Expression Research Of Targeting Multifunctional Anti-thrombosis Fusion Protein

Thrombotic disease is seriously disserving the health to a large extent. Researchers have paid attentions to prevention and regulation of thrombus. However, the research of anti-thrombotic was confined to the subject of hemolysis, anti-coagulated blood and thrombosis. Little attention has been given to the interrelationship between inflammation or infection and pathological process of thrombus.The unique human Cathelicidin was often referred to as LL-37. In addition to its immunomodulatory properties, LL-37 was recently proved to be provided a link between inflammation and atherosclerosis. Hirudin has various advantages including the potent direct anti-thrombin, the stable and sustained anti-thrombus activity, rare side effects, little toxicity. AAP peptide was derived from Agkistrodon acutus Guenther venom, which had proved its activity in three aspects– its ability to inhibit the process of coagulation and fibrin formation, its effects on the plasma fibrinolysis system and vascular endothelium and platelets. The RGD sequence could target the activated platelet at thrombotic sites, serving as a special ligand for GpIIb/IIIa.In order to increase activity and prevent the formation of thrombus in the coagulation pathway, we designed a novel recombinant fusion protein, namely LHAD composed of LL-37, Hirudin-12-residue and AAP, RGD peptide. Meantime, a FXa recognition sequence was fused to the N-terminal of Hirudin-12-residue to obtain specific targeting. LL-37 and Hirudin-12-residue were blanked by FXa recognize sequence to maintain each activity of the two peptides and increased the anticoagulant activity with low immunogenicity and toxicity, avoiding bleeding side effect. LHAD was expressed in the Pichia pastoris GS115 strain. Functional assays indicated that the anti-thrombin capability was enhanced to a certain scope compared to that of natural Hirudin. And the bioactivity of anti-platelet aggregation and anticoagulation was favorable. This strategy may supply a novel approach to inhibit thrombus formation, prevent and manage thrombosis.