Functional characterization of single-chain factor X from rat liver

14C-Labeled single-chain factor X prepared by vitamin K-dependent carboxylation in vitro was partially purified by adsorption to BaSO 4 and chromatography on DEAE-Sephacel. Known activators of factor X were analyzed for their effect on the single-chain molecule. 14C-Labeled factor X antigens were recovered immunochemically from incubation mixtures and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation with trypsin resulted in the generation of factor Xa clotting activity, and the 14C-labeled product migrated after reduction with an apparent molecular weight of 22,500 ± 1500 (mean ± 1 SD). The light chain produced by factor Xa was similar to that produced by trypsin ( M r 24,500 ± 1500; mean ± 1 SD). Incubation of single-chain factor X with factor VII and thromboplastin, factor IXa, or the factor X activating enzyme from Russell's viper venom gave a reducible product with a light chain of higher apparent molecular weight ( M r 37,000–38,000). Incubation with factor VII and thromboplastin also resulted in the generation of factor Xa clotting activity. Incubation of single-chain factor X with platelets resulted in the binding of about 20% of the 14C. The bound 14C-labeled factor X antigen released by freezing and thawing in the presence of EDTA was reduced to give a 14C-labeled polypeptide with M r 31,000. Walker 256 tumor cells bound about 30% of the 14C. The bound material, after reduction, gave a 14C-labeled polypeptide with M r 23,000.