Characterization of a chimeric plasminogen activator consisting of a single-chain Fv fragment derived from a fibrin fragment D-dimer-specific antibody and a truncated single-chain urokinase.
An Mr 57,000 single-chain chimeric plasminogen activator, K12G0S32, consisting of a variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 and of a 33-kDa (amino acids Ala132 to Leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-PA-33k) was studied. K12G0S32, secreted by infected Spodoptera frugiperda insect cells at a rate of 1.5 micrograms/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography and gel filtration. It was obtained essentially as a single-chain molecule with a Ka = 5.5 x 10(9) M-1 for immobilized fragment D-dimer, similar to that of MA-15C5. The specific activity of both its single-chain and two-chain forms on fibrin plates was 100,000 IU/mg of urokinase-type plasminogen activator (u-PA) equivalent. Activation of plasminogen by two-chain K12G0S32 obeyed Michaelis-Menten kinetics with Km = 2.9 +/- 0.6 microM and a k2 = 3.7 +/- 0.6 s-1 (mean +/- S.D.; n = 3), as compared to Km = 12 microM and k2 = 4.8 s-1 for rtcu-PA-32k (recombinant low Mr two-chain u-PA consisting of amino acids Leu144 to Leu411). Single-chain K12G0S32 induced a dose- and time-dependent lysis of a 125I-fibrin-labeled human plasma clot immersed in citrated human plasma; 50% lysis in 2 h was obtained with 0.70 +/- 0.07 micrograms/ml (mean +/- S.D.; n = 5), as compared with 8.8 +/- 0.1 micrograms/ml for rscu-PA-32k (recombinant low Mr single-chain u-PA consisting of amino acids Leu144 to Leu411) (mean +/- S.D.; n = 3). With two-chain K12G0S32, 50% clot lysis in 2 h required 0.25 +/- 0.03 micrograms/ml (mean +/- S.D.; n = 3), as compared with only 0.62 +/- 0.04 micrograms/ml (mean +/- S.D.; n = 2) for rtcu-PA-32k. These results indicate that low Mr single-chain u-PA can be targeted to a fibrin clot with a single-chain Fv fragment of a fibrin-specific antibody, resulting in a 13-fold increase of the fibrinolytic potency of the single-chain form and a 2.5-fold increase of the potency of the two-chain form.
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
P
P
Holvoet
Y
Y
Laroche
H R
HR
Lijnen
R
R
Van Cauwenberge
E
E
Demarsin
E
E
Brouwers
G
G
Matthyssens
D
D
Collen
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D002678:Chimera; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004226:Dithioerythritol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000276:immunology*; D005342:Fibrinolysis / Q000187:drug effects; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010960:Plasminogen Activators / Q000235:genetics*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D000577:Amides; D000911:Antibodies, Monoclonal; D004226:Dithioerythritol; D005337:Fibrin; D004247:DNA; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator