The activation of factor X by hepatocyte plasma membranes.

Synthesis and secretion of blood coagulation factor X was studied during incubations of hepatocytes prepared by perfusion of rat livers with collagenase. The apparent molecular weight of factor X isolated from the incubation medium was about 14,000 less than factor X isolated from rat plasma. The extracellular form of factor X was a two-chain polypeptide and the observed difference in molecular weight was reflected in the heavy chain. Since these properties were more characteristic of factor Xa than factor X, experiments were designed to determine if factor X activation occurred during the incubations. Clotting factor assays indicated that factor X secreted by hepatocytes was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes the added factor X was converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium-dependent reaction. The results suggest that the activity is due to the presence of thromboplastin (tissue factor) and factor VII in the membrane preparations.

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 BaSO4 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 (Mr 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 (Mr 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 Mr 31,000. Walker 256 tumor cells bound about 30% of the 14C. The bound material, after reduction, gave a 14C-labeled polypeptide with Mr 23,000.

Vitamin K requirement and the concentration of vitamin K in rat liver.

The concentration of vitamin K was determined in the liver of different strains of rats, and in male and female warfarin-resistant rats by feeding 3H-vitamin K in a purified diet. In each case, the level of vitamin K in the liver correlated approximately with the amount of vitamin K fed. The results indicate that differences in the requirement for vitamin K between the sexes and between strains of rats are due principally to different required concentrations of vitamin K in liver and not to differences in absorption or turnover of the vitamin. The results of the determination of vitamin K epoxide levels in male and female warfarin-resistant rats, and other data, suggest that the amount of vitamin K required in liver may be in part due to differences in the activity of the enzyme, vitamin K epoxide reductase.

Metabolism and biological activity of cis- and trans-phylloquinone in the rat.

The separation of sufficient cis and trans forms of vitamin K for feeding and metabolic studies was accomplished on silica gel columns eluted with solvent containing n-butyl ether. The lack of biological activity of the cis isomer of phylloquinone was observed. The cis isomer was retained longer in liver, particularly in mitochondria, but had low retention in that portion of the endoplasmic reticulum isolated as the rough membrane fraction. The cis isomer of phylloquinone was a poor substrate for 2,3-epoxidation in vivo and in vitro. These data are consistent with the hypothesis that epoxidation of vitamin K is coupled to the biological activity of the vitamin, and that microsomes are the site of metabolism and function of vitamin K.

Changes in phylloquinone epoxidase activity related to prothrombin synthesis and microsomal clotting activity in the rat.

The oxidation of phylloquinone to the 2,3-epoxide (by phylloquinone epoxidase) was studied in liver from control and warfarin-resistant rats. The reaction requires microsomal fraction, soluble protein, a heat-stable soluble factor and O(2). It is not inhibited by CO or CN(-). Epoxidase activity was stimulated if plasma prothrombin was lowered either by anticoagulants or the absence of vitamin K. The activity of the enzyme rapidly returned to normal values after the administration of vitamin K to hypoprothrombinaemic rats. These differences in the activity of the enzyme occur in the microsomal fraction and not the cytosol. A thrombin-generating polypeptide that accumulates in microsomal fraction of hypothrombinaemic rats correlated directly with epoxidase activity. These data support the view that enzymic interconversion of phylloquinone and its 2,3-epoxide participates in the biological activity of vitamin K.