Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase.
K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)
Holvoet
P
P
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Laroche
Y
Y
Lijnen
H R
HR
Van Hoef
B
B
Brouwers
E
E
De Cock
F
F
Lauwereys
M
M
Gansemans
Y
Y
Collen
D
D
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005337:Fibrin / Q000276:immunology* / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010960:Plasminogen Activators / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D015183:Restriction Mapping; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D004247:DNA; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator