Argatroban inhibition of thrombin present in aged plasma clots treated with streptokinase. Plasma clots (50 μL each) were formed from PRP and aged for 3 hours (as described in the legend to Fig5). Then, 50 μL of either streptokinase (250 IU/mL in HBS, pH 7.4) or buffer was layered on each aged plasma clot and incubated for 30 minutes to induce lysis. Control plasma samples (ie, no thrombin added) were also incubated for 3 hours, and then streptokinase was added to their wells. In each case, argatroban (0 to 65 μmol/L) was added to the wells and incubated for an additional 30 minutes before the addition of substrate S-2238 and determination of thrombin activity (as described in the legend to Fig 5). Note that excess liquid was not removed at any step in this assay to recover both clot-bound thrombin and that released into the supernatant during clot lysis. Argatroban dose-response data obtained with streptokinase-treated plasma clots (•) and with intact plasma clots (○) were analyzed with equation 2 by nonlinear regression to obtain the IC50 and maximum inhibition parameters (cited in Table 3 and used to calculate the solid lines). Data obtained with plasma samples treated with streptokinase are presented as △ and provide a measure of the amidolytic activity due to plasmin generation in plasma, as described in the text. (Insert) Time course of streptokinase lysis of aged plasma clots. Clots formed from PPP (100 μL) were aged for 3 hours and then overlaid with an equal volume of either buffer (control trace) or streptokinase (SK trace; 250 IU/mL in HBS, pH 7.4). The change in optical density at 405 nm was recorded as a function of time (Vmax Kinetic Microtiter Plate Reader). The extent clot lysis of was determined from the difference in optical density between the control and SK-treated data, at selected time points as described in the text.
