Characterization of bifunctional chimeric molecule of PRGDWR containing pro-urokinase

In order to obtain the bifunctional chimeric molecule of single-chain urokinase-type plasminogen activator (scu-PA) which can inhibit platelet aggregation, PRGDWR peptide was inserted into the site between Gly¹¹⁸ and Leu¹¹⁹ (called insertion mutant B, InB). The recombinant gene of InB was expressed by Pichia pastoris. The secreted protein was purified by metal chelate affinity and strong cation exchange chromatography. The amidolytic ability of mutant InB is 5 900 IU/mg, the kinetic constants is: K_m,plg^InB = 56.8 μmol·L^-1, k_cat,plg^InB = 0.33 s^-1. The kinetic constants of plasminogen activation reaction is: K_m,plg^InB = 0.397 μmol·L^-1, k_cat,plg^InB = 0.0164 s^-1. Fibrin inhibit the catalytiv ability of InB during plasminogen activation, the influence factor is 0.463 (means InB remain 46.3% of the catalytic ability when fibrin was involved in the reaction system). The mutant not only has almost the same catalytic ability as wild type scu-PA, but also has strong ability of anti-platelet aggregation (compared with scu-PA), IC₅₀ of InB is 12.7 μmol·L^-1.