Recombinant　tissue-type　plasminogen　activator　(r-tPA)　was　approved　by　U.S.　Food　and　Drug　Administration　as　a　thrombolytic　drug.　However,　a　high　dose　of　r-tPA　(up　to　100　mg/person)　is　typically　used　in　clinical　applications.　Such　high　dosage　leads　to　severe　side　effects　including　haemorrhage　and　neurotoxicity,　which　can　be　fatal.　To　improve　the　proteolytic　properties　of　tPA　to　enhance　thrombolytic　therapy,　we　designed　a　series　of　mutants　in　tPA　serine　protease　domain　(tPA-SPD)　based　on　the　crystal　structure　of　tPA-SPD:plasminogen　activators　inhibitor-1　(PAI-1)　complex　that　we　determined　recently.　We　found　that　the　A146Y　substitution　in　tPA-SPD(A146Y)　enhanced　resistance　to　PAI-1　inactivation　by　30-fold　compared　with　original　tPA-SPD.　Interestingly,　the　tPA-SPD(A146Y)　variant　showed　fivefold　higher　activation　for　plasminogen　compared　with　tPA-SPD.　The　variant　also　demonstrated　thrombolytic　activity　stronger　than　tPA-SPD　in　a　clot　lysis　assay.　In　vivo,　we　showed　tPA-SPD(A146Y)　possessed　higher　thrombolytic　efficacy　in　a　pulmonary　embolism　model　compared　with　original　tPA-SPD.　Furthermore,　a　mouse　tail　bleeding　assay　showed　that　tPA-SPD(A146Y)　did　not　increase　bleeding　risk　compared　with　clinical　drug　r-tPA.　Together,　our　findings　reveal　novel　functions　of　A146Y　variant,　which　not　only　increases　the　catalytic　efficiency　of　the　enzyme,　but　also　enhances　resistance　to　PAI-1　inhibition,　and　demonstrating　that　tPA-SPD　(A146Y)　variant　is　a　much　improved　agent　for　thrombolytic　therapy.