Argatroban inhibition of thrombin bound to aged plasma clots (clot permeation assay). Plasma clots (50 μL each) were formed by the addition of thrombin (to 0.5 NIH units/mL) and CaCl₂(to 20 mmol/L) to citrated human plasma (PRP, solid symbols; PPP, open symbols) in the wells of a microtiter plate and then incubated for either 3 hours (upper panel) or 6 hours (lower panel) before permeation with argatroban (0 to 300 μmol/L) in HBS, pH 7.4. After the removal of excess inhibitor, thrombin activity was determined with chromogenic substrate S-2238 and the results were expressed as the initial rate of color development at 405 nm in units of milli-OD per minute (in a Vmax Kinetic Microtiter Plate Reader). Data obtained as a function of argatroban concentration were analyzed with equation 2 to determine the parameters shown in the inserts, IC₅₀, and percentage of maximum inhibition (defined as 100 * [1 − noninhibited fraction]). The solid lines were calculated from the resultant parameters obtained with PRP clots at each clot age. Table 2 presents the complete set of inhibition parameters obtained in these assays.