title: Human hepatoma cells secrete single chain factor X, prothrombin, and antithrombin III.
abstract: The human hepatoma cell line, Hep G2, was analyzed for the ability to synthesize and secrete several coagulation proteins. Using specific radioimmunoassays, factor X, prothrombin, and antithrombin III were present in 8-day culture supernatants at 62, 405, and 1,220 ng/mL, respectively. Factor IX was not detected, either in supernatants or in cell extracts. Intrinsically labeled factor X was secreted as a single-chain polypeptide of 66,000 daltons, as measured by sodium dodecylsulfate-polyacrylamide gels under nonreduced and reduced conditions. Immunoblots of Hep G2 supernatants and normal human plasma also indicate the presence of single-chain factor X. These findings support the hypothesis of a postsecretion proteolytic cleavage of factor X into the two-chain form. Prothrombin and antithrombin represented their plasma protein counterparts structurally, with molecular weights of 73,000 and 61,000, respectively. Secreted factor X, prothrombin, and antithrombin III were biologically active, as determined in coagulation or chromogenic assays, and all three activities were neutralized by monospecific antibodies. Vitamin K increased the quantity of prothrombin secreted by twofold, without affecting the rate of secretion over a five-day culture period, and had an apparent transient inhibitory effect on secretion of antithrombin III. Warfarin caused a three to fourfold decrease in the rate and quantity of secreted prothrombin, but did not affect intracellular concentrations. The intracellular and extracellular concentrations and rate of secretion of antithrombin III were not modulated by warfarin. These data suggest that the Hep G2 cell line may provide a useful model for assessing the regulation of biosynthesis and secretion of human coagulation proteins.
mesh_terms: D000990:Antithrombin III / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002460:Cell Line; D002863:Chromogenic Compounds; D005170:Factor X / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms; D011516:Prothrombin / Q000378:metabolism*; D014020:Tissue Extracts / Q000032:analysis; D014812:Vitamin K / Q000494:pharmacology; D014859:Warfarin / Q000494:pharmacology
chemical_list: D002863:Chromogenic Compounds; D014020:Tissue Extracts; D014812:Vitamin K; D014859:Warfarin; D000990:Antithrombin III; D011516:Prothrombin; D005170:Factor X