Summary

A simplified and fully functional deletion mutant of plasminogen was created in which the middle portion of the molecule was removed, resulting in kringle 1 attachment to the serine protease domain. This recombinant plasminogen deletion mutant, Δ(K2-K5)Pg,was produced in the form of inclusion bodies at the yield of up to 200 mg/l in an Escherichia coli T7 expression system. Following protein refolding and purification on lysine-Sepharose, the conversion of the recombinant molecule Δ(K2-K5)Pg to the active enzyme mutant Δ(K2-K5)Pm by plasminogen activators was evaluated, and functional characteristics of the simplified plasmin were studied. Properties of Δ(K2-K5)Pg were similar to native, human plasma-derived plasminogen. Δ(K2-K5)Pg effectively bound ε-aminocaproic acid (Kd = 11.3 ± 2.3 μM) and fibrin (C50 ≈ 0.3 μM).The plasminogen activators streptokinase, urokinase, and tissue plasminogen activator effectively converted the recombinant zymogen Δ(K2-K5)Pg to the active recombinant enzyme, Δ(K2-K5)Pm. Additionally, Δ[K2-K5]Pm was rapidly inhibited by α2-antiplasmin (1.1 ± 0.1 x 107 M-1s-1) and α2-macroglobulin (7.6 ± 0.6 x 105 M-1s-1). In an in-vitro model, Δ(K2-K5)Pm demonstrated fibrinolytic potency comparable to human plasma-derived plasmin. Because of their unique biochemistry,including fibrin-binding properties and rapid inhibition by α2-antiplasmin, both native plasmin and a simplified deletion mutant of plasmin are potentially safe and effective direct thrombolytic agents for various thrombotic conditions. Further studies evaluating the in-vivo pharmacologic safety and clinical efficacy of this simplified plasmin (i.e. Δ[K2-K5]Pm) are warranted.