Development of the Plasmin-independent Thrombolytic Agent for the Treatment of Acute Ischemic Stroke

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Project Summary Thrombosis caused by intravascular blood clots is the most common underlying pathology of the three major cardiovascular disorders: ischemic heart disease, stroke, and venous thromboembolism. The significantly high incidence rate, as well as its fatal consequences, has called the search for safe and effective thrombolytic enzymes, which remained extensive throughout the past several decades. Despite the effort, current thrombolytics have significant drawbacks, including bleeding complications. All the current thrombolytic agents are involved in the activation of plasminogen to plasmin. Unfortunately, the non-specific proteolytic activity of plasmin degrades essential proteins involved in hemostasis and wound healing. Thus, the clinical use of plasmin- dependent thrombolytic therapy has been limited, requiring the initiation of treatment within 3 hours from stroke onset to avoid fatal hemorrhagic complications. A need for new thrombolytics remains urgent for improved treatment without concerns of hemorrhage and tissue injury complications. SNJ Pharma, Inc. identified HtrA1 (high-temperature requirement A1, named as thrombase) that appears to exert strong thrombolytic activity on intravascular clots while permitting normal wound healing. Unlike all currently available thrombolytics, HtrA1 protease does not activate plasminogen to plasmin, a non-specific protease responsible for fatal hemorrhagic complications from thrombolytic therapy. The high specificity of HtrA1 enzyme activity enables selective degradation of pathological thrombi while preserving hemostatic clots, removing the risk of massive bleeding. In vivo murine models, including wound excision and tail bleeding assays, demonstrated that animals treated with HtrA1 exhibited no damage in wound healing in contrast to current thrombolytics. In addition, HtrA1 administration completely dissolved blood thrombi in mice with tail thrombosis and intravascular thrombi in mice with pulmonary embolism when administrated intravenously, resulting in rescued thromboembolism. Here, upon confirming the specificity of HtrA1 activity and preservation of beneficial hemostatic clots, we aim to evaluate the therapeutic efficacies of HtrA1 protease and its potential for the treatment of acute ischemic stroke (AIS) using in vitro thrombectomy model and in vivo photothrombotic AIS model. Our successful completion of this proposal with the first plasmin-independent thrombolytic enzyme will present HtrA1 protease as a revolutionary therapeutic candidate for AIS without the drawback of a limited treatment window in the absence of hemorrhage and tissue injury complications.