Design And Molecular Pharmacological Studies Of Long-acting PAI-1 Inhibitors

Plasminogen activator inhibitor type-1(PAI-1)is a major inhibitor of fibrinolysis by its capacity to inhibit urokinase-type plasminogen activator(uPA)and tissue-type plasminogen activator(tPA).Elevated circulating levels of PAI-1 is associated with thrombosis,myocardial infarction,cancer and systemic inflammation.Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis,indicating that PAI-1 inhibitors may be used as effective antithrombotic agents.This thesis contains two parts.The first part focuses on the development of a long-acting PAI-1 inhibitor.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 increase plasma half-life of the 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 optimization to enhance inhibitory potency for PAI-1.The new PAItrap,denominated PAItrap(H37R)-HSA,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 IC50 value of 12 nM,allowing the inhibitor to be evaluated in murine models.Using an intravital microscopy,we demonstrated that PAItrap(H37R)-HS A 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 demonstrate the potential benefit of PAItrap(H37R)-HSA to prevent thrombosis and accelerates fibrinolysis.In the second part of this dissertation,based on the crystal structure of the tPA·PAI-1Michaelis complex and the principles which its successfully design a PAI-1 inhibitor—PAItrap,we have developed a new type tPA-based PAI-1 inhibitor.The inhibitor was fused with SA to increase the overall molecular weight to improve pharmacokinetics.The PAItrap2-HSA as a novel PAI-1 inhibitor was demonstrated to be highly specific and potent for inhibiting active PAI-1 by a chromogenic assay.In vitro,PAItrap2-HSA showed significantly improved thrombolytic activity by inhibiting exogenous or endogenous PAI-1.In vivo,using thrombosis model,we here demonstrated that PAItrap2-HSA prevented the development and progression of thrombosis by target endogenous PAI-1.It is important to note that PAItrap2-HSA as a specific PAI-1 inhibitor can significantly reduce inflammatory response and PAI-1 protein expression for enhance survival in LPS-induced inflammatory model.In addition,we also demonstrated that PAItrap2-HSA did not induced significantly impairing normal hemostasis.These results demonstrated that PAItrap2-HSA is a new strategy to reduce thrombus formation and inflammatory response by inhibiting active PAI-1.