HUMAN HEPATOMA-CELLS SECRETE SINGLE CHAIN FACTOR-X, PROTHROMBIN, AND ANTITHROMBIN-III

  • 1 January 1984
    • journal article
    • research article
    • Vol. 64  (1) , 194-204
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 1220 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. The hypothesis of a postsecretion proteolytic cleavage of factor X into the 2-chain form was supported. Prothrombin and antithrombin represented their plasma protein counterparts structurally, with MW 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 3 activities were neutralized by monospecific antibodies. Vitamin K increased the quantity of prothrombin secreted by 2-fold, without affecting the rate of secretion over a 5 day culture period, and had an apparent transient inhibitory effect on secretion of antithrombin III. Warfarin caused a 3- to 4-fold 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. The Hep G2 cell line may provide a useful model for assessing the regulation of biosynthesis and secretion of human coagulation proteins.