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 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.
Division of Cellular, Molecular and Microbial Biology, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Qun
Q
Lian
Steven J
SJ
Szarka
Kenneth K S
KK
Ng
Sui-Lam
SL
Wong
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects; D004274:DNA, Recombinant / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D015195:Drug Design; D005337:Fibrin / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000494:pharmacology*; D006629:Hirudins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D015394:Molecular Structure; D015202:Protein Engineering; D013917:Thrombin / Q000378:metabolism*; D013927:Thrombosis / Q000097:blood / Q000188:drug therapy*
D004274:DNA, Recombinant; D004220:Disulfides; D005343:Fibrinolytic Agents; D006629:Hirudins; D005337:Fibrin; D013917:Thrombin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus