In situ formed depot of elastin- like polypeptide- hirudin fusion protein for long- acting antithrombotic therapy

Thrombosis, induced by abnormal coagulation or fibrinolytic systems, is the most common pathology associated with many life- threatening cardio- cerebrovascular diseases. However, first - line anticoagulant drugs suffer from rapid drug elimination and risk of hemorrhagic complications. Here, we developed an in situ formed depot of elastin-like polypeptide (ELP)- hirudin fusion protein with a prodrug-like feature for long - term antithrombotic therapy. Highly secretory expression of the fusion protein was achieved with the assistance of the Ffu312 tag. Integration of hirudin, ELP, and responsive moiety can customize fusion proteins with properties of adjustable in vivo retention and controllable recovery of drug bioactivity. After subcutaneous injection, the fusion protein can form a reservoir through temperature- induced coacervation of ELP and slowly diffuse into the blood circulation. The biological activity of hirudin is shielded due to the N- terminal modification, while the activated key proteases upon thrombus occurrence trigger the cleavage of fusion protein together with the release of hirudin, which has antithrombotic activity to counteract thrombosis. We substantiated that the optimized fusion protein produced long - term antithrombotic effects without the risk of bleeding in multiple animal thrombosis models. Significance Anticoagulant drugs used in clinics for the prevention of thrombosis are subject to frequent administration and risk of bleeding complications. In this study, we present a long- acting and safe antithrombotic treatment strategy by integrating hirudin, elastin- like peptide, and responsive moiety through genetic engineering. The customized fusion protein can form an in situ depot for sustained release. Importantly, the fusion protein remains inactive in the circulation, while the proteases activated during thrombosis can degrade the fusion protein to generate active hirudin, potentiating antithrombotic efficacy against thrombosis.