Inhibition of plasminogen activation by tPA variant depends on the conformation of plasminogen. (A) tPA-S⁴⁸¹A inhibits lysis of plasma clots. Plasma clots were lysed by WT-tPA (10 nM) or WT-uPA (10 nM)²⁴  in the absence or presence of tPA-S⁴⁸¹A (50 nM). (B) Clot lysis by WT-tPA and WT-uPA was performed as in panel A in the absence or presence of the indicated concentrations of tPA-S⁴⁸¹A or tranexamic acid (TA). The data are expressed as percent lysis compared to WT-tPA and WT-uPA alone. The mean ± SD of 3 experiments is shown. (C) Lysis of clots prepared from fresh whole human blood by WT-tPA (10 nM) was monitored by thromboelastography³⁰  in the absence (Control) or presence of 50 nM or 500 nM tPA-S⁴⁸¹A. Data represent results from 1 of 3 independent experiments. (D) Effect of tPA variants on PA activity of tPA and uPA in the absence of fibrin. Plasminogen (500 nM) was incubated alone (Cont) or with WT-tPA (5 nM) or uPA (5 nM) in the absence or presence of tPA-S⁴⁸¹A (100 nM) or tranexamic acid (1 µM) plus a plasmin chromogenic substrate (100 µM) (TA). The OD at 405 nm was measured continuously over the next 20 minutes.²²  In another set of experiments, tranexamic acid was added together with tPA-S⁴⁸¹A (100 nM). The data are expressed as the change in OD per minute. The figure shows that tPA-S⁴⁸¹A had no effect on plasminogen activation by tPA or uPA in the absence of fibrin and that the effect of tranexamic acid mimics the stimulatory effect of fibrin on plasminogen activation by uPA and inhibition by the tPA variant. Comparisons between groups were analyzed using the Student t test. The mean ± SD of 3 experiments is shown. SD, standard deviation.