The human plasminogen activator inhibitor-1 gene.

Abstract

This thesis focuses on the human gene for plasminogen activator inhibitor-1 (PAI-1). The generation of the active serine protease plasmin plays an important role in many biological processes including fibrinolysis, cell migration, tumor metastasis, tissue remodeling, and ovulation. Plasmin is first synthesized as the proenzyme plasminogen, which is activated by either tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). PAI-1 inhibits this activation via its interaction with both uPA and tPA. The first portion of the thesis describes the isolation and characterization of the structural gene for human PAI-1. This gene was found to span approximately 12 kb and contains eight introns. The start point of transcription was localized to 142 nucleotides upstream from the start codon. A segment containing 730 nucleotides of 5$\sp\prime$ upstream region was shown to function as a promoter in COS cells. The second section of the dissertation is a detailed comparison of PAI-1 gene structure with other members of the serine protease inhibitor (serpin) gene family. Although the locations of the human and rat PAI-1 introns are identical, little conservation of intron positions was observed when compared to other serpins. It is proposed that intron location among gene family members may be a better predictor of relatedness than amino acid sequence. Finally, a novel method for quantitating RNA from small numbers of cells using the polymerase chain reaction (PCR) was developed in order to examine the regulation and expression of PAI-1. Using this procedure the previously observed decrease of PAI-1 mRNA levels in human umbilical vein endothelial cells (HUVEC) in the presence of ECGF and heparin was confirmed. An estimate for PAI-1 mRNA abundance of approximately 1700 molecules per cell was obtained for 6th passage HUVEC. Comparing PAI-1 mRNA levels in the nucleus with those present in the cytoplasm suggests that the effect of ECGF/heparin on mRNA level may occur at the nuclear rather than post-nuclear level. This approach may prove to be of general utility for the study of gene expression in a number of other instances.