“The Engineering of Serpins for Vascular Applications” contributes to the understanding of serine protease inhibitors (serpins) in cardiovascular disease, the relationship between their structures and activities, and the manipulations of those structures for the benefit of medicine. Chapter 1 presents a comprehensive overview of serpins’ structure-activity relationships, pathophysiology and their roles in thrombosis, hemostasis and fibrinolysis. Chapter 2 establishes the co-localization of (pro)thrombin with heparin cofactor II (HCII) in the core of atherosclerotic plaques and demonstrates a positive correlation between the presence of HCII, (pro)thrombin or antithrombin (AT) and lesion severity. Additionally, these results indicate that atheromas may act as a non-specific watershed for plasma proteins and should serve as a warning against assumptions that (co-)localization of protein in atheromas implies a pathophysiologic role in atherosclerosis. Chapter 3 utilizes a gain-of-function approach to examine glycosaminoglycan (GAG)-binding in serpins. An α1-protease inhibitor (α1PI) mutant, α1PIPitt-GAG, containing five basic residues homologous to the HCII D-helix was shown to have HCII-like GAGaccelerated thrombin inhibition. Results confirm the benefit of using a gain-of-function approach to the study of serpins. The finding that GAG-binding and associated functionality iv can be added to a non-GAG binding serpin has exciting implications for the engineering of serpins for biomedical purposes. In Chapter 4, a chimeric serpin, HATpin, was engineered with intent to create a potent anti-thrombotic, anti-inflammatory, anti-atherogenic protein. HATpin was designed to comprise 1) the reactive center loop of AT for specificity of thrombin and factor Xa over activated protein C; 2) the N-terminal acidic domain of HCII to utilize thrombin exosite 1 for specific thrombin inhibition; 3) the GAG-binding region of HCII to target it to areas of vascular injury; 4) on an α1PI backbone. Although HATpin was successfully created, its inhibitory profile failed to meet expectations. The results from Chapter 4 illustrate our need for a better understanding of serpin structural domain interactions and challenge the concept that serpin domains are strictly modular building blocks that can be exchanged without a contextual effect. Finally, Chapter 5 outlines additional future studies that can be pursued based on questions that arose during the completion of this dissertation.