Thrombin inhibition by 2c in the presence of UFH (A) and HirP (B), and by 2i in the presence of UFH (C) and HirP (D). Residual thrombin activity was measured by determining Spectrozyme TH hydrolysis in 20 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, 2.5 mM CaCl2, and 0.1%PEG8000 at 25 °C in the presence of 0 to 50 nM HirP and 0–50 μM of UFH or 0–30 nM of HirP. Solid lines represent fits using a logistic equation (see Supporting Information) to obtain the apparent IC50, as described in experimental procedures.