Argatroban inhibition of thrombin bound to aged fibrin clots (clot permeation assay). Fibrin clots (50 μL each) were formed in the wells of a microtiter plate by addition of thrombin (to 0.5 NIH units/mL) to fibrinogen (1 mg/mL) in HBS, pH 7.4, and then aged for 0.5 hours (○), 3 hours (△), or 6 hours (•) at 23°C before permeation with argatroban (0 to 30 μmol/L). After removal of excess inhibitor, thrombin activity was determined with chromogenic substrate S-2238, and the results were presented as the initial rate of color development at 405 nm (expressed as milli-OD per minute determined in a Vmax Kinetic Microtiter Reader). These data were fit to equation 1 to determine the IC₅₀, ie, the concentration of argatroban that reduced the clot-bound thrombin activity to 50% of its maximum value. Resultant IC₅₀ values and the maximum inhibition are presented in Table 1. Data shown as △ were obtained by clotting fibrinogen in HBS, pH 7.4, containing 2 mmol/L CaCl₂ and 3 mg/mL bovine serum albumin, and then incubating for 3 hours before permeation with argatroban (0 to 300 μmol/L), followed by removal of excess inhibitor and addition of chromogenic substrate S-2238. The solid line was calculated from equation 2, using the IC₅₀(0.8 ± 0.1 μmol/L) and noninhibited fraction (0.11 ± 0.01) determined by nonlinear regression analysis. This procedure also yields the uncertainty in each fitted parameter, expressed as its standard deviation. (Insert) Time-dependent changes in the activity of thrombin, in solution and bound to fibrin clots. Thrombin (0.5 NIH units/mL) was incubated either in HBS, pH 7.4 (□) or in fibrin clots (▪) (1 mg/mL) for the indicated period before the addition of S-2238. Thrombin activity is expressed as the initial rate parameter in milli-OD per minute.