In an in vitro clot lysis model in human plasma, carboxypeptidase U (CPU) is generated by thrombin following the coagulation and by plasmin at the later stage of clot lysis. CPU is able to slow down clot lysis by suppressing the cofactor activity of partially degraded fibrin in the plasminogen activation by tissue-type plasminogen activator (t-PA). Making use of thrombomodulin and a thrombin inhibitor, the generation of CPU during the in vitro clot lysis can be manipulated both in terms of magnitude and time course. The data obtained demonstrate that CPU affects the clot dissolution through a threshold-dependent mechanism: as long as the CPU activity remains above the threshold value, lysis is prevented from proceeding into the propagation phase. From the moment the CPU activity drops below this threshold value, the rate of lysis accelerates. This threshold value for CPU activity is dictated by the t-PA concentration: increasing the t-PA concentration increases the CPU threshold and vice versa. This implies that the effect of the CPU pathway will become more apparent at a lower fibrinolytic capacity. Our threshold-based hypothesis indicates that the time course of proCPU activation, the stability of CPU and the t-PA concentration all play a crucial role in determining the result of the in vitro clot lysis experiment. Furthermore, this hypothesis provides us with new insights into previously published data on the effects of CPU on in vitro clot lysis by high and low t-PA concentrations.