Argatroban inhibition of thrombin bound to aged fibrin clots (clot perfusion assay). (Insert) Time-course of clot perfusion assay. Clots were formed by the addition of thrombin (0.5 NIH units/mL) to fibrinogen (1 mg/mL) in HBS, pH 7.4, containing CaCl₂ (2 mmol/L) and bovine serum albumin (3 mg/mL). The solution was then (before the gel point) transferred to an absorbance flow-cell (path length, 1 cm) in a spectrophotometer. Relative intensity, ie, the absorbance at 405 nm expressed as a fraction of the clotted fibrin signal, is presented as a function of time (1). After 180 minutes of incubation at 23°C, clots were perfused with either HBS, pH 7.4 (upper trace), or argatroban (100 μmol/L in buffer; lower trace) for the period indicated by the bracket (2). Thirty minutes after the start of that perfusion, each clot was perfused with chromogenic substrate S-2238 (560 μmol/L in water), as indicated by the bracket (3). After this perfusion (during which the absorbance increases by ∼10%), the initial rate of color development was determined from the digitally stored data. Argatroban dose-response data. (△) Thrombin activity (initial rate of color development at 405 nm) obtained as a function of argatroban concentration in the perfusate. The solid line was determined with the IC₅₀ value of 8.0 ± 2.4 μmol/L obtained by fitting the data to equation 1.