The uncatalyzed (k_uncat) and pentasaccharide (k_H5) catalyzed second-order rate constants were determined by the incubation of wild-type and mutant proteases (1-2 nM) with AT (0.025-10 μM) in the absence and presence of saturating concentrations of H₅ (1-20 μM) at room temperature in TBS/Ca²⁺. The full-length heparin (k_H70) catalyzed values for both wild-type and mutant APC were determined by the same procedures except that enzymes were incubated with 0.25-5 μM AT and catalytic levels of H₇₀ (0.01-2.5 μM). In the case of thrombin, both wild-type and mutant protease (0.5 nM each) were incubated with 200 nM AT and catalytic levels of heparin (0.5-2.0 nM). The second-order rate constants were calculated from the remaining chromogenic substrate activities as described in “Materials and methods.” The fold conformational effect of H₅ was calculated from the ratio of the second-order rate constant in the presence of H₅ to the same value in the absence of H₅ (k_H5/k_uncat) as described. ¹⁶  The fold template effect of the full-length heparin (H₇₀) was calculated from the ratio of the second- order rate constant in the presence of H₇₀ to the same value in the presence of H₅ (k_H70/k_H5) as described.¹⁶ 

ND indicates not determinable since the incubation of APC with AT (10 μM) for 5 hours resulted in no decline in the chromogenic substrate activity of the enzyme.