Plasminogen activator inhibitor 1 (PAI-1), an essential regulatory protein of the fibrinolytic system, harbors interaction sites for plasminogen activators (tissue-type [t-PA] and urokinase-type [u-PA]) and for fibrin. In this study, anti-PAI-1 monoclonal antibodies (MoAbs) were used to identify interaction sites of PAI-1 with these components. The binding sites of 18 different MoAbs were established and are located on five distinct "linear" areas of PAI-1. MoAbs, binding to two distinct areas of PAI-1, are able to prevent the inhibition of t-PA by PAI-1. In addition, two interaction sites for fibrin were identified on PAI-1. The area located between amino acids 110 and 145 of PAI-1 contains a binding site for both components and its significance is discussed in the context of the t-PA inhibition by fibrin-bound PAI-1. Subsequently, the MoAbs were used to assess the role of platelet-PAI-1 in clot-lysis. An in vitro clot-lysis system was used to demonstrate that clot-lysis resistance is dependent on the presence of activated platelets and that PAI-1 is a major determinant for lysis-resistance. We propose that, upon activation of platelets, PAI-1 is fixed within the clot by binding to fibrin and retains its full capacity to inhibit t-PA and u-PA.