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Abstract

Cardiovascular diseases (CVDs)—such as heart attack, stroke, and pulmonary embolism—continue to be a leading cause of death globally, representing a major health concern. Thrombosis, the condition of excessive clot formation inside arteries or veins, is the primary cause of CVDs. In clinical settings, heparin-based anticoagulants are generally administered to manage and treat these thrombotic conditions. Heparin is a fast-acting anticoagulant and is particularly a suitable anticoagulant for emergency scenarios to manage clot formation. It is available in many different forms, ranging from its natural occurring high-molecular-weight form, unfractionated heparin (UFH), to low-molecular-weight heparins (LMWH) and synthetic derivatives. However, despite its therapeutic significance, the use of heparin-based anticoagulants is associated with serious side effects, such as heparin-induced thrombocytopenia (HIT), excessive bleeding, and acute allergic reactions. This underscores an urgent need for an effective antidote capable of neutralizing all forms of heparin. Recently a class of synthetic molecules known as Universal Heparin Reversal Agents (UHRAs) has shown promising results for safely neutralizing all forms of heparin. However, the binding affinity of these molecules is currently limited to micromolar range. In order to tailor the design, there is a need to understand the driving forces for binding. This thesis presents the first detailed investigation of the structure and the thermodynamics of UHRA and heparin interaction using a combination of Nuclear Magnetic Resonance spectroscopy, Isothermal Titration Calorimetry, and Molecular Dynamics Simulations. From this study, a new understanding of both the structural characteristics of UHRAs and the thermodynamic behaviour of their interaction with heparin has emerged which differs from the original hypothesis that guided the design of these molecules. This study reveals that UHRA size is insensitive to salt conditions and temperature which is important for the storage and therapeutic use. Furthermore, it is found that the binding between UHRA and heparin is driven by ion-pair formation. This study also reveals that there is no significant hydrogen bond formation between UHRA and heparin. This knowledge could play a critical role in improving the affinity and specificity of these molecules, potentially leading to the development of more effective heparin reversal agents.