Uncatalyzed second-order inhibition rate constants (M⁻¹ second⁻¹) were determined from the residual chromogenic activities of factor Xa or thrombin (1.0 nM) after incubation at room temperature with antithrombin (250-1000 nM) for 5 to 10 minutes in TBS buffer, containing 1 mg/mL BSA, 0.1% PEG 8000, and 2.5 mM Ca²⁺, as described in “Materials and methods.” Except for the reaction of thrombin with the mutant of antithrombin, the pentasaccharide (H₅) and approximately 70-saccharide (H₇₀) high-affinity heparin-catalyzed values were determined by the same procedures, except that 0.2 nM enzyme was incubated with 6.25 to 25 nM H₅ or 0.4 to 10 nM H₇₀ in complex with 100 nM antithrombin.

BSA indicates bovine serum albumin; PEG, polyethylene glycol; ND, not determinable.

The rate of inhibition of thrombin by the mutant antithrombin-H₇₀ complex was measured by incubating thrombin (1.0 nM) with antithrombin (1.0 μM) in complex with 200 nM H₇₀ for 30 minutes at room temperature. Not determinable after incubation of thrombin (1.0 nM) with the antithrombin mutant (1.0 μM) for 60 minutes at room temperature in either the absence or the presence of pentasaccharide (1.0 μM).