title: Biochemical, thrombolytic and pharmacokinetic properties of rt-PA P47G, K49N, a substitution variant of human tissue-type plasminogen activator.
abstract: rt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahern et al. (J Biol Chem 1990; 265:5540-5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse). The specific fibrinolytic activities on fibrin plates were 160,000 +/- 17,000, 210,000 +/- 88,000 and 460,000 +/- 72,000 IU/mg (mean +/- SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k2/Km) in the absence of fibrin were comparable (1.1 to 1.7 x 10(-3) microM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 +/- 0.14 micrograms/ml rt-PA P47G, K49N, 0.36 +/- 0.01 micrograms/ml rt-PA and 0.17 +/- 0.01 micrograms/ml alteplase, respectively (mean +/- SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
authors.forename: L
authors.initials: L
authors.lastname: Nelles
authors.forename: X K
authors.initials: XK
authors.lastname: Li
authors.forename: I
authors.initials: I
authors.lastname: Vanlinthout
authors.forename: F
authors.initials: F
authors.lastname: De Cock
authors.forename: H R
authors.initials: HR
authors.lastname: Lijnen
authors.forename: D
authors.initials: D
authors.lastname: Collen
mesh_terms: D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011655:Pulmonary Embolism / Q000188:drug therapy; D011994:Recombinant Proteins / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000493:pharmacokinetics / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D005340:Fibrinogen; D004247:DNA; D010959:Tissue Plasminogen Activator