Recombinant　tissue-type　plasminogen　activator　(rtPA,　or　Alteplase)　is　the　first　approved　thrombolytic　drug　for　acute　ischemic　stroke,　but　suffers　from　a　short　half-life　and　poor　resistance　to　plasminogen　activator　inhibitor　(PAI-1),　limiting　its　clinical　use.　The　development　of　novel　thrombolytic　agents　with　improved　benefit/risk　balance　has　always　been　of　great　significance.　In　this　study,　we　identified　a　mutant　of　serine　protease　domain　of　tPA　(named　ΔtPAA146V)　capable　of　escaping　the　inhibition　by　endogenous　PAI-1　with　66-fold　increased　resistance　compared　to　the　wild　type　tPA.　Based　on　this　mutant,　we　generated　a　triple　fusion　ΔtPA　(TriF-ΔtPA)　containing　albumin　and　fibrin　binding　peptide(FBP).　The　fusion　with　albumin　effectively　prolonged　the　plasma　half-life　of　ΔtPA　in　mice　to　144　min,　which　is　much　longer　than　ΔtPA　and　did　not　affect　its　thrombolytic　activity.　Furthermore,　FBP　rendered　fibrin　specificity　of　the　fusion　protein,　giving　a　dissociation　constant　of　∼　25　±　0.9　μM.　In　a　novel　murine　carotid　embolism-induced　stroke　(CES)　model,　i.v.　administration　of　TriF-ΔtPA　promoted　vascular　recanalization,　reduced　infarct　volume,　and　mitigated　neurobehavioral　deficits　more　significantly　compared　to　ΔtPA-HSA　or　Alteplase,　showing　little　bleeding　risk.　Together,　this　long-acting　PAI-1-resistant　thrombolytic　agent　holds　great　potential　for　clinical　applications.　©　2023