The Conversion of Bovine Abnormal Prothrombin into Prothrombin

In order to characterize the enzyme system which converts abnormal prothrombin (PIVKA-II) in prothrombin we prepared liver cell-free extracts from normal healthy cows. The enzyme system devoid of either precursor or prothrombin was isolated by differential centrifugation and partly purified with the aid of column chromatography.The carboxylation of purified PIVKA-II to prothrombin was detected by means of the one-stage coagulation assay and with 14CO2 incorporation. The reaction was dependent upon microsomal supernatant, vitamin K, ATP, Mg++ ions, O2, pH and temperature. Inhibitors were: warfarin, 2.4-dinitrophenol and factor II. From our experiments we concluded that PIVKA-II can act as a precursor of prothrombin and that PIVKA-II can be converted into the normal clotting factor in an in vitro cell-free system. In contrast to the systems published by others, here the converting enzymes are added to separately purified precursor proteins, so the system provides a way to study the nature of this reaction in more detail.