title: Biochemical and biologic properties of rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistant to plasminogen activator inhibitor-1.
abstract: A mutant of recombinant tissue-type plasminogen activator (rt-PA), obtained by deletion of residues Lys296 to Gly302 [rt-PA del(K296-G302)], was previously shown to be resistant to inhibition by plasminogen activator inhibitor-1 (PAI-1) (Madison et al, Nature 339:721, 1989). This mutant was obtained by expression of its cDNA in Chinese hamster ovary cells and purification to homogeneity from conditioned cell culture medium. It was obtained as a single chain molecule with amidolytic activity, specific fibrinolytic activity, and binding to fibrin and lysine, which were comparable or somewhat lower than those of wild-type rt-PA obtained in the same expression system. The plasminogen-activating potential of rt-PA del(K296-G302) in the presence of CNBr-digested fibrinogen was about twofold lower than that of wild-type rt-PA. The inhibition rate of rt-PA del(K296-G302) by recombinant PAI-1 (rPAI-1) was more than 500-fold lower than that of wild-type rt-PA. In a human plasma milieu in vitro, rt-PA del(K296-G302) induced dose-dependent lysis of a 125I-fibrin-labeled plasma clot; equi-effective concentrations (causing 50% clot lysis in 2 hours) were 0.28 micrograms/mL and 0.36 micrograms/mL for mutant and wild-type rt-PA, respectively. In this system, addition of rPAI-1 to the plasma resulted in a concentration-dependent reduction of the fibrinolytic potency of rt-PA del(K296-G302) and of rt-PA; a 50% reduction required 2.4 micrograms/mL and 0.15 micrograms/mL rPAI-1, respectively. Continuous infusion of mutant or wild-type rt-PA over 60 minutes in hamsters with a 125I-labeled plasma clot in the pulmonary artery resulted in dose-dependent clot lysis, with a thrombolytic potency (percent clot lysis per milligram of compound administered per kilogram of body weight) and a specific thrombolytic activity (percent clot lysis per microgram per milliliter steady state rt-PA-related antigen level in plasma) that were not significantly different. Bolus injection in hamsters of 1 mg/kg rPAI-1 followed by bolus injection of 1 mg/kg rt-PA del(K296-G302) or wild-type rt-PA resulted in neutralization of the thrombolytic potency of wild-type rt-PA, while the mutant retained approximately half of its thrombolytic potency. These results indicate that rt-PA del(K296-G302), with a known resistance to inhibition by rPAI-1 in purified systems, maintains this property both in a plasma milieu in vitro and in an experimental animal model of thrombolysis in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
authors.forename: X K
authors.initials: XK
authors.lastname: Li
authors.forename: H R
authors.initials: HR
authors.lastname: Lijnen
authors.forename: L
authors.initials: L
authors.lastname: Nelles
authors.forename: B
authors.initials: B
authors.lastname: Van Hoef
authors.forename: J M
authors.initials: JM
authors.lastname: Stassen
authors.forename: D
authors.initials: D
authors.lastname: Collen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002863:Chromogenic Compounds; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis; D015870:Gene Expression; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D015849:Plasminogen Inactivators / Q000494:pharmacology; D011655:Pulmonary Embolism / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D002863:Chromogenic Compounds; D009842:Oligopeptides; D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; C041319:isoleucyl-prolyl-arginine-4-nitroanilide; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D004247:DNA; D010959:Tissue Plasminogen Activator