Fibrinogen is a plasma glycoprotein, produced by hepatocytes, that plays a central role in the final phases of blood coagulation. It is a symmetrical molecule composed of two sets of three non-identical polypeptides. The component polypeptide chains have molecular weights of 65,000 (A Alpha), 55,000 (B Beta) and 47,000 (Gamma). It contains four carbohydrate chains, one on each of the BBeta and Gamma chains. The two sets of identical half-molecules are held together by symmetrical bonds between two A Alpha and two Gamma chains. In addition, fibrinogen contains other inter- and intra-chain disulfide linkages. The complete molecule appears to form a trinodal structure linked by rope-like strands. Our objectives are to determine how the three component polypeptides of fibrinogen are assembled into a functional dimeric molecule and to understand how the production and secretion of fibrinogen is regulated. Pulse-chase experiments with a human hepatocellular carcinoma (Hep-G2) have shown that unequal synthesis of the 3 component chains of fibrinogen leads to intracellular pools of free Gamma chains and of an A Alpha-Gamma complex. Fibrinogen assembly occurs by sequential disulfide interactions between chains. The first step is the addition of preformed A Alpha and Gamma chains to nascent B Beta chains. A number of precursor forms of fibrinogen were observed in Hep-G2 cells. Our immediate aims are to characterize these precursors and to determine the sequence in which the disulfide linkages are formed. To understand the reasons for the unequal synthesis of the 3 chains we shall measure, using hybridization methods with cDNA for the individual chains, the levels of mRNA in the whole cells and in polysomes. Attempts will be made to stimulate fibrinogen production by Hep-G2 cells. The synthesis of the individual chains, pool sizes of free Gamma chains and of the A Alpha-Gamma complex, and the rate of assembly and secretion of fibrinogen in unstimulated and stimulated cells, will be determined. In addition, fibrinogen mRNA will be injected into Xenopus oocytes and the oocytes will be used as a model system to study the mechanisms of fibrinogen assembly.
| Yu, S; Kong, V; Kudryk, B et al. (1987) Endogenous forms of fibrinogen in Hep G2 cells. Thromb Res 46:281-93 |
| Liu, M C; Yu, S; Sy, J et al. (1985) Tyrosine sulfation of proteins from the human hepatoma cell line HepG2. Proc Natl Acad Sci U S A 82:7160-4 |
