Tissue extract thromboplastin: Quantitation, fractionation and characterization of protein components

Abstract

Bovine brain and lung tissue thromboplastins were extracted with absolute ethanol into an ethanol soluble lipid fraction and an insoluble protein fraction. The lipid fraction functioned as a partial thromboplastin, while the protein fraction was devoid of activity. The protein fraction was further purified by deoxycholate extraction and column chromatography on Bio-Gel A-1.5 m in the presence of deoxycholate. Through this procedure, residual lipids in the protein fraction were removed. The sedimentation coefficient in the presence of 0.1% SDS was 2.9. The molecular weight as determined by SDS gel electrophoresis was 56,000. Protein aggregates with molecular weights greater than 1.5 million occurred in the absence of the detergents or bile salts. The purified protein, after addition of phospholipid, regained clotting activity. Recombination of the protein and the lipid fractions was achieved in ethanol. Recombination was also achieved in imidazole buffered saline at pH 7.2 in the presence of 0.025% deoxycholate. The recombined material had full thromboplastic activity. The protein components isolated from brain and lung tissue had similar biochemical and biological characteristics. Combining lung lipids with brain protein and brain lipids with lung protein gave essentially the same results observed when the recombined protein and lipids were derived from the same tissue source.