Inhibition of thrombin by PAI-1 in the presence of heparin. (A) Initial rates of thrombin (36.6 nmol/L) inhibition in the presence of heparin and PAI-1 (concentrations as indicated), were determined. Initial rates were corrected for the presence of DAPA (182.9 nmol/L) as described under Experimental Procedures. (▴), 190 nmol/L PAI-1; (•), 570 nmol/L PAI-1; (▪), 1,476 nmol/L PAI-1. (B) Initial rates of thrombin (30 nmol/L) inhibition by increasing concentrations of PAI-1 (100 to 1,800 nmol/L) were measured at a fixed concentration of heparin (0.17 U/mL; 60 nmol/L) and corrected for the presence of DAPA (370.4 nmol/L) as described under Experimental Procedures. (C) Initial reaction rates were measured at fixed concentrations of heparin (0.17 U/mL; 60 nmol/L), PAI-1 (1 μmol/L), and DAPA (150 nmol/L) at various concentrations of thrombin. Rates were corrected for the presence of DAPA, according to equation (2), as described under Experimental Procedures. The linear increase in the double-logarithmic plot of rate versus thrombin exhibits a slope of 1.0, indicating that the reaction is first-order with respect to thrombin. The inset shows the relation between fractional lifetime (t_1/3 ) as a function of thrombin concentration. The inflection point indicates a transition from first-order to zero-order kinetics with respect to thrombin concentration, as a result of saturation of the heparin template. For further details see Experimental Procedures.