title: Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain.
abstract: To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa. The one-chain mt-PA-6 produced by cultured Syrian hamster cells was purified in high yield by affinity and size exclusion chromatography. The purified mt-PA-6 displayed the same two types of microheterogeneity observed for t-PA. NH2-terminal amino acid sequencing demonstrated that one-chain mt-PA-6 existed in both a GAR and a des-GAR form. Purified mt-PA-6 also existed in two glycosylation forms that accounted for the 40- and 42-kDa doublet of activity produced by the cultured Syrian hamster cells. Separation of these two forms by hydrophobic interaction chromatography and subsequent tryptic peptide mapping demonstrated that both forms contained N-linked glycosylation at Asn448; in addition, some mt-PA-6 molecules were also glycosylated at Asn184. Plasmin treatment of one-chain mt-PA-6 converted it to a two-chain molecule by cleavage of the Arg275-Ile276 bond. This two-chain mt-PA-6, like t-PA, had increased amidolytic activity. The fibrinolytic specific activities of the one- and two-chain forms of mt-PA-6 were similar and twice that of t-PA. The plasminogen activator activity of one-chain mt-PA-6 was enhanced greater than 80-fold by CNBr fragments of fibrinogen, and the one-chain enzyme lysed human clots in vitro in a dose-dependent manner. The ability to produce and purify a structurally simple plasminogen activator with desirable fibrinolytic properties may aid in the development of a superior thrombolytic agent for the treatment of acute myocardial infarction.
authors.affiliation: Department of Molecular Genetics Research, Eli Lilly and Company, Indianapolis, Indiana 46285.
authors.forename: P J
authors.initials: PJ
authors.lastname: Burck
authors.forename: D H
authors.initials: DH
authors.lastname: Berg
authors.forename: M W
authors.initials: MW
authors.lastname: Warrick
authors.forename: D T
authors.initials: DT
authors.lastname: Berg
authors.forename: J D
authors.initials: JD
authors.lastname: Walls
authors.forename: S R
authors.initials: SR
authors.lastname: Jaskunas
authors.forename: R M
authors.initials: RM
authors.lastname: Crisel
authors.forename: B
authors.initials: B
authors.lastname: Weigel
authors.forename: C J
authors.initials: CJ
authors.lastname: Vlahos
authors.forename: D B
authors.initials: DB
authors.lastname: McClure
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D005091:Exons; D005784:Gene Amplification; D006801:Humans; D007438:Introns; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011487:Protein Conformation; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
chemical_list: D046911:Macromolecular Substances; D010959:Tissue Plasminogen Activator