Prothrombin is a vitamin K-dependent protein that is synthesized in the liver. Due to the central role that prothrombin plays in coagulation and fibrinolysis (through proteins C and S) a thorough understanding of how the gene for prothrombin is regulated is of interest. Although the functions of prothrombin and the other vitamin K-dependent coagulation factors have been well characterized, little is known about the regulation of their expression or the molecular mechanisms which limit their synthesis primarily to hepatocytes. The study of the regulation of expression of prothrombin, the most abundant of these evolutionarily- related factors, may provide insight into the molecular mechanisms regulating tissue-specific expression of this family of genes. The role that vitamin K plays in the regulation process is not completely established, and little is known about hormonal regulation of prothrombin synthesis. The experiments proposed in this application are an attempt to further understand the process of regulation of prothrombin synthesis and hopefully can be extrapolated to the regulation of other coagulation proteins. Ultimately, a better understanding of how genes for coagulation proteins are regulated may lead to new treatments for diseases or conditions where abnormal bleeding or clotting occurs. We have identified four regions in the 5' flanking region of the human prothrombin gene to be potentially important for its regulation and expression in a liver cell line. A unique feature is the presence of several repressor sequences, one of which appears to be responsible for suppression of prothrombin expression in HeLa cells and possibly other non-liver cells. The regulatory regions will be further characterized to determine whether the repressor regions can be activated by titration of nuclear proteins that bind to these regions and to correlate the repressor phenotype in HeLa cells with tissue-specific extinction observed in somatic cell hybrids. Enhancer and repressor regions identified to be functional in HepG2 cells will be further characterized in primary rat hepatocytes. The protein binding ability of these four regions and the exact sequences responsible for binding will be determined. Competition experiments with well characterized protein-binding sequences will determine whether these regions bind previously identified nuclear factors or as yet unidentified protein(s). Mutagenesis of protein binding regions will determine the exact sequences required for binding. Any newly identified nuclear binding protein will be further characterized by isolation of cDNA clones from expression libraries using the ligand-based screening method. A trans-activation or repression assay will be used to test the function of a cDNA purported to code for a regulatory protein.
