Structure and function of human tissue-type plasminogen activator (t-PA).
Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004727:Endothelium / Q000032:analysis; D005337:Fibrin / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010958:Plasminogen / Q000378:metabolism; D015849:Plasminogen Inactivators; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000502:physiology*
D006023:Glycoproteins; D015849:Plasminogen Inactivators; D005337:Fibrin; D010958:Plasminogen; D004247:DNA; D010959:Tissue Plasminogen Activator