Simplified recombinant plasmin: production and functional comparison of a novel thrombolytic molecule with plasma-derived plasmin

• Published in: Thrombosis and haemostasis Vol. 100; no. 3; p. 413
• Main Authors: Hunt, Jennifer A, Petteway, Jr, Stephen R, Scuderi, Philip, Novokhatny, Valery
• Format: Journal Article
• Language: English
• Published: Germany 01.09.2008
• Subjects: Amino Acid Sequence; Dose-Response Relationship, Drug; Escherichia coli - metabolism; Fibrin - chemistry; Fibrinolysin - chemistry; Fibrinolytic Agents - pharmacology; Humans; Molecular Sequence Data; Mutation; Plasma - metabolism; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Spectrometry, Fluorescence - methods; Time Factors; Urokinase-Type Plasminogen Activator - chemistry
• ISSN: 0340-6245
• DOI: 10.1160/TH08-04-0225

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, Delta(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 Delta(K2-K5)Pg to the active enzyme mutant Delta(K2-K5)Pm by plasminogen activators was evaluated, and functional characteristics of the simplified plasmin were studied. Properties of Delta(K2-K5)Pg were similar to native, human plasma-derived plasminogen. Delta(K2-K5)Pg effectively bound epsilon-aminocaproic acid (K(d)=11.3+/-2.3 microM) and fibrin (C(50) approximately 0.3 microM). The plasminogen activators streptokinase, urokinase, and tissue plasminogen activator effectively converted the recombinant zymogen Delta(K2-K5)Pg to the active recombinant enzyme, Delta(K2-K5)Pm. Additionally, Delta[K2-K5]Pm was rapidly inhibited by alpha(2)-antiplasmin (1.1+/-0.1 x 10(7) M(-1) s(-1)) and alpha(2)-macroglobulin (7.6+/-0.6 x 10(5) M(-1) s(-1)). In an in-vitro model, Delta(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 alpha(2)-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. Delta[K2-K5]Pm) are warranted.