Biochemical characterization of single‐chain chimeric plasminogen activators consisting of a single‐chain Fv fragment of a fibrin‐specific antibody and single‐chain urokinase

K 12 G 0 S 32 is a 57‐kDa recombinant single‐chain chimeric plasminogen activator consisting of scFv‐K 12 G 0 , a single‐chain variable‐region antigen‐binding fragment (Fv) of the monoclonal anti‐body 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 K 12 G 0 S 32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K 12 G 0 S 32 , determined in a system composed of a 125 I‐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 K 12 G 0 S 32 T, with an intact thrombin‐cleavage site, was 6–15‐fold higher than that of rscu‐PA. Conversion of 1 μM single‐chain K 12 G 0 S 32 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 K 12 G 0 S 32 T and rscu‐PA, respectively. Purified fragment D‐dimer of human cross‐linked fibrin inhibited the fibrinolytic potency of single‐chain K 12 G 0 S 32 T, but not of two‐chain K 12 G 0 S 32 T, in a dose‐dependent manner. Furthermore, the fibrinolytic potencies of two‐chain K 12 G 0 S 32 and K 12 G 0 S 32 T were not significantly higher than those of recombinant two‐chain u‐PA (rtcu‐PA) or of rtcu‐PA(M). These findings suggest that the 50‐fold increase in fibrinolytic potency of K 12 G 0 S 32 T, 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 K 12 G 0 S 32 T 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. K 12 G 0 S 45 which contains the scu‐PA kringle domain, and K 12 G 0 S 54 which contains intact scu‐PA, had a similar fibrinolytic potency as K 12 G 0 S 32 , indicating that the fibrin‐targeting in the Fv domain of K 12 G 0 S 32 is not hampered by spatial constraints in the molecule.