Effect of heparin on thrombin inhibition by l-ANT. Shown are the discontinuous measurements for the rates of thrombin inhibition by wild type l-ANT (A) or P1R (C) without or with 0.1 μg/μl LMW or HMW heparin. The apparent second rate constants (kapp) were determined from the slope of the plot of kobs
versus inhibitor concentrations, which are listed in Table 4. Apparent first order rate constants (kobs) for wild type (B) or P1R (D) were measured with LMW or HMW heparin ranging from 10−5 to 10 μg/μl under pseudo-first-order conditions containing 5 nm thrombin and 1.0 μm l-ANT or 0.05 μm P1R in PBS with 0.1 mg/ml BSA and 0.1% PEG 8000. The kobs values were obtained from the slope of semilog plots of residual enzyme activity measured from initial velocities of S-2238 hydrolysis (A405/min) versus time. Filled inverted triangles, l-ANT; open circles, l-ANT + LMW; filled squares, l-ANT + HMW; filled triangles, P1R; open squares, P1R + LMW; filled circles, P1R + HMW.