thrombin and antithrombin function of human serum. Klin. Wschr. 21: 627 1942;
(23) Volkert M. The antithrombin content of the blood and its relation to heparin. Biochem. Z. 314: 34 1943;
(24) Waugh D. F, Fitzgerald M. A. Quantitative aspects of antithrombin and heparin in plasma. Amer. J. Physiol. 184: 627 1956;
(25) Wöhlisch E, Grüning W. Antithrombin action of the serum proteins and their relation to metathrombin formation. Biochem. Z 305: 183 1940;
(26) Wöhlisch E, Köhler V. Serum proteins and their significance for clotting physiology. Naturwissenschaften 28: 550 1940;

Isolation of Antithrombin Globulin from Human Blood Plasma[*)]

Isolation of Antithrombin Globulin from Human Blood Plasma^[*)]