Platelet-rich clots are inefficiently lysed by current fibrinolytic agents. Platelets contain a great deal of plasminogen activator inhibitor 1 (PAI-1), the principal endogenous inhibitor of tissue-type plasminogen activator (t-PA). We have tested whether PAI-1 resistant t-PAs would be more effective thrombolytic agents in an in vitro model of platelet-rich clots. Clots were formed with recalcified human plasma without or with the addition of platelets. The lysis of these clots was followed by the release of incorporated 125I-fibrinogen. Mutant and wild-type t-PA were almost equally effective against clots lacking platelets but the mutant was twice as effective at lysing platelet-rich clots. A mechanism for this effect is suggested by the demonstration that a complex between wild-type t-PA and extruded platelet contents resembles that between purified t-PA and PAI-1 and that the PAI-1 resistant t-PA does not interefer with formation of this adduct. Because of its enhanced ability to lyse platelet-rich clots in vitro, further in vivo work may find that PAI-1 resistant t-PA is a more efficacious therapeutic agent than wild-type t-PA in situations where platelets contribute to the failure of thrombolysis.