Administration　of　recombinant　tPA　(rtPA,　or　trade　name　Alteplase®)　is　an　FDA-approved　therapy　for　acute　ischemic　stroke　(AIS),　but　poses　the　risk　of　hemorrhagic　complications.　Recombinant　tPA　can　be　rapidly　inactivated　by　the　endogenous　inhibitor,　plasminogen　activator　inhibitor　1　(PAI-1).　In　this　work,　we　study　a　novel　treatment　approach　that　combines　a　PAI-1　inhibitor,　PAItrap4,　with　a　reduced　dose　of　rtPA　to　address　the　hemorrhagic　concern　of　rtPA.　PAItrap4　is　a　highly　specific　and　very　potent　protein-based　inhibitor　of　PAI-1,　comprising　of　a　variant　of　uPA　serine　protease　domain,　human　serum　albumin,　and　a　cyclic　RGD　peptide.　PAItrap4　efficiently　targets　and　inhibits　PAI-1　on　activated　platelets,　and　also　possesses　a　long　half-life　in　vivo.　Our　results　demonstrate　that　PAItrap4　effectively　counteracts　the　inhibitory　effects　of　PAI-1　on　rtPA,　preserving　rtPA　activity　based　on　amidolytic　and　clot　lysis　assays.　In　an　in　vivo　murine　stroke　model,　PAItrap4,　together　with　low-dose　rtPA,　enhances　the　blood　perfusion　in　the　stroke-affected　areas,　reduces　infarct　size,　and　promotes　neurological　recovery　in　mice.　Importantly,　such　treatment　does　not　increase　the　amount　of　cerebral　hemorrhage,　thus　reducing　the　risk　of　cerebral　hemorrhage.　In　addition,　PAItrap4　does　not　compromise　the　normal　blood　coagulation　function　in　mice,　demonstrating　its　safety　as　a　therapeutic　agent.　These　findings　highlight　this　combination　therapy　as　a　promising　alternative　for　the　treatment　of　ischemic　stroke,　offering　improved　safety　and　efficacy.　Copyright　©　2023　Elsevier　B.V.　All　rights　reserved.