Activation of Purified Prothrombin in Ammonium Sulfate Solutions: Purification of Autoprothrombin C

Abstract

SummaryFor the generation of autoprothrombin C activity from prothrombin preparations in concentrated ammonium sulfate solution the optimum conditions were found to be near 2 M at pH 7. In addition to thrombin and autoprothrombin C an inhibitor was obtained. Methods were developed to obtain the autoprothrombin C consistently with a specific activity of about 4,700 units per mg protein, and a yield of one mg per liter of bovine plasma. At an intermediate stage of purification the stability of autoprothrombin C was far better than in the final product which lost activity even by simple freezing and thawing. Preservation of activity in 50% glycerol solutions at pH 7.2 was found to be the most convenient procedure. The alterations produced in purified prothrombin with DEAE cellulose chromatography are discussed with respect to possible significance for thrombin and autoprothrombin C.

Differential Interaction of Clotting Factors II, VII, IX and X with Sephadex and Dextran Blue

“…These considerations are based upon the observations of Seegers [23] and Seegers el al. [24] that pro thrombin complex can undergo activation in 25 % sodium citrate in the absence or presence of traces of thrombin and that ammonium sulphate in high concentrations can have the same effect [25] as citrate.…”

VIII. Protein C and Autoprothrombin II-A

“…The main component of each fraction was eventually carried forward to purity. Activation of prothrombin complex with thrombin in 25% sodium citrate solution also made it possible to separate the inhibitor (Seegers, Heene and Marciniak 1966).…”

Inhibitor of Blood Clotting Derived from Prothrombin

“…In previous experiments on the conversion of purified bovine prothrombin to thrombin, the formation of an inhibitor of blood clotting was observed (1,2). Under suitable conditions it retarded the generation of thrombin from prothrombin.…”