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Abstract

Thrombosis is a common pathology that underpins ischemic heart disease, ischemic stroke, and venous thromboembolism and has been linked to sepsis and cancer. Polyanionic molecules such as polyphosphates, cell-free DNA, Neutrophil Extracellular Traps (NETs), and extracellular RNA have been shown to be the mediators of thrombosis. NETs are released from neutrophils when activated by microbial or inflammatory stimuli. These web-like structures are composed of cell-free DNA, histones, and antimicrobial proteins. They have been shown to trap and kill microorganisms, playing a critical role in host defense. The presence of NETs has been observed in sepsis and is shown to promote Disseminated Intravascular Coagulation (DIC) and multiorgan failure. This thesis presents a strategy for inhibiting nucleic acid-based mediators of thrombosis with Polycationic Nucleic Acid Binding Inhibitors (PNBIs). The general architecture of these PNBIs is similar to the Universal Heparin Reversal Agent (UHRA) class of molecules previously reported from our lab. They have a core of hyperbranched polyglycerol (HPG) with cationic binding groups (R) and a brush layer of methoxy polyethylene glycol (mPEG) to protect the R groups from non-specific interactions. We screened a library of such polycationic compounds developed in our lab with different R group types, R group numbers, and molecular weights of HPG core and identified effective candidates that target polyanionic nucleic acids and reverse their procoagulant potential in vitro. We also showed that lead PNBIs could reverse the altered clot properties caused by nucleic acids. Next, we demonstrated the inhibition of NET-associated immunothrombosis by PNBIs in the murine Cecal Ligation and Puncture (CLP) polymicrobial sepsis model. In addition to promoting thrombosis, NETs have been shown to increase hyperinflammation in sepsis. We showed that PNBIs could reduce the cytokine storm and the elevated coagulation parameters in this model while remaining well tolerated in mice. Besides demonstrating the inhibition of nucleic acids in both in vitro and in vivo, this work also provides new insights into the mechanism of coagulation activation by nucleic acids. The findings described in this thesis may pave the way for developing non-toxic molecules that target nucleic acids as new antithrombotic therapy.