Endogenous forms of fibrinogen in hep g2 cells

• Published in: Thrombosis research Vol. 46; no. 2; pp. 281 - 293
• Main Authors: Yu, Sharon, Kong, Victoria, Kudryk, Bohdan, Redman, Colvin, Kimball, Lindsley F.
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Ltd 15.04.1987; Elsevier Science
• Subjects: Biological and medical sciences; Blood coagulation. Blood cells; Carcinoma, Hepatocellular - metabolism; Cell Line; Coagulation factors; disulfide interactions and biosynthesis; Fibrinogen - metabolism; Fibrinogen pools; Fundamental and applied biological sciences. Psychology; Humans; Iodoacetamide - metabolism; isoforms; Liver Neoplasms - metabolism; Molecular and cellular biology; precursors; Protein Conformation; Protein Precursors - metabolism; Fibrinogen pools; isoforms; disulfide interactions and biosynthesis; precursors; Cell culture; Molecular form; Glycoproteins; Coagulation factor; Thiohydroxyl group; Fibrinogen
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/0049-3848(87)90290-8

The three polypeptide chains of fibrinogen, Aα, Bβ and γ 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 Aα and gg 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 Aα-γ complex and,to a smaller degree, of fully-formed fibrinogen (13–33%). Furthermore,the Hep G2 cell also contains endogenous pools of free γ chain (11–26%). Other fibrinogen precursors (namely, the Bβ-Aα, Bβ-γ 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 heterogeneiety 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 Aα-γ complex and the free γ chains.