The effect of human activated protein C (APC) on t-PA dependent fibrinolysis was studied in vitro using plasma (and whole blood) clot lysis techniques. Clot lysis was monitored by measuring the release of soluble 125I-labelled fibrin degradation products from the clot over time. It was demonstrated that the stimulatory effect of APC on plasma and blood clot lysis was specific for APC and depended on the presence of its active site and Ca2+ ions. Furthermore, the effect depended on the presence of phospholipids in plasma or cells in blood. The presence of pro-urokinase, factor XIII or alpha 2-antiplasmin was not required for the expression of the profibrinolytic effect of APC. Subsequent experiments revealed that the profibrinolytic effect of APC was only observed when thrombin was formed through the coagulation pathway during the initial phase of the clot lysis experiment. It was also shown that the addition of increasing concentrations of thrombin itself could delay the t-PA dependent lysis of clots prepared from Al(OH)3 adsorbed plasma via a mechanism not yet understood. Based on these findings we propose that (a) t-PA dependent lysis of clots prepared from pooled normal plasma is delayed by thrombin generated through the coagulation system, and (b) that by its anticoagulant properties APC blocks this thrombin generation and thereby prevents the delay in clot lysis. Because in this model the profibrinolytic effect of APC is directly related to its anticoagulant properties we predicted and confirmed that other anticoagulants--like heparin--also have profibrinolytic activity. Conversely, procoagulants such as phospholipids can be antifibrinolytic.