title: Characterization of human tissue-type plasminogen activator variants with amino acid mutations in the kringle 1 domain.
abstract: Recombinant variants of tissue-type plasminogen activator (t-PA) were constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Five variants were designed to improve the function of t-PA by mutagenesis in the kringle 1 (K1) domain. The amino acids were replaced with the corresponding residues present in the kringle 2 (K2) domain of native t-PA. The t-PA mutants expressed were as follows: variant E94V.D95G with point mutations in Glu94----Val and Asp95----Gly; variant N115P.S119M, Asn115----Pro and Ser119----Met; variant P125A.R129Q.R13OS, Pro125----Ala, Arg129----Gln and Arg130----Ser; variant G161R.K162R.-S165W, Gly161----Arg, Lys162----Arg and Ser165----Trp; and variant N115P, Asn115----Pro, respectively. The half-life following intravenous bolus injection in rabbits was prolonged in all variants except P125A.R130S. This was particularly true for N115P.S119M. The kinetic parameters for plasminogen activation were improved in t-PA G161R.K162R.S165W which showed a 0.6-fold decrease in Km, and a 1.8-fold increase in Vmax, thus promoting a 2.7-fold increase in kcat/Km compared to native t-PA. For a similar degree of thrombolysis in the rabbit jugular vein thrombosis model, the thrombolytic activity of G161R.K162R.S165W, at the dose tested, was four-fold greater than that of native t-PA. Thus, the substitution of the amino acids in the K1 domain with those corresponding in the K2 domain significantly enhanced the enzymatic activity of t-PA and improved the plasma survival.
authors.affiliation: Biochemical Research Laboratories, Kaneka Corporation, Takasago, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ikenaka
authors.forename: K
authors.initials: K
authors.lastname: Yajima
authors.forename: H
authors.initials: H
authors.lastname: Yahara
authors.forename: H
authors.initials: H
authors.lastname: Maruyama
authors.forename: K
authors.initials: K
authors.lastname: Matsumoto
authors.forename: K
authors.initials: K
authors.lastname: Okada
authors.forename: S
authors.initials: S
authors.lastname: Ueshima
authors.forename: O
authors.initials: O
authors.lastname: Matsuo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D005337:Fibrin / Q000378:metabolism; D005342:Fibrinolysis; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics*; D010958:Plasminogen / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D010446:Peptide Fragments; D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator