title: A long-acting PAI-1 inhibitor reduces thrombus formation.
abstract: 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 IC50(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 IC50value 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.
authors.forename: Shuangzhou
authors.initials: S
authors.lastname: Peng
authors.forename: Guangpu
authors.initials: G
authors.lastname: Xue
authors.forename: Lihu
authors.initials: L
authors.lastname: Gong
authors.forename: Chao
authors.initials: C
authors.lastname: Fang
authors.forename: Jingfei
authors.initials: J
authors.lastname: Chen
authors.forename: Cai
authors.initials: C
authors.lastname: Yuan
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Chen
authors.forename: Lishan
authors.initials: L
authors.lastname: Yao
authors.forename: Bruce
authors.initials: B
authors.lastname: Furie
authors.affiliation: Mingdong Huang, State Key Laboratory of Structural Chemistry and, Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian, China, E-mail: mhuang@fjirsm.ac.cn or HMD_lab@fzu.edu.cn.
authors.forename: Mingdong
authors.initials: M
authors.lastname: Huang
mesh_terms: D000818:Animals; D001760:Bleeding Time; D004195:Disease Models, Animal; D015195:Drug Design; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D006207:Half-Life; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000097:blood / Q000737:chemistry / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000097:blood / Q000737:chemistry / Q000494:pharmacology; D058258:Serpin E2 / Q000037:antagonists & inhibitors; D013927:Thrombosis / Q000517:prevention & control*; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology
chemical_list: D005343:Fibrinolytic Agents; C000621142:PAItrap peptide; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins; C051321:SERPINE1 protein, human; D015842:Serine Proteinase Inhibitors; D058258:Serpin E2; C511869:Serpine2 protein, mouse; D014568:Urokinase-Type Plasminogen Activator
keywords: PAI-1 inhibitor; clot lysis; human serum albumin (HSA); thrombosis