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However, the use of tPA is limited due to hemorrhagic transformation (HT) and the narrow therapeutic time window. Previous studies demonstrated that asparagine endopeptidase (AEP), a widely expressed pH\u2010dependent endo\u2010lysosomal cysteine protease, can induce neuronal death during ischemia\u2010reperfusion injury. But whether AEP is engaged in HT during ischemia\u2010reperfusion injury is unclear. In the current study, we expanded the role of AEP on HT after delayed tPA administration.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>In order to investigate the effects of AEP on HT after delayed tPA administration following ischemic stroke, the middle cerebral artery occlusion\/reperfusion (MCAO\/R) was performed in wild\u2010type (WT) and AEP knockout (KO) transgenic mice, followed by delayed administration of tPA (10\u2009mg\/kg, 3\u2009h after occlusion). Additionally, we explored the potential of R13, a specific TrkB agonist with a strong inhibitory impact on AEP, to mitigate injury induced by tPA. 24\u2009h after tPA administration, the following parameters were assessed: infarct volume, behavioral tests, hemorrhagic levels, Evans blue leakage, tight and adherens junction protein expression, blood\u2013brain barrier (BBB) function, cerebral vascular structure, matrix metalloproteinases (MMPs), and BBB\u2010regulated protein low\u2010density lipoprotein receptor\u2010related protein 1 (LRP\u20101) expression. To construct an in\u00a0vitro model to examine the effects of AEP on ischemia\u2010reperfusion injury after tPA treatment, human umbilical vein endothelial cells (HUVECs) were exposed to 4\u2009h of oxygen\u2013glucose deprivation (OGD), followed by treatment with tPA (500\u2009ng\/mL). 7,8\u2010dihydroxyflavone (7,8\u2010DHF), a natural TrkB agonist with an inhibitory effect on AEP, was applied before OGD.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Compared with tPA\u2010treated WT mice, AEP KO mice treated with tPA showed improved infarct volume, neurological function, brain edema, brain hemoglobin levels, Evans blue leakage, vascular tight junctions, and basement membrane structure combined with reduced AEP expression and activity within the peri\u2010infarct area. In addition, the mice treated with R13 exhibited protective effects on the BBB. Furthermore, we found that the expression of MMP2, MMP9, and LRP\u20101 in the brain was inhibited by both AEP knockout and R13 treatment. Moreover, HUVECs treated with 7,8\u2010DHF showed improvements in tight and adherens junction proteins and suppressed levels of MMP2, MMP9, and LRP\u20101.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>Our findings demonstrate that AEP exacerbates HT induced by delayed tPA treatment in acute ischemic stroke by activating LRP\u20101, MMP2, and MMP9, which disrupts BBB integrity. We further confirmed R13 as a preventive therapy to attenuate HT induced by delayed tPA treatment in acute ischemic stroke. The present study suggests AEP inhibition may serve as a promising strategy to enhance the safety of delayed tPA thrombolysis for ischemic stroke.<\/jats:p><\/jats:sec>","DOI":"10.1111\/cns.70345","type":"journal-article","created":{"date-parts":[[2025,3,23]],"date-time":"2025-03-23T10:41:47Z","timestamp":1742726507000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Asparagine Endopeptidase Inhibition Attenuates Tissue Plasminogen Activator\u2010Induced Brain Hemorrhagic Transformation After Ischemic Stroke"],"prefix":"10.1111","volume":"31","author":[{"given":"Guanfeng","family":"Xie","sequence":"first","affiliation":[{"name":"Department of Neurology Renmin Hospital of Wuhan University  Wuhan Hubei Province China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gege","family":"Jiang","sequence":"additional","affiliation":[{"name":"Department of Neurology Renmin Hospital of Wuhan University  Wuhan 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