Effect of avidin on EP217609-accelerated antithrombin inhibition of factor Xa. (A) EP217609-accelerated antithrombin inhibition of factor Xa activity is shown as a function of EP217609 concentration in the absence and presence of avidin. Reactions contained 200nM antithrombin, 5nM factor Xa, the indicated EP compound concentration, and 0 (●), 12nM (▴), or 24nM (▵) avidin as indicated for a fixed reaction time of 300 seconds. Solid lines indicate fits by a single exponential function from which k_obs was obtained. (B) Kinetic titrations of the binding of EP217609 to 50nM antithrombin in the absence (●) and presence (▴) of 1000nM avidin followed from the enhancement by the EP compound of the rate of antithrombin inhibition of 5nM factor Xa. Plotted are observed pseudo-first-order rate constants (k_obs) calculated from the extent of antithrombin inhibition of factor Xa in the presence of the indicated concentrations of EP217609 in the absence or presence of avidin for 1-minute reactions. Solid lines indicate fits to the quadratic binding equation from which K_D for the antithrombin–EP217609 interaction and the maximal rate constant for the antithrombin–EP217609 reaction with factor Xa were obtained. It should be noted that reciprocal effects of antithrombin binding on avidin binding are insignificant because avidin binds biotin with a femtomolar K_D.