Biochemical and functional characterization of human tissue-type plasminogen activator variants with mutagenized kringle domains.

The cDNA encoding full-length human tissue-type plasminogen activator (t-PA) and five variant cDNAs, constructed by in vitro site-directed mutagenesis, were cloned and expressed in Chinese hamster ovary cells. The variant cDNAs were designed to increase the fibrin affinity of t-PA by mutagenesis in the kringle domains of specific amino acids which are assumed to constitute the lysine-binding site. These amino acids were replaced with the corresponding amino acids present in kringle 1 of plasminogen, which has a high affinity for lysine analogues. The mutants included: rt-PA-Arg125 with a Pro125----Arg mutation; rt-PA-Arg164,Tyr165 with Ser164,Ser165----Arg,Tyr; rt-PA-Arg125,Arg164,Tyr165 with Pro125,Ser164,Ser165----Arg,Arg,Tyr; rt-PA-Arg213 with Val213----Arg; and rt-PA-Arg252 with Thr252----Arg. Compared to wild-type recombinant t-PA (rt-PA), the catalytic efficiency for plasminogen activation was enhanced 4-fold for rt-PA-Arg125, and 3-fold for rt-PA-Arg252 while stimulation of plasminogen activation by CNBr-digested fibrinogen was comparable to wild-type rt-PA for rt-PA-Arg125 and 2-fold enhanced for rt-PA-Arg252. All rt-PA moieties showed a similar concentration-dependent and nearly quantitative binding to fibrin as well as to lysine-Sepharose and induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma. Equieffective concentrations (causing 50% clot lysis in 2 h) were 0.17 micrograms/ml for rt-PA-Arg125, and 0.31 micrograms/ml for rt-PA-Arg252 as compared to 0.55 micrograms/ml for rt-PA. The initial plasma half-life following intravenous bolus injection of 0.25 mg/kg in hamsters was 1.2-2.6 min, not significantly different from wild-type rt-PA (2.4 min). Continuous infusion over 60 min in hamsters with a 125I-fibrin-labeled pulmonary embolus produced 50% clot lysis over background with a dose of 0.9-1.8 mg/kg, which is not markedly superior to wild-type rt-PA (2.1 mg/kg). It is concluded that these variants, designed to mimic the high affinity fibrin-binding site of plasminogen, are not endowed with a markedly improved thrombolytic potency.