HUMAN FIBRINOGEN: MECHANISM OF ASSEMBLY

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Human fibrinogen is a plasma glycoprotein which plays major roles in hemostasis and thrombotic disorders and has implied roles in a number of other biological processes such as cell adhesion and aggregation, promoter of cell growth, angiogenesis, developmental processes and wound healing. Fibrinogen is a dimer with each half-molecule composed of 3 non-identical chains (A alpha, B beta, and tau). The half-molecules of the dimer are held together by symmetrical disulfide bonds between 2 A alpha and 2 tau chains. In addition fibrinogen contains a number of inter- and intrachain disulfide linkages. Our aim is to understand the mechanism by which this multi-chain protein is synthesized, assembled, processed and secreted. Our previous studies gave a detailed view of the sequential steps by which the individual chains are assembled and indicated that, in Hep G 2 cells, synthesis of the B beta chain is a rate-limiting factor in the production of fibrinogen. Transfection of Hep G2 cells with B beta cDNA caused increased production of fibrinogen by specifically elevating, not only B beta chain synthesis, but also the synthesis of the other two component chains. We now have the following specific aims 1) Determining how enhancement of the synthesis of one of the fibrinogen chains affects the expression of the other two chains. 2) To study interactions and transport of fibrinogen chains, we shall transfect heterologous secretory cells with individual cDNAs for each of the fibrinogen chains and with a combination of cDNAs for the A alpha, B beta and tau chains. The produce expressed will be characterized and their assembly, intracellular transport and secretion determined. 3) An in vitro system will be developed using specific mRNAs for each of the fibrinogen chains and a reconstituted microsomal system capable of translation and translocation of secretory proteins. The redox potential will be varied, by the addition of glutathione, and we will study in detail the early steps in fibrinogen chain interaction. 4) The role of resident endoplasmic reticulum proteins on fibrinogen chain assembly, sequestration and degradation will be determined. 5) Modify fibrinogen cDNAs by site specific mutagenesis to determine the effects on fibrinogen chain assembly and secretion.