A New Method for the Preparation of Thrombin

Abstract

In the preparation of thrombin (or prothrombin) 4 different methods have been followed. The method of Schmidt 1 starts with fresh serum which is precipitated by a large excess of alcohol, the precipitate subsequently being extracted with water or saline solution. The method of Buchanan as modified subsequently by Gamgee 2 and by Howell 3 starts with the fibrin of the clot from which the thrombin is extracted by a strong saline solution and purified by acetone precipitation. The method of Mellanby, 4 as modified by Bleibtreu, 5 and by Mellanby, 6 starts with oxalated plasma, from which, after strong dilution, the fibrinogen and prothrombin are precipitated together by the addition of weak acid. The separation of the prothrombin from the fibrinogen is effected in different ways. The method of Bordet 7 also starts with oxalated plasma from which the prothrombin (serozyme) is precipitated by adsorption upon a suspension of tricalcium phosphate, or, as suggested by Fuchs, by adsorption upon magnesium hydroxide. It is well known that the original method of Schmidt usually gives a feeble preparation of thrombin which is not wholly satisfactory for purposes of experiment or demonstration. The methods described by Howell, Mellanby, Bleibtreu and Bordet, on the other hand, give, under favorable conditions, very potent preparations which cause rapid and strong coagulation of oxalated plasma or fibrinogen solutions. Experience in this laboratory has shown that the method of Howell does not always give a satisfactory product. With some specimens of fibrin, the yield is excellent, while with others it is poor. The principal object of the work here reported was to restudy and improve this method so as to obtain a more uniform preparation of thrombin and if possible a preparation free from admixture with impurities. The attempt to improve the method has proved to be a more difficult task than was anticipated. After many unsuccessful attempts, modifications have been developed which it is believed, are actual improvements and insure a good yield of very active thrombin in a state of considerable purity.