Influence of N-terminal peptide and oligosaccharide on the clearance of t-PA

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 activa tor (t-PA). In order to intensify the influence of these structural feature s, Gln117 t-PA, which is a mutant tissue plasminogen activator (mt-PA) expr essed 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 aci d from Asn117 to Gln. We isolated 4 variants of Gln117 t-PA by cation-excha nge 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 va riants were investigated after single intravenous administration to male ra ts at a dose of 250 mu g/kg. Significant differences in pharmacokinetic par ameters were observed among these variants, but there was no considerable d ifference in fibrin clot lysis time (FCLT) activity. Cly-Ala-Arg peptide at the N-terminus increased the CLt, whereas the oligosaccharide at Asn184 de creased the CLt. Moreover, the effects of the N-terminal peptide and the ol igosaccharide on the CLt were independent of each other. Our study with Gln 117 t-PA revealed the role of the N-terminal peptide found in the L-chain p roduced during the processing of t-PA precursor.