Bleeding complications, especially intracranial hemorrhage (ICH), are the major concerns of tissue plasminogen activator (tPA) in the treatment of patients with thrombotic conditions, such as heart attack, stroke and venous thromboembolism. The short half-life of tPA requires intravenous infusion of a large dose of tPA over several hours in order to achieve restoration of blood flow. As a result, an excessive lytic state in circulation is evident due to systemic plasmin activity that seriously depletes important clotting factors, thereby increasing the risk of bleeding. In addition, tPA itself can act as a cytokine stimulating production of matrix degrading enzymes, such as matrixmatalloprotease-9 (MMP-9) that significantly contributes to blood-brain barrier integrity, leading to ICH. Therefore, a strategy for targeted and triggered plasminogen activation is desired in order to achieve 1) controlled activation of tPA for reduction of systemic activation of plasminogen; and 2) diminished production of MMPs at the brain capillary endothelial cells. This strategy involves reversible complexes of tPA and serum albumin that are joined by biotin-avidin binding. Biotin, a nontoxic nutrient, will be used to locally trigger tPA release by removing the bulky targeting complex by breaking biotin/avidin interaction. The goal of this project is to design an inactive prodrug construct oftPA that can be controlled by a safe nutrient. It is anticipated that such system can be constructed and can demonstrate controlled activation of tPA, as well as reduction of MMP-9 production by brain endothelial cells. Successful completion of this project will further pave the way for a new paradigm for a safer and effective therapeutic modality for this type of drug by overcoming some of the major drawbacks of current thrombolytic treatment strategies.