title: A specific plasminogen activator inhibitor-1 antagonist derived from inactivated urokinase.
abstract: Fibrinolysis is a process responsible for the dissolution of formed thrombi to re-establish blood flow after thrombus formation. Plasminogen activator inhibitor-1 (PAI-1) inhibits urokinase-type and tissue-type plasminogen activator (uPA and tPA) and is the major negative regulator of fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis. However, a specific antagonist of PAI-1 is currently unavailable for therapeutic use. We screened a panel of uPA variants with mutations at and near the active site to maximize their binding to PAI-1 and identified a potent PAI-1 antagonist, PAItrap. PAItrap is the serine protease domain of urokinase containing active-site mutation (S195A) and four additional mutations (G37bR-R217L-C122A-N145Q). PAItrap inhibits human recombinant PAI-1 with high potency (Kd = 0.15 nM) and high specificity. In vitro using human plasma, PAItrap showed significant thrombolytic activity by inhibiting endogenous PAI-1. In addition, PAItrap inhibits both human and murine PAI-1, allowing the evaluation in murine models. In vivo, using a laser-induced thrombosis mouse model in which thrombus formation and fibrinolysis are monitored by intravital microscopy, PAItrap reduced fibrin generation and inhibited platelet accumulation following vascular injury. Therefore, this work demonstrates the feasibility to generate PAI-1 inhibitors using inactivated urokinase.
authors.affiliation: 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.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Lihu
authors.initials: L
authors.lastname: Gong
authors.affiliation: Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Valerie
authors.initials: V
authors.lastname: Proulle
authors.affiliation: Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Chao
authors.initials: C
authors.lastname: Fang
authors.affiliation: 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.
authors.forename: Zebin
authors.initials: Z
authors.lastname: Hong
authors.affiliation: 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.
authors.forename: Zhonghui
authors.initials: Z
authors.lastname: Lin
authors.affiliation: 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.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.affiliation: 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.
authors.forename: Guangpu
authors.initials: G
authors.lastname: Xue
authors.affiliation: 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.
authors.forename: Cai
authors.initials: C
authors.lastname: Yuan
authors.affiliation: Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.affiliation: Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Furie
authors.affiliation: Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Flaumenhaft
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Andreasen
authors.affiliation: Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Bruce
authors.initials: B
authors.lastname: Furie
authors.affiliation: 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. mhuang@fjirsm.ac.cn.|University of Chinese Academy of Sciences, Beijing, China. mhuang@fjirsm.ac.cn.|Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. mhuang@fjirsm.ac.cn.
authors.forename: Mingdong
authors.initials: M
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004195:Disease Models, Animal; D005342:Fibrinolysis; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011485:Protein Binding; D013927:Thrombosis / Q000473:pathology; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D050505:Mutant Proteins; C000621142:PAItrap peptide; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D014568:Urokinase-Type Plasminogen Activator
keywords: PAI-1; antithrombotic agent; fibrinolysis; protein structure; urokinase variants