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

Jennifer A. Hunt; Stephen R. Petteway Jr; Philip Scuderi; Valery Novokhatny

doi:10.1160/TH08-04-0225

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2008; 100(03): 413-419
DOI: 10.1160/TH08-04-0225

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

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

Jennifer A. Hunt

¹Talecris Biotherapeutics, Research Triangle Park, North Carolina, USA

,

Stephen R. Petteway Jr

¹Talecris Biotherapeutics, Research Triangle Park, North Carolina, USA

,

Philip Scuderi

¹Talecris Biotherapeutics, Research Triangle Park, North Carolina, USA

,

Valery Novokhatny

¹Talecris Biotherapeutics, Research Triangle Park, North Carolina, USA

› Author Affiliations

Abstract

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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 coliT7 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 (K_d = 1 1.3 ± 2.3 μ. M) 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-antiplas-min(1.1±0.1x10⁷M⁻¹s⁻¹)andα₂-macroglobulin(7.6±0.6x10⁵ 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.

Keywords

Fibrinolysis - peripheral arterial occlusion - plasmin - plasminogen - thrombosis

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References

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