title: Biochemical and functional characterization of human tissue-type plasminogen activator variants obtained by deletion and/or duplication of structural/functional domains.
abstract: Five cDNA encoding human tissue-type plasminogen activator (t-PA) variants with deletion and/or duplication of structural/functional domains were cloned and expressed in Chinese hamster ovary cells. The mutants included: rt-PA-delta FE (where r represents recombinant), with deletion of the finger (F) and growth factor (E) domains; rt-PA-delta K1 delta K2, with replacement of kringle 1 (K1) by a second copy of kringle 2 (K2); and rt-PA-delta FK1 delta K2, rt-PA-delta EK1 delta K2, and rt-PA-delta FEK1 delta K2, with deletions in rt-PA-delta K1 delta K2 of the finger or growth factor domain or both, respectively. The variant rt-PAs, purified to homogeneity, were obtained essentially as single-chain molecules. CNBr-digested fibrinogen enhanced plasminogen activation between 110-fold with rt-PA-delta EK1 delta K2 and 150-fold with rt-PA-delta FEK1 delta K2 as compared to 140-fold with rt-PA. All rt-PA moieties showed a comparable concentration-dependent binding to fibrin, except rt-PA-delta FE, which had significantly reduced binding that was, however, partially restored by additional replacement of K1 with K2. All the rt-PA variants with two copies of K2 showed increased binding to lysine-Sepharose as compared to rt-PA, whereas rt-PA-delta FE had reduced binding. All rt-PA moieties induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma. Equally effective concentrations (causing 50% clot lysis in 2 h) ranged between 1.0 microgram/ml for rt-PA-delta K1 delta K2 and 1.6 micrograms/ml for rt-PA-delta FE as compared to 0.5 microgram/ml for rt-PA. Thus, replacement in rt-PA of K1 by a second copy of K2, which is known to contain a lysine-binding site, significantly enhances its affinity for lysine, with maintenance of its affinity for intact fibrin. Deletion of the finger and growth factor domains results in decreased fibrin affinity and fibrinolytic potency in a plasma milieu, which are partially restored by replacement of K1 by K2.
authors.affiliation: Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
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: F
authors.initials: F
authors.lastname: De Cock
authors.forename: D
authors.initials: D
authors.lastname: Collen
mesh_terms: D000345:Affinity Labels; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003488:Cyanogen Bromide; D004247:DNA / Q000235:genetics; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis; D015870:Gene Expression; D006801:Humans; D009154:Mutation; D010957:Plasmids; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000502:physiology*
chemical_list: D000345:Affinity Labels; C030675:lysine-sepharose; D005337:Fibrin; D005340:Fibrinogen; D004247:DNA; D012685:Sepharose; D004262:DNA Restriction Enzymes; D010959:Tissue Plasminogen Activator; D003488:Cyanogen Bromide