Time course of the pentasaccharide-catalyzed inhibition of factor Xa or prothrombinase by the mutant of antithrombin.

(A) Factor Xa (1 nM) was incubated with 500 nM mutant antithrombin in complex with 25 nM pentasaccharide in the absence (○) or the presence of 50 μM PC/PS minus (●) or plus 5 nM factor Va (■) in TBS buffer containing 1 mg/mL BSA, 0.1% PEG 8000, and 2.5 mM Ca⁺⁺. At the indicated time points, aliquots of the reaction were removed to 500 μM SpFXa containing 1 mg/mL Polybrene (Sigma), and the remaining enzyme activity was determined by an amidolytic activity assay using Spectrozyme FXa (American Diagnostica), as described in “Materials and methods.” (B) Factor Xa (1 nM) in the absence (○) or the presence of 50 μM PC/PS (●) or 0.2 pM factor Xa in complex with 5 nM factor Va on 50 μM PC/PS (■) was incubated with 1.5 μM prothrombin in the same TBS buffer system. Time course of thrombin generation in the absence and the presence of 2.3 μM antithrombin mutant in complex with 25 nM (○, ●) or 1000 nM (■) pentasaccharide was monitored from hydrolysis of S2238. Thrombin generation in the absence of heparin was normalized to 100, and the percentage inhibition of thrombin generation (y-axis) was plotted as a function of varying concentrations of the antithrombin-heparin complex (x-axis), as described in “Materials and methods.” Solid lines are nonlinear regression fit of data to a pseudo first-order rate equation.