Purification and some properties of the protein component of tissue thromboplastin from human brain

Abstract

The protein component of tissue thromboplastin (Factor III) from human brain was purified by extraction of a microsomal fraction with sodium deoxycholate, gel filtration of the extract on Sephadex G-100 and preparative polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. The product, apoprotein III, was homogeneous by analytical polyacrylamide-gel electrophoresis, and it induced monospecific antibodies in rabbits and goat as shown by immunodiffusion and immunoelectrophoresis. Amino acid- and carbohydrate-analysis data for apoprotein III are presented. The carbohydrate moiety of the protein consists of fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminate, amounting to a total content of 6.3 g/100 g. The apoprotein alone had no procoagulant activity. When Factor III was reconstituted by combining the pure apoprotein with a purified lipid fraction from the deoxycholate extract of crude Factor III, a high and optimal procoagulant activity was obtained at a phospholipid/protein ratio of 1.1 g/g. Phosphatidylethanolamine alone had a weak but significant ability to restore activity, but phosphatidylcholine and phosphatidylserine separately had almost none. Two-component mixtures were on average more effective and 3-component mixtures far more effective, than the single phospholipids. The inclusion of a small amount of phosphatidylserine was very important for high activity.