Abstract 305: Development of an Antidote-Controlled RNA Probe for Molecular Thrombi Imaging

Vascular thrombosis is a major underlying factor in many cardiovascular diseases worldwide and is also a major post-surgical complication. Early detection and treatment of thrombi improves outcomes for patients since the responsiveness of thrombi to fibrinolytic treatment decreases with thrombus age. The serine protease thrombin plays a central role in thrombogenesis and remains associated with the thrombus thereby facilitating further activation of coagulation factors and platelets after initial clot formation. Therefore, targeting and imaging clot-bound thrombin may provide a way of distinguishing newly formed thrombi from older, constituted ones. The goal of this study was to determine whether a thrombin-binding RNA aptamer conjugated to a near-infrared fluorophore could detect clot-bound thrombin and if a complementary antidote oligonucleotide could remove clot-bound aptamer thereby creating a subtraction image. The thrombin-binding aptamer conjugate, Tog25t-AF680, was assessed for its in vitro binding capability to newly formed human plasma clots using fluorescence reflectance imaging. In comparison to a labeled non-binding control RNA, we observed a greater than 6-fold increase in near-infrared fluorescence (NIRF) target-to-background ratio (TBR) with Tog25t-AF680. Upon treatment with the antidote oligonucleotide (AO4), we observed an approximately 57% reduction in fluorescence signal after 10 minutes of incubation. Our results suggest that thrombin is a suitable target for imaging newly formed clots and that a RNA aptamer conjugate binds to clot-bound thrombin with high specificity. This thrombin-targeting near-infrared probe has the potential to be used as a diagnostic tool for arterial and venous thrombosis and may further advance our understanding of the role of thrombin in vivo.