Endogenous forms of fibrinogen in Hep G2 cells

The three polypeptide chains of fibrinogen, A alpha, B beta and gamma chain, are synthesized on separate polysomes. Fully formed fibrinogen is a six chain, disulfide-linked, dimeric molecule with a molecular weight of 340kDa. Previous pulse-chase studies with L-35 S methionine using the human hepatocellular carcinoma cell line, Hep-G2, showed that the three chains are not immediately disulfide-linked and that there exist intermediate precursors as well as pools of A alpha and gamma chains (J. Biol. Chem. 259, 10574-10581, 1984). In this study the endogenous levels of fibrinogen and its precursors are measured by two different methods; pulse and steady-state labelling with L-35 S-methionine and immunoblotting. In Hep-G2 cells intracellular fibrinogen-related antigen is primarily (30-53%) composed of an A alpha-gamma complex and, to a smaller degree, of fully-formed fibrinogen (13-33%). Furthermore, the Hep G2 cell also contains endogenous pools of free gamma chain (11-26%). Other fibrinogen precursors (namely, the B beta-A alpha, B beta-gamma complexes as well as the fibrinogen half-molecule) do not appear to accumulate intracellularly. Most, if not all, of these precursors occur as isoforms but this heterogeneity is not due to varying degrees of glycosylation. In all the intracellular fibrinogen forms identified thus far, free sulfhydryl groups, detected by 14C-iodoacetamide incorporation, occur only in the A alpha-gamma complex and the free gamma chains.