Browsing by Author "Nallan Chakravarthula, Tanmaye"

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    Fluorescently conjugated annular fibrin clot for multiplexed real-time digestion analysis
    (Royal Society of Chemistry, 2021-12) Zeng, Ziqian; Nallan Chakravarthula, Tanmaye; Muralidharan, Charanya; Hall, Abigail; Linnemann, Amelia K.; Alves, Nathan J.; Emergency Medicine, School of Medicine
    Impaired fibrinolysis has long been considered as a risk factor for venous thromboembolism. Fibrin clots formed at physiological concentrations are promising substrates for monitoring fibrinolytic performance as they offer clot microstructures resembling in vivo. Here we introduce a fluorescently labeled fibrin clot lysis assay which leverages a unique annular clot geometry assayed using a microplate reader. A physiologically relevant fibrin clotting formulation was explored to achieve high assay sensitivity while minimizing labeling impact as fluorescence isothiocyanate (FITC)-fibrin(ogen) conjugations significantly affect both fibrin polymerization and fibrinolysis. Clot characteristics were examined using thromboelastography (TEG), turbidity, scanning electron microscopy, and confocal microscopy. Sample fibrinolytic activities at varying plasmin, plasminogen, and tissue plasminogen activator (tPA) concentrations were assessed in the present study and results were compared to an S2251 chromogenic assay. The optimized physiologically relevant clot substrate showed minimal reporter-conjugation impact with nearly physiological clot properties. The assay demonstrated good reproducibility, wide working range, kinetic read ability, low limit of detection, and the capability to distinguish fibrin binding-related lytic performance. In combination with its ease for multiplexing, it also has applications as a convenient platform for assessing patient fibrinolytic potential and screening thrombolytic drug activities in personalized medical applications.
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    Multivalent Benzamidine Molecules for Plasmin Inhibition: Effect of Valency and Linker Length
    (Wiley, 2022) Nallan Chakravarthula, Tanmaye; Zeng, Ziqian; Alves, Nathan J.; Emergency Medicine, School of Medicine
    There is an emerging interest in utilizing synthetic multivalent inhibitors that comprise of multiple inhibitor moieties linked on a common scaffold to achieve strong and selective enzyme inhibition. As multivalent inhibition is impacted by valency and linker length, in this study, we explore the effect of multivalent benzamidine inhibitors of varying valency and linker length on plasmin inhibition. Plasmin is an endogenous enzyme responsible for digesting fibrin present in blood clots. Monovalent plasmin(ogen) inhibitors are utilized clinically to treat hyperfibrinolysis‐associated bleeding events. Benzamidine is a reversible inhibitor that binds to plasmin's active site. Herein, multivalent benzamidine inhibitors of varying valencies (mono‐, bi‐ and tri‐valent) and linker lengths (∼1–12 nm) were synthesized to systematically study their effect on plasmin inhibition. Inhibition assays were performed using a plasmin substrate (S‐2251) to determine inhibition constants (Ki). Pentamidine (shortest bivalent) and Tri‐AMB (shortest trivalent) were the strongest inhibitors with Ki values of 2.1±0.8 and 3.9±1.7 μM, respectively. Overall, increasing valency and decreasing linker length, increases effective local concentration of the inhibitor and therefore, resulted in stronger inhibition of plasmin via statistical rebinding. This study aids in the design of multivalent inhibitors that can achieve desired enzyme inhibition by means of modulating valency and linker length.