Plasminogen　activator　inhibitor　1　(PAI-1)　is　the　main　inhibitor　of　tissue-type　and　urokinase-type　plasminogen　activators　(t/uPA)　and　plays　an　important　role　in　fibrinolysis.　Inhibition　of　PAI-1　activity　prevents　thrombosis　and　accelerates　fibrinolysis,　indicating　that　PAI-1　inhibitors　may　be　used　as　effective　antithrombotic　agents.　We　previously　designed　a　PAI-1　inhibitor　(PAltrap)　which　is　a　variant　of　inactivated　urokinase　protease　domain.　In　the　present　study,　we　fused　PAltrap　with　human　serum　albumin　(HSA)　to　develop　a　long-acting　PAI-1　inhibitor.　Unfortunately,　the　fusion　protein　PAltrap-HSA　lost　some　potency　compared　to　PAltrap　(33　nM　vs　10　nM).　Guided　by　computational　method,　we　carried　out　further　optimisation　to　enhance　inhibitory　potency　for　PAI-1.　The　new　PAltrap,　denominated　PAltrap(H37R)-HSA,　which　was　the　H37R　variant　of　PAltrap　fused　to　NSA,　gave　a　six-fold　improvement　of　IC50　(5　nM)　for　human　active　PAI-1　compared　to　PAIt-rap-NSA,　and　showed　much　longer　plasma　half-life　(200-fold)　compared　to　PAltrap.　We　further　demonstrated　that　the　PAltrap(H37R)-1　HSA　inhibited　exogenous　or　endogenous　PAI-1　to　promote　fibrinolysis　in　fibrin-clot　lysis　assay.　PAltrap(H37R)-HSA　inhibits　murine　PAI-1　with　IC50　value　of　12　nM,　allowing　the　inhibitor　to　be　evaluated　in　murine　models.　Using　an　intravital　microscopy,　we　demonstrated　that　PAIt-rap(H37R)-HSA　blocks　thrombus　formation　and　platelet　accumulation　in　vivo　in　a　laser-induced　vascular　injury　mouse　model.　Additionally,　mouse　tail　bleeding　assay　showed　that　PAltrap(H37R)-HSA　did　not　affect　the　global　haemostasis.　These　results　suggest　that　PAltrap(H37R)HSA　have　the　potential　benefit　to　prevent　thrombosis　and　accelerates　fibrinolysis.