Linkage effects on inhibitor potency of EP compounds. (A) Shown are the decrease in initial velocities of substrate hydrolysis for thrombin, aPC, and factor XIa measured as a function of increasing EP217609 concentration in the absence (solid symbols) or presence (open symbols) of 0.1, 0.5, and 1μM antithrombin, respectively. Solid and dashed lines indicate fits to the competitive binding equations in the text for titrations in the absence or presence of antithrombin, respectively. Values of K_I* obtained from fits were corrected for competitive substrate binding for aPC and factor XIa. (B) Progressive inhibition of factor Xa by increasing concentrations of antithrombin–EP42675 (triangles) and antithrombin–EP217609 (circles) complexes was measured in the absence (open symbols) and presence (closed symbols) of thrombin levels equimolar with EP compound for reactions containing 200nM antithrombin, 0.5nM factor Xa, and the indicated EP concentrations for a fixed reaction time of 300 seconds. Inverted triangles indicate controls in which antithrombin was omitted from factor Xa reactions with equimolar EP42675 and thrombin. Solid lines indicate fits by a single exponential decay function, which yielded k_obs values. These were used to calculate the dissociation constant for antithrombin binding to the EP-thrombin binary complex as described in “Linkage effects.”