Fibrinolysis is regulated, in part, by a balance between tissue-type plasminogen activator (tPA) and its specific, rapidly acting inhibitor, plasminogen activator inhibitor (PAI-1). PAI-1 synthesis occurs in a number of tissues including the vascular endothelial cell and is regulated via a complex process involving multiple factors. Abnormal plasma levels of PAI-1 have been associated with a variety of thromboembolic disorders in man. The studies outlined in this proposal aim to determine the structure of the human PAI-1 gene, characterize the regulation of PAI-1 gene expression at the molecular level, investigate the relationship between PAI-1 structure and function, and begin to study the molecular genetic basis of hereditary abnormalities in PAI-1. In preliminary studies, full-length human PAI-1 cDNA has been isolated from a lambda gt11 cDNA library and its complete sequence determined. The human PAI-1 gene has been localized to chromosome 7. The PAI-1 sequence shows extensive homology to other members of the serine protease inhibitor (SERPIN) supergene family. Preliminary studies of PAI-1 gene regulation show PAI-1 mRNA levels to be decreased approximately 10-fold in the presence of heparin and endothelial cell growth factor. If this heparin affect also occurs in vivo, this could add significantly to heparin's clinical affect by increasing tPA activity with a subsequent increase in fibrinolysis. In this proposal, these observations will be further characterized at the molecular level by quantitative mRNA studies and introduction of recombinant vectors to assess the role of specific regulatory sequences. The detailed structure of the human PAI-1 gene will be determined by the study of PAI-1 specific clones isolated from a recombinant human lambda phage genomic DNA library. Comparison of the deduced exon/intron structure and promotor sequences to those of other genes, particularly the SERPIN gene family, may have major implications for coordinate control of related genes and the evolution of gene families. One PAI-1 gene restriction length polymorphism (RFLP) has been identified in preliminary studies and additional ones will be sought. These tools can eventually be applied to genetic linkage analysis in families with hereditary thromboembolic disease.
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