Recombinant　tissue-type　plasminogen　activator　(rtPA)　is　the　clot　lysis　drug　approved　for　clinical　use,　and　is　characterised　by　a　short　half-life　and　substantial　inactivation　by　plasminogen　activator　inhibitor-1　(PAI-1).　We　previously　discovered　that　a　tPA　mutation　(A419Y)　at　the　protease　domain　led　to　enhanced　fibrinolysis　activity.　In　the　present　study,　we　studied　the　mechanism　of　such　mutation　in　enhancing　the　proteolytic　activity,　and　whether　such　enhancement　persists　in　reteplase,　an　United　States　Food　and　Drug　Administration-approved　tPA　truncated　variant.　We　constructed　and　expressed　a　series　of　reteplase-based　mutants,　including　rPA(G)　(glycosylated　rPA),　rPA(G)-Y　(with　A419Y　mutant　at　rPA(G)),　rPA(G)-A4　(tetra-alanine　mutation　at　37-loop　of　rPA(G)),　and　rPA(G)-A4/Y　(with　both)　and　evaluated　their　plasminogen　activation　and　PAI-1　resistance.　Surface　plasmon　resonance　analysis　showed　that　the　rPA(G)　had　fibrin　affinity　comparable　to　full-length　tPA.　Moreover,　rPA(G)-Y　had　8　center　dot　5-fold　higher　plasminogen　activation　and　stronger　tolerance　to　PAI-1　compared　to　rPA(G).　We　also　found　that　the　mutations　containing　tetra-alanine　(rPA(G)-A4　and　rPA(G)-A4/Y)　had　dramatically　reduced　plasminogen　activation　and　impaired　clot　lysis.　In　a　pulmonary　embolism　murine　model,　rPA(G)-Y　displayed　a　more　efficient　thrombolytic　effect　than　rPA(G).　These　results　identified　a　novel　mutant　reteplase　variant　of　tPA　with　increased　fibrinolytic　activity,　laying　the　foundation　for　the　development　of　a　new　potent　fibrinolytic　agent.