Synergistic Thrombolysis–Antiplatelet Therapy of Engineered tPA Variant citPA5 and Tirofiban in a Mouse Stroke Model

Acute ischemic stroke is a leading cause of global disability and mortality. Current therapies are limited by a narrow 4.5-hour treatment window, high risk of bleeding, frequent reocclusion post-thrombolysis, and poor neuroprotective/repair capacity. The thrombolytic activity of citPA5 with or without tirofiban was first assessed using in vitro clot lysis assays. Therapeutic efficacy and safety were then evaluated in a murine carotid embolism-induced stroke model. Mice received citPA5 plus tirofiban at 0.5 hours after occlusion. Cerebral blood flow, infarct volume, neurological function, platelet accumulation, microglial activation, and cerebral hemorrhage were assessed. In vitro, tirofiban did not impair citPA5-mediated thrombolysis. In vivo, citPA5 plus tirofiban improved cerebral blood flow recovery to 71.7% at 3 hours and 89.2% at 24 hours, reduced infarct volume to 4.2%, and accelerated neurological recovery, with significant improvement sustained over a 7-day observation period. Immunofluorescence analysis showed modest reduction of platelet accumulation and marked attenuation of CD68+Iba1+ microglial activation, with partial restoration of ramified microglial morphology. No significant cerebral hemorrhage was observed. Combined citPA5 and tirofiban therapy enhances reperfusion, mitigates thrombo-inflammatory injury, and promotes both acute and longer-term neurological recovery in a mouse ischemic stroke model. These findings support this dual thrombolytic–antiplatelet strategy as a potentially safe and effective therapeutic approach for ischemic stroke.