A long-acting PAI-1 inhibitor reduces thrombus formation

• Published in: Thrombosis and haemostasis Vol. 117; no. 7; p. 1338
• Main Authors: Peng, Shuangzhou, Xue, Guangpu, Gong, Lihu, Fang, Chao, Chen, Jingfei, Yuan, Cai, Chen, Zhuo, Yao, Lishan, Furie, Bruce, Huang, Mingdong
• Format: Journal Article
• Language: English
• Published: Germany 2017
• Subjects: Animals; Bleeding Time; Disease Models, Animal; Drug Design; Fibrinolysis - drug effects; Fibrinolytic Agents - blood; Fibrinolytic Agents - chemistry; Fibrinolytic Agents - pharmacology; Half-Life; Humans; In Vitro Techniques; Mice; Mice, Inbred C57BL; Models, Molecular; Mutagenesis, Site-Directed; Peptide Fragments - pharmacology; Plasminogen Activator Inhibitor 1 - blood; Plasminogen Activator Inhibitor 1 - chemistry; Plasminogen Activator Inhibitor 1 - pharmacology; Protein Engineering; Recombinant Fusion Proteins - blood; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - pharmacology; Serine Proteinase Inhibitors - blood; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - pharmacology; Serpin E2 - antagonists & inhibitors; Thrombosis - prevention & control; Urokinase-Type Plasminogen Activator - pharmacology; thrombosis; PAI-1 inhibitor; human serum albumin (HSA); clot lysis
• ISSN: 2567-689X
• DOI: 10.1160/TH16-11-0891

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain. In the present study, we fused PAItrap with human serum albumin (HSA) to develop a long-acting PAI-1 inhibitor. Unfortunately, the fusion protein PAItrap-HSA lost some potency compared to PAItrap (33 nM vs 10 nM). Guided by computational method, we carried out further optimisation to enhance inhibitory potency for PAI-1. The new PAItrap, denominated PAItrap(H37R)-HSA, which was the H37R variant of PAItrap fused to HSA, gave a six-fold improvement of IC (5 nM) for human active PAI-1 compared to PAItrap-HSA, and showed much longer plasma half-life (200-fold) compared to PAItrap. We further demonstrated that the PAItrap(H37R)-HSA inhibited exogenous or endogenous PAI-1 to promote fibrinolysis in fibrin-clot lysis assay. PAItrap(H37R)-HSA inhibits murine PAI-1 with IC value of 12 nM, allowing the inhibitor to be evaluated in murine models. Using an intravital microscopy, we demonstrated that PAItrap(H37R)-HSA blocks thrombus formation and platelet accumulation in vivo in a laser-induced vascular injury mouse model. Additionally, mouse tail bleeding assay showed that PAItrap(H37R)-HSA did not affect the global haemostasis. These results suggest that PAItrap(H37R)-HSA have the potential benefit to prevent thrombosis and accelerates fibrinolysis.