Effect of mutations in the heparin-binding exosite of factor IXa on the ability of DHG to inhibit factor X activation by intrinsic tenase. Increasing concentrations of DHG were added to a reaction mixture containing 0.2 nM factor IXa wild type (•), H92A (○), R170A (▪), R233A (□), or K241A (▴) with 1.0 nM factor VIIIa, 200 nM factor X, and 50 μM PC/PS vesicles in 0.15 M NaCl, 20 mM HEPES (pH 7.4), 2 mM CaCl₂, 1 mg/mL BSA, and 0.1% PEG-8000. The rate of factor X activation (nM/min) by the intrinsic tenase complex was determined as described in “Materials and methods.” The rate of factor X activation (nM/min) by intrinsic tenase was normalized to activity present in the absence of DHG. Mean values (% control; no DHG) were plotted with error bars representing ± SD (n = 3 to 6). The inhibition constant (K_i) for DHG was determined by fitting the data by nonlinear regression to the equation for partial, noncompetitive inhibition. The K_i values ± SE for the recombinant proteins were as follows: WT, 1.6 ± 0.1 nM; H92A, 6.5 ± 0.5 nM; R170A, 13.9 ± 2.1 nM; R233A, 113.9 ± 29.3 nM; and K241A, 4.6 ± 0.4 nM.