Fibrinolysis: An International Journal of Fibrinolysis and Thrombolysis vol:6 issue:3 pages:149-159
rt-PA I276G, a mutant of recombinant human tissue-type plasminogen activator (rt-PA) with altered plasmin cleavage site, was obtained by site-directed mutagenesis of Ile276 to Gly. rt-PA I276G, purified to homogeneity from the conditioned medium of transfected Chinese hamster ovary cells, was obtained as a single chain molecule, which was quantitatively converted to a two chain moiety by cleavage of the Lys277-Gly278 peptide bond with plasmin. The specific activities on fibrin films of the single chain and two chain forms of rt-PA I276G were 8900 and 15000 IU/mg respectively, as compared to 210000 and 130000 IU/mg respectively for the single chain and two chain forms of wild-type rt-PA obtained in the same expression system. The amidolytic activity of the rt-PA I276G moieties was 3- to 5-fold lower and their catalytic efficiency for plasminogen activation 20- to 50-fold lower than those of the wild-type rt-PA moieties. Both single chain and two chain rt-PA I276G induced concentration-dependent lysis of a I-125-fibrin labelled plasma clot submersed in human plasma; equi-effective concentrations (causing 50% clot lysis in 2 h) were 0.55 and 1.40-mu-g/ml respectively, as compared to 0.36 and 0.60-mu-g/ml for single chain or two chain wild-type rt-PA respectively. Continuous infusion over 60 min of single chain rt-PA I276G or wild-type rt-PA in hamsters with a pulmonary embolus, revealed an approximately 2-fold lower thrombolytic potency (clot lysis versus dose) for the mutant, but a comparable specific thrombolytic activity (clot lysis versus steady state plasma antigen level). It is concluded that replacement of Ile276 by Gly in single chain rt-PA significantly reduces the intrinsic enzymatic activity in purified systems. In a plasma milieu in the presence of fibrin the fibrinolytic potential of the mutant is, however, only 2-fold lower than that of wild-type rt-PA obtained in the same expression system.