Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the 4th, 5th, and most of the 6th EGF-like domain (TM456m) has been prepared that has much improved solubility, thrombin-binding capacity, and anticoagulant activity over previous TM456 constructs. In the present work we compare backbone amide exchange of human α-thrombin in three states: apo, PPACK-bound, and TM456m-bound. Beyond causing decreased amide exchange at their binding sites, TM and PPACK both cause decreased amide exchange in regions more distant from the active site, within the γ-loop and the adjacent N-terminus of the heavy chain. The decreased amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the β-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, HDXMS results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation.