Neurotoxicity of the anticoagulant-selective E149A-activated protein C variant after focal ischemic stroke in mice

Wild type (WT) activated protein C (APC) and cytoprotective-selective APC variants such as 3K3A-APC (< 10% anticoagulant but normal cytoprotective activity) are neuroprotective in murine focal ischemic stroke models. Here we compared the neuroprotective effects of the anticoagulant-selective E149A-APC variant (> 3-fold increased anticoagulant activity but defective cytoprotective activities) to those of the cytoprotective-selective 5A-APC variant (< 10% anticoagulant activity). After transient distal middle cerebral artery occlusion, mice received a vehicle, E149A-APC or 5A-APC at 0.2 mg/kg at 4 h after stroke. Treatment with 5A-APC was neuroprotective, as it improved performance on forelimb use asymmetry test and foot fault test (P < 0.05), reduced by 48% and 50% the infarct and edema volumes, respectively (P < 0.05), and was not associated with an increased risk of bleeding as indicated by normal hemoglobin levels in the ischemic brain at day 7. In contrast, E149A-APC treatment worsened neurological outcome determined by foot fault tests and forelimb use asymmetry tests, and increased significantly by 44% and 60% infarct and edema volume, respectively (P < 0.05). At 7 days after treatment, E149A-APC compared to vehicle or 5A-APC notably increased by ~ 5-fold the hemoglobin level in the ischemic hemisphere suggesting it provoked significant intracerebral bleeding. Thus, the enhanced anticoagulant activity of E149A-APC increased post-ischemic accumulation of neurotoxic erythrocyte-derived hemoglobin which likely worsened the neurological and neuropathological outcomes after stroke. Our data emphasize that APC's cytoprotective activities, but not its anticoagulant activity, are key for APC neuroprotection after transient ischemic stroke.