UC San Diego

The final step of the blood coagulation cascade is the activation of thrombin. Active thrombin cleaves fibrinogen to create fibrin which polymerizes into clots. Regulation of thrombin is imperative to maintaining normal hemostasis. The blood contains a high concentration of prothrombin, which must be proteolytically cleaved at two sites to generate active [alpha]-thrombin. Very little [alpha]-thrombin is ever generated, and this is rapidly captured by either thrombomodulin (TM) and/or antithrombin III. This work investigates the dynamics of thrombin by ITC and H/D exchange mass spectrometry, characterizes new and improved fragments of TM, and beings preliminary work with potential biologically useful TM fusion proteins for in vivo use. In Chapter I, I introduce the coagulation cascade and parts that thrombin and thrombomodulin play in maintaining hemostasis. Chapter II describes the work that I did using ITC and DSC to characterize the affects of various ligands binding to thrombin and how that binding allosterically altered the active site, ABEI, and even change the thermal stability of thrombin. In Chapter III, I summarize the work done trying to make new constructs of TM with improved activity and binding affinity toward thrombin without the negative impact on protein stability and present two new constructs that show significant improvement overall. Chapter IV is our most recent HD exchange mass spectrometry work looking at the effects of TM456t binding to both human and bovine thrombin, and how much this binding changes regions all throughout the protein. The results there show significant dynamic change in the entire molecule. And finally, Chapter V highlights our first attempts at using TM as part of a fusion protein designed to target activated platelets by the & alpha]IIb[beta]3 integrin, potentially bringing a strong anticoagulant to sites of blood clots