title: The effector roles of kringle 1 and kringle 2 in the enzymatic properties of recombinant tissue-type plasminogen activator as revealed by generation of recombinant molecules containing each kringle linked to the protease domain.
abstract: Recombinant DNA technology has been employed to construct and express in human kidney 293 cells cDNAs encoding deletion-mutant recombinant (r) tissue-type plasminogen activators (tPA) retaining only the kringle 1 ([K1tPA]) and serine protease (P) functional domains (r-[K1tPA]P), and the kringle 2 ([K2tPA]) and P domains (r-[K2tPA]P), along with a variant of r-tPA containing a W253S mutation (r-tPA/W253S). Of these mutants, only r-[K2tPA]P retained its ability to interact with omega-amino acid effector molecules. The Km for single-chain wild-type (wt) r-tPA toward the synthetic substrate H-D-Ile-L-Pro-L-Arg-p-nitroanilide (S2288) was decreased by approximately 3-fold in the presence of a saturating concentration of human fibrinogen (Fg), along with a small (1.14-fold) increase in the kcat for this same reaction. The kinetic activation (dissociation) constant (KA) for Fg was 2.4 microM. Fg did not influence the steady-state amidolytic properties of two-chain wtr-tPA. Similar effects of Fg on the Km for hydrolysis of S2288 were displayed for single-chain forms of r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S, with additional small effects of Fg on the kcat of this reaction. The KA for Fg toward these proteins ranged from 2.4 microM for wtr-tPA to 5.2 microM for r-[K1tPA]P. The amidolytic properties of the two-chain forms of these variants were also unaffected by Fg. The activation rate of [Glu1]-plasminogen ([Glu1]Pg) by wtr-tPA was stimulated approximately 7-fold by Fg and approximately 139-fold by the same concentration (in Fg equivalents) of human fibrin (Fn) (Fn/Fg stimulatory ratio = 19.9). The Fn/Fg ratio was 10.6, 20.9, and 18.0 for r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S, respectively. Quantitative [Glu1]Pg-enriched clot lysis assays revealed that r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S were approximately 18, 72, and 54%, respectively, as effective as wtr-tPA in catalyzing the plasminogen activation event leading to lysis. The antifibrinolytic agent epsilon-aminocaproic acid, inhibited clot lysis with approximately equal effectiveness in [Glu1]Pg-enriched clots when wtr-tPA, r-[K1tPA]P, r-[K2tPA]P, or r-tPA/W253S were employed as the activators. These studies demonstrated that Fg- and Fn-based stimulatory effects on the enzymatic properties of r-tPA and its variants were generally not proportional to the macroscopic binding abilities of these proteins with omega-amino acids or with Fg and Fn.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
authors.forename: A
authors.initials: A
authors.lastname: Rydzewski
authors.forename: F J
authors.initials: FJ
authors.lastname: Castellino
mesh_terms: D000596:Amino Acids / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
chemical_list: D000596:Amino Acids; D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator