A fusion protein with improved thrombolytic effect and low bleeding risk.
To resolve the therapeutic dilemma between efficacy of thrombolysis and bleeding risk associated with the use of a combination of thrombolytic and anticoagulant treatments, we created a fusion protein. Staphylokinase was fused to the N-terminus of hirudin using thrombin recognition sequence as linker peptide, resulting in a fusion protein STH. We hypothesised that STH would be cleaved by thrombin at the thrombus site, releasing staphylokinase and hirudin to perform bifunctionally, and attenuating bleeding risk. SDS-PAGE and Western blot analyses indicated that the linker peptide could be specially recognised and cleaved by thrombin. Amidolytic and thromboelastogram assays showed that the N-terminus of hirudin in STH was blocked by staphylokinase and linker peptide, impeding hirudin's anticoagulant activity. Once cleaved, STH displayed 35.7% of the anticoagulant activity of equimolar hirudin and exhibited anticoagulant effects in the fibrin clot lysis assay. Thrombin-binding and fibrin clot lysis assays showed that the C-terminus of hirudin retained its high affinity for thrombin. Moreover, STH showed improved thrombolytic effects and a lower bleeding risk in animals. Thus, STH may have the capacity to perform bifunctionally and release anticoagulant activity in a thrombus-targeted manner in vivo, which may reduce the bleeding risk that often accompanies high thrombolytic efficacy in the treatment of thromboembolic diseases.
Wang
Xiudong
X
Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing, China.
Zhang
Guangman
G
Wang
Lisheng
L
Zhang
Qinglin
Q
Qin
Yide
Y
Wu
Chutse
C
Yu
Aiping
A
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D004195:Disease Models, Animal; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000494:pharmacology*; D006470:Hemorrhage / Q000517:prevention & control; D006629:Hirudins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D051379:Mice; D010958:Plasminogen / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013916:Thrombelastography; D013917:Thrombin / Q000378:metabolism; D013923:Thromboembolism / Q000188:drug therapy; D015912:Thrombolytic Therapy / Q000009:adverse effects / Q000379:methods; D014682:Vena Cava, Inferior
D017931:DNA Primers; D005343:Fibrinolytic Agents; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D010958:Plasminogen; D013917:Thrombin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus