HUMAN FIBRINOGEN: MECHANISMS OF ASSEMBLY

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Human fibriogen is a plasma glycoprotein which plays major roles in hemostasis and thrombotic disorders. It is a dimer with each half-molecule composed of three non-identical chains (Ayield, BBeta and Gamma). The half-molecules of the dimer are held together by symmetrical disulfide bonds between two Ayield chains and two Gamma chains. In addition, fibrinogen contains a number of inter- and interchain disulfide linkages. It is our aim to elucidate how this multichain protein is assembled and secreted and to understand how the process is regulated. The three chains are synthesized by separate mRNA's and are assembled into dimeric fibrinogen in the rough endoplasmic reticulum. A pool of Gamma chains and of an Ayield-Gamma complex have been detected in hepatocytes and fibrinogen assembly is thought to begin by the independent attachment of preformed Ayield and Gamma chains to nascent, polysome-bound BBeta chains. A number of precursor intermediate forms have been identified including BBeta-Ayield, BBeta-Gamma, and the half-molecule (Ayield-BBeta-Gamma). To study how these intermediates are formed and to learn the steps which lead to the formation of functional fibrinogen we will: 1) Isolate fibrinogen chains and measure interchain disulfide bond formation under various in vitro conditions in the presence of protein disulfide isomerase. In addition separated fibrinogen chains will be introduced into hepatic and non-hepatic microsomes and interchain interactions will be measured. 2) mRNA's for each of the chains will be incubated with a reconstituted microsomal system which contains endogenous protein disulfide isomerase and is capable of protein translation, translocation and processing. At various times the fibrinogen procursors will be isolated, characterized and compared to those obtained previously from intact hepatocytes. 3) To study intracellular transport and secretion of fibrinogen and other fibrinogen-related precursors, surrogate secretory cells will be transfected with genes for each of the chains. The various intracellular forms, their transport and eventual fate will be determined. 4) In the final phase of the study, the fibrinogen will be modified at critical points involved in the junction of the half-molecules (cysteines at Gamma8, Gamma9 and Ayield 28) by site specific mutagenesis of the corresponding cDNA's. These genes will be introduced into host secretory cells and the assembly and secretion of modified fibrinogen will be determined.