RECOMBINANT VARIANTS OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR CONTAINING AMINO-ACID SUBSTITUTIONS BETWEEN POSITION-37 AND POSITION-42

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 refin ements in this mutant would reduce the clearance rate and maintain hig h affinity for fibrin. Two variants, t-PA N37S.S38V.G39V.R40E.A41S.Q42 S and t-PA N37S.S38V.G39V.R40E.A41F.Q42S had a 5-fold increased half-l ife, 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 m odifications 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 de creased fibrin binding. Two of them, t-PA N37S.S38V.G39V.R40I.A41V.Q42 S and t-PA N37S.S38V.G39V.R40I.A41V.Q42L, bound to fibrin stronger tha n the variants, t-PA N37S.S38V.G39V.R40E.A41S.Q42S and t-PA N37S.S38V. G39V.R40E.A41F.Q42S, accompanied by a prolonged half-life in vivo. The other objective of this study was to estimate the effects of the amin o acid substitution at position 42. Two variants, t-PA N37S.S38V.G39V. R40E.A41S and t-PA N37S.S38V.G39V.R40E.A41F had about a 6-fold longer half life in vivo. These data suggest that changes in the pharmacokine tic characteristics of the 37-42 variants are mainly due to modificati ons at amino acid residues 37-41.