title: Influence of N-terminal peptide and oligosaccharide on the clearance of t-PA.
abstract: We have studied the influence of Gly-Ala-Arg peptide at the N-terminus and the oligosaccharide at Asn184 on the clearance of tissue plasminogen activator (t-PA). In order to intensify the influence of these structural features, Gln117 t-PA, which is a mutant tissue plasminogen activator (mt-PA) expressed in mouse C127 cells, was used for the investigation. It is altered to remove a high mannose type oligosaccharide by the mutation of an amino acid from Asn117 to Gln. We isolated 4 variants of Gln117 t-PA by cation-exchange chromatography, which are abbreviated as S-I, S-II, L-I and L-II. These variants originated from the heterogeneity of the peptide chains (S-chain, 527 amino acids, L-chain, 530 amino acids) and oligosaccharide (Type I, 2 oligosaccharides, Type II, 1 oligosaccharide). Pharmacokinetics of these variants were investigated after single intravenous administration to male rats at a dose of 250 microg/kg. Significant differences in pharmacokinetic parameters were observed among these variants, but there was no considerable difference in fibrin clot lysis time (FCLT) activity. Gly-Ala-Arg peptide at the N-terminus increased the CLt, whereas the oligosaccharide at Asn184 decreased the CLt. Moreover, the effects of the N-terminal peptide and the oligosaccharide on the CLt were independent of each other. Our study with Gln117 t-PA revealed the role of the N-terminal peptide found in the L-chain produced during the processing of t-PA precursor.
authors.affiliation: Product Technology Development Laboratory, Tanabe Seiyaku Co., Ltd., Osaka, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Aoki
authors.forename: N
authors.initials: N
authors.lastname: Shimizu
authors.forename: J
authors.initials: J
authors.lastname: Koyama
authors.forename: Y
authors.initials: Y
authors.lastname: Karo
authors.forename: M
authors.initials: M
authors.lastname: Kitagawa
authors.forename: K
authors.initials: K
authors.lastname: Okumura
authors.forename: Y
authors.initials: Y
authors.lastname: Tanigawara
mesh_terms: D000818:Animals; D019540:Area Under Curve; D008297:Male; D009842:Oligopeptides / Q000493:pharmacokinetics*; D009844:Oligosaccharides / Q000493:pharmacokinetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000493:pharmacokinetics; D010959:Tissue Plasminogen Activator / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics*
chemical_list: D009842:Oligopeptides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator