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J. Biol. Chem., Vol. 278, Issue 29, 26677-26686, July 18, 2003

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

Qun Lian, Steven J. Szarka , Kenneth K. S. Ng  and Sui-Lam Wong ¶

From the Division of Cellular, Molecular and Microbial Biology, Division of Biochemistry, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

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.

Received for publication, March 28, 2003 , and in revised form, May 7, 2003.

^* This work was supported by research grants from the Heart and Stroke Foundation of Canada (Alberta) and Natural Sciences and Engineering Research Council of Canada (NSERC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: SemBioSys Genetics, Calgary, Alberta T1Y 7L3, Canada.

^¶ To whom correspondence should be addressed: Division of Cellular, Molecular and Microbial Biology, Dept. of Biological Sciences, University of Calgary, 2500 University Drive, N.W., Alberta T2N 1N4, Canada. Tel.: 403-220-5721; Fax: 403-289-9311; E-mail: slwong{at}ucalgary.ca.

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