Schematic comparison of reactions of thrombin with natural and engineered serpins. The upper panel depicts the reaction of thrombin (yellow) with heparin-activated HCII or HCII-API fusion protein HAPI M358R (blue). (1) Thrombin (IIa, with pie-shaped active site canyon opening and labelled exosite 1 (exo) encounters heparin-activated HCII or fusion protein HAPI-M358R, both containing HCII 1-75 (1-75, blue teardrop-shaped extension) and a Reactive Centre Loop (RCL, blue triangle) on a serpin scaffold (blue large oval). Interactions between HCII 1-75 and exosite 1 and the serpin RCL with thrombin’s active site guide the serpin into productive encounter complex formation (2). Aligned encounter complex formation leads to final serpin-enzyme complex formation, with translocation of thrombin; the RCL is no longer visible due to its conversion into a β-strand inserted into the body of the serpin (3). The lower panel depicts the reaction of thrombin (yellow) with HV3API M358R (blue), in which the C-terminal triskaidecapeptide of HV3 (blue circular extension) was substituted for HCII 1-75. For this fusion protein, initial complexes form which comprise either a sub-optimally aligned complex in which exosite 1 and HV3 interact without engagement of the RCL with thrombin’s active site (2a), or the appropriately aligned encounter complex forms directly (2b). Suboptimally aligned complexes from (2a) can proceed to optimal alignment (2b) but misalignment is predicted to be more likely with HV3API M358R than with HAPI M358R or heparin-activated HCII. Apart from the greater size of the serpins with respect to thrombin, diagrams are not to scale.