Aims:　Alteplase　is　a　cornerstone　thrombolytic　agent　in　clinical　practice　but　presents　a　potential　bleeding　risk.　Stroke　patients　need　pre-screening　to　exclude　haemorrhagic　stroke　before　using　alteplase.　In　this　study,　we　develop　a　new　thrombolytic　agent　citPA5,　characterized　by　an　enhanced　safety　profile　and　minimal　bleeding　tendency.　Methods　and　results:　A　clot　lysis　agent,　named　citPA5,　is　developed　based　on　rtPA　with　point　mutations　to　completely　suppress　its　proteolytic　activity　in　the　absence　of　fibrin.　In　the　presence　of　fibrin,　citPA5　exhibited　significantly　higher　fibrinolytic　activity　(a　15.8-fold　increase　of　kcat/Km).　Furthermore,　citPA5　showed　resistance　to　endogenous　fibrinolysis　inhibitor,　PAI-1,　resulting　in　enhanced　potency.　In　a　series　of　safety　evaluation　experiments,　including　thrombelastography　assay,　mice　tail　bleeding　assay,　and　a　murine　intracerebral　haemorrhage　(ICH)　model,　citPA5　did　not　cause　systemic　bleeding　or　worsen　ICH　compared　with　alteplase.　This　highlights　the　low　risk　of　bleeding　associated　with　citPA5.　Finally,　we　found　that　citPA5　effectively　improved　cerebral　blood　flow　and　reduced　infarct　volume　in　a　carotid　embolism-induced　stroke　model.　Conclusion:　This　clot　lysis　agent,　citPA5,　not　only　exhibits　a　low　risk　of　bleeding　but　also　demonstrates　highly　effective　thrombolysis　capabilities.　As　a　result,　citPA5　shows　great　potential　for　administration　prior　to　the　classification　of　stroke　types,　making　it　possible　for　use　in　ambulances　at　the　onset　of　stroke　when　symptoms　are　identified.　The　findings　presented　in　this　study　also　suggest　that　this　strategy　could　be　applied　to　develop　a　new　generation　of　fibrinolytic　drugs　that　offer　greater　safety　and　specificity　in　targeting　fibrin.　　©　2024　The　Author(s).　Published　by　Oxford　University　Press　on　behalf　of　the　European　Society　of　Cardiology.　All　rights　reserved.