A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

Abstract

To develop a fast-acting clot dissolving agent, a clottargeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein in Bacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced from B. subtilis was improperly folded because of its complicated disulfidebond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flowcell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues.

Integrin αMβ2 Orchestrates and Accelerates Plasminogen Activation and Fibrinolysis by Neutrophils

“…PMAstimulated PMNs, but not resting cells, mediated clot lysis over a 5-h period. The observed time course of clot lysis time is consistent with previous reports (5,30). Thus, activation of the PMNs is essential for efficient fibrinolysis.…”

High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris

“…Recombinant tissue plasminogen activator (tPA), urokinase, streptokinase (SK) and their derivatives are some of the approved thrombolytic drugs (Wu et al 2003;Tsikouris and Tsikouris 2001;Sinnaeve and Van de Werf 2001). These bloodclot dissolving agents are plasminogen activators, which convert inactive plasminogen to plasmin that eventually dissolve fibrin in the blood clot (Su et al 2004).…”

“…Thrombolytic therapy serves as an attractive means to treat acute myocardial infarction, one of the predominant causes of death (Wu et al 2003). Recombinant tissue plasminogen activator (tPA), urokinase, streptokinase (SK) and their derivatives are some of the approved thrombolytic drugs (Wu et al 2003;Tsikouris and Tsikouris 2001;Sinnaeve and Van de Werf 2001).…”

The evolution of fibrin-specific targeting strategies

“…12,61,62 However, the molecular weight of the Kringle-1 domain, at 10 kDa is much more manageable than plasminogen's at 88 kDa. 61 Kringle-1 has been successfully employed in the targeted delivery of thrombolytics 12 and as a method of prolonging growth factor retention in wound matrices to improve neovascularization and overall healing in rat models. 63 …”