Thrombin is a serine protease that has both procoagulant and anticoagulant functions in the blood clotting cascade. The blood contains a high concentration of prothrombin, which must be proteolytically cleaved at two sites to generate active α-thrombin. Active thrombin cleaves fibrinogen to create fibrin which polymerizes into clots. Very little α-thrombin is ever generated, and this is rapidly captured by either thrombomodulin (TM) and/or antithrombin III. Regulation of thrombin is imperative to maintaining normal hemostasis and to restrict clot extension. TM binds at anion binding exosite I (ABE I) of thrombin, which is the recognition site for fibrinogen and hirudin. TM acts as a molecular switch, changing the specificity of thrombin from procoagulant activity, defined as cleavage of fibrinogen, to anticoagulant activity, defined as cleavage of protein C. This work investigates the allosteric influence TM has on the active site of thrombin by fluorescence spectroscopy and isothermal titration calorimetry (ITC).

Chapter II describes the expression, purification, and inhibition activity of TMEGF56 which binds ABE I of thrombin but lacks EGF4 and therefore does not have cofactor activity toward protein C. We show that we have produced large quantities (20 mg/fermentation) of protein from Pichia pastoris that has the same binding affinity as TMEGF56 made from Chinese hamster ovary cells. In Chapter III several mutations in TMEGF45 and TMEGF456 were studied that affect protein C activation but do not contact thrombin according to the crystal structure of the thrombin-TMEGF456 complex. We measure changes in fluorescence of a covalently bound active site fluorophore when thrombin is titrated with TM. TM variants with the most cofactor activity exhibit the largest affect on fluorescence of the active site fluorophores. The results show that mutations in TM that do not contact thrombin directly decrease cofactor activity and decrease the way TM alters the active site of thrombin. Chapter IV presents a study of the thermodynamics of thrombin in the presence of its cofactor, TM, by ITC. Here we measured the binding thermodynamics of a non-covalent thrombin active site ligand. When various molecules were bound at ABE I of thrombin, an active TM cofactor (TMEGF45), inactive TM cofactor (TMEGF56), or a DNA aptamer, the thermodynamic profile observed for the active site ligand changed. We suggest that TM is more than a docking site for PC. It allosterically influences the active site of thrombin, without an overall change in ΔG. This enthalpy-entropy compensation may be a hallmark of dynamic allostery.