Modulating Fibrinolysis Dynamics by Leveraging Multivalent Avidity to Control Enzyme Activity

点击翻译按钮获取中文摘要

PROJECT SUMMARY Blood clots kill more humans than any other single pathogenic cause. A new or recurrent diagnosis of venous thromboembolism (VTE), which encompasses both pulmonary embolism (PE) and deep vein thrombosis (DVT), affects >900,000 people each year in the US alone. Current PE standard of care includes the delivery of acute clot digesting enzymes such as recombinant tissue Plasminogen Activator (tPA), administered IV or by catheter. All currently FDA cleared clot digesting enzymes rely on activation of the patient’s endogenous plasminogen converting nearly all of it to active clot digesting plasmin. This widespread activation massively depletes plasmin regulatory proteins, causing unchecked digestion of both pathogenic and beneficial blood clots resulting in a 5-10% rate of major bleeding complications, including intracranial hemorrhage. Due to safety concerns and contraindications nearly 50% of patients that would benefit from active clot digesting interventions are not eligible to receive the current therapy. Direct infusion of exogenous active plasmin to digest blood clots is limited due to the nearly instantaneous inactivation by circulating α2-antiplasmin (serpin protein) and α2-macroglobulin (steric inhibitor). To address the current limitations of plasmin infusion as a direct fibrinolytic therapeutic intervention, a novel clot digesting delivery strategy leveraging multivalent enzymatic control is necessary. Preliminary data demonstrates that reversible competitive inhibitors can be used to deliver active plasmin with reduced inactivation from regulatory proteins in-vitro while still allowing efficient clot digestion; however, there is critical need to determine the underlying characteristics responsible for protection and delivery of plasmin as a direct fibrinolytic in-vivo. This proposal will identify critical relationships that contribute to multivalent control of active enzymes in-vivo for the novel clinical translation of therapeutic interventions to digest blood clots with an improved safety profile. Aim 1: Determine how homo- and hetero-multivalent inhibitors impact inhibition of plasmin and its interactions with regulatory proteins in- vitro; Aim 2: Evaluate the impact of reversible multivalent enzyme inhibition on clot digestion efficiency of standardized ex-vivo blood clots under shear; and, Aim 3: Examine the impact of multivalent inhibitor delivered plasmin on clot targeting and digestion in-vivo. Overall, these experiments will identify key multivalent enzyme delivery principles and provide lead compounds to be further developed. A safer direct fibrinolytic therapeutic will have expanded indications for use that include: ischemic stroke, myocardial infarction, limb ischemia, PE, DVT, and occluded vascular catheters.