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
XK Li, HR Lijnen, L Nelles, B Van Hoef, JM Stassen and D Collen
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
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)
Volume 79,
Issue 2,
pp. 417-429,
01/15/1992
Copyright © 1992 by The American Society of Hematology
