A specific plasminogen activator inhibitor‐1 antagonist derived from inactivated urokinase |
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Authors: | Zebin Hong Zhonghui Lin Min Liu Guangpu Xue Cai Yuan Lin Lin Barbara Furie Robert Flaumenhaft Peter Andreasen Bruce Furie Mingdong Huang |
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Institution: | 1. 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;2. University of Chinese Academy of Sciences, Beijing, China;3. Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA;4. Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark |
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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. |
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Keywords: | PAI‐1 urokinase variants protein structure fibrinolysis antithrombotic agent |
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