REGULATION OF PLASMA PROTEIN BIOSYNTHESIS

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Fibrinogen and plasminogen are two hepatically derived plasma protein that play critical but opposite roles in hemostasis. Activated fibrinogen (fibrin) is clot forming and activated plasminogen (plasmin) is clot dissolving. Even though much is known about the chemistry of these two proteins little is known about how their synthesis is regulated or how the polypeptides are assembled and processed into functional proteins. The long range goal of this proposal is to elucidate at the cellular and molecular level these regulatory processes. Fibrinogen is a plasma protein whose synthesis is greatly increased during an inflammatory reaction. We have isolated a factor from monocytes that causes hepatocytes in culture to greatly increase their synthesis of fibrinogen thus mimicing the acute-phase response in culture. This cell culture will be used as a model system to investigate the intracellular events which leads to increased transcription of the fibrinogen mRNA's. We will use well characterized highly specific cDNA's as probes to determine both the kinetics of transcription of the fibrinogen mRNA's and the mRNA half-lives. We will also use the hepatocyte culture and stimulating factor to investigate what intracellular signals are operant that leads to the increased transcription of fibrinogen. We will attempt to determine exactly how the six polypeptides of fibrinogen are assembled into a functional protein. These experiments will be carried out by translating fibrinogen mRNA's in vitro and using very specific (monoclonal) antibodies as probes to determine the way the subunits link together. Elucidating the molecular origin of the two forms of plasminogen is one of the goals of this proposal. Plasminogen mRNA will be isolated and used in an in vitro translation system and the products analyzed to determine if two different translational forms of plasminogen exist. These results should give information on whether two mRNA's code for the two forms. We will use the purified mRNA to construct cDNA clones. Selected clones will be isolated and characterized as a first step to understanding the plasminogen gene structure. It is anticipated that information derived from these studies will significantly increase our understanding of how these important molecules are regulated. Once the control systems are fully understood then there is a possibility of being able to regulate their synthesis which can be of enormous therapeutic value in vascular diseases.