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.

Complete nucleotide sequence, origin of isoform and functional characterization of the mouse hepsin gene

“…HepG2 cells, a human hepatoma cell line [17–19], were used for transient expression assay as described previously [20]. Cells were grown and maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics (penicillin and streptomycin) at 37 °C under 5% CO 2 in a humidified incubator.…”

Deletion analysis of a unique 3' splice site indicates that alternating guanine and thymine residues represent an efficient splicing signal

“…Hep-G2 [32][33][34][35] cells were grown at 37°C under 5% CO2 in MEM medium supplemented with 10% FCS and 2 mM glutamine. DMEM medium supplemented with 2 mM glutamine and 10% or 20% FCS was used to grow HeLa [36] cells.…”

Lymphoid procoagulant response to bacterial endotoxin in the rat

“…Cells cultivated with warfarin as well as those cultured with vitamin K expressed lower basal PCA than untreated cells. There was no evidence of decreased viability under these conditions previously utilized for analyses of cellular biosynthesis of the gamma-carboxylated coagulation proteases (5). On stimulation with LPS, however, the drug-treated cells responded equally well as untreated cells, giving stimulation indices of about 2.…”

“…Pharmacological studies. Under conditions previously used to analyze biosynthesis (5) At this pH the half-life of p-APMSF is 6 min. After incubation the samples were tested for PCA.…”