Recombinant variants of tissue-type plasminogen activator containing amino acid substitutions between positions 37 and 42

Abstract

The finger domain deletion mutant of tissue type plasminogen activator (t-PA) has a prolonged half-life in vivo but tends to be accompanied by a decrease in affinity for fibrin. We considered that further refinements in this mutant would reduce the clearance rate and maintain high affinity for fibrin. Two variants, t-PA N37S.S38V G39V R40E.A41S.Q42S and t-PA N37S.S38V G39V R40E.A41F Q42S had a 5-fold increased half-life, but the amount of these variants that bound fibrin was lower than that of wild-type t-PA. To further investigate the effects of these modifications and create a t-PA variant with higher affinity for fibrin, we produced six variants in which residues 37–42 were substituted by more hydophobic amino acids. These variants had various degrees of decreased fibrin binding. Two of them, t-PA N37S.S38VG39VR40I.A41VQ42S and t-PA N37S.S38V.G39V R40I.A41V.Q42L, bound to fibrin stronger than the variants, t-PA N37S.S38VG39VR40E.A41S.Q42S and t-PA N37S.S38VG39VR40E.A41FQ42S, accompanied by a prolonged half-life in vivo. The other objective of this study was to estimate the effects of the amino acid substitution at position 42. Two variants, t-PA N37S.S38VG39VR40E.A41S and t-PA N37S.S38VG39VR40E.A41F had about a 6-fold longer half life in vivo. These data suggest that changes in the pharmacokinetic characteristics of the 37–42 variants are mainly due to modifications at amino acid residues 37–41.