Fig. (3) A discontinuous assay to measure thrombin inhibition rate in the absence (A) and presence (B) of heparin. A fixed amount of thrombin (0.125 NIH units) was incubated with Serp-1 (500 ng) at room temperature. At indicated time points (1, 3, 5, 10, 15, and 20 minutes), the chromogenic substrate (S-2238) containing 4 mg/ml Polybrene was added to a final concentration of 0.2 mM. Absorbance at 405 nm (Y axis) was monitored by kinetic measurement every 1 minute for 1800 seconds (X axis) at room temperature.Slopes represent the initial reaction velocity from residual thrombin and are used to calculate Ka value using the equation described in “Materials and Methods”