This grant proposes to study the mechanism of induction of a biologically important inflammatory mediator, a macrophage/monocyte procoagulant (a prothrombinase). This molecule is an important mediator in the pathogenesis of hepatic lesions produced during mouse hepatitis virus (MHV) infection. cDNAs representing the MHV-induced procoagulant gene will be synthesized, cloned, and sequenced. These cDNA clones will be used to directly determine if up-regulation of the procoagulant gene occurs at the level of transcription. The possibilities of translational up-regulation and post-translational activation of a procoagulant precursor protein will also be investigated. Genomic clones of the MHV-induced procoagulant gene will be isolated. These will be and characterized as to their organization by Southern blot hybridization and S1 nuclease mapping as well as sequenced. Sequences upstream from the procoagulant coding sequences will be characterized as to their ability to function as promoter element for this gene. A deletion analysis of the promoter region will be performed to determine the contribution different sequence motifs make to the procoagulant promoter, and as to their inducibility by MHV infection. DNA footprinting will further define cis-acting regulatory elements present in this gene. Studies to determine which MHV genes are important to the induction of procoagulant will be initiated. Molecular clones representing individual MHV genes will be expressed in macrophages and their effect on procoagulant activity determined. MHV gene products which bind to putative procoagulant regulatory elements will be sought through DNA binding assays.
