Engineering of a Staphylokinase-based Fibrinolytic Agent with Antithrombotic Activity and Targeting Capability toward Thrombin-rich Fibrin and Plasma Clots

Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylo...

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Bibliographic Details
Published in:The Journal of biological chemistry 2003-07, Vol.278 (29), p.26677-26686
Main Authors: Lian, Qun, Szarka, Steven J., Ng, Kenneth K.S., Wong, Sui-Lam
Format: Article
Language:English
Subjects:
Bacillus subtilis
Bacillus subtilis - genetics
Bacillus subtilis - metabolism
Base Sequence
Blood Coagulation - drug effects
Disulfides - chemistry
DNA, Recombinant - genetics
Drug Design
Fibrin - metabolism
Fibrinolytic Agents - chemistry
Fibrinolytic Agents - pharmacology
Hirudins - chemistry
Hirudins - genetics
Hirudins - pharmacology
Humans
In Vitro Techniques
Mass Spectrometry
Metalloendopeptidases - chemistry
Metalloendopeptidases - genetics
Metalloendopeptidases - pharmacology
Molecular Structure
Protein Engineering
Thrombin - metabolism
Thrombosis - blood
Thrombosis - drug therapy
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Summary:Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain. This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach. After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules. This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity. The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase. In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase. Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M303241200