A well-characterized in vitro model system composed of thrombin- stimulated gel-filtered human platelets, fibrin-(ogen), plasminogen, and recombinant tissue plasminogen activator (rt-PA) was used to examine the relationship between platelet-fibrin adhesive interactions and the lytic resistance of a platelet-rich thrombus. Laser light scattering kinetic experiments demonstrated that the ligand-mimetic peptide D-RGDW and an anti-alpha IIb beta 3 monoclonal antibody both inhibited clot retraction, but neither integrin-targeted reagent affected the overall delay in lysis of “bulk” fibrin caused by thrombin- stimulated platelets. However, lysis of the model platelet-rich thrombus did proceed some 30% more quickly when treated with a plasminogen activator inhibitor (PAI)-resistant t-PA variant. Taken together, these results confirm that platelet-released PAI-1 is a major determinant of global lytic resistance. Next events occurring during fibrinolysis in the unique microenvironment near the platelet surface were monitored by scanning electron microscopy and quantitative fluorescence microscopy. Scanning electron micrographs of the partially lysed model thrombus in the presence of 200 mumol/L of D-RGDW showed no platelet aggregates, and fibrin was attached by fewer strands to the platelets. Quantitative fluorescence microscopy, using fluorescein- labeled fibrin, showed that fibrin adherent to the surface of thrombin- stimulated platelets lysed 20% to 50% more slowly than bulk fibrin (monitored in parallel by laser light scattering). Furthermore, this microspectroscopic technique showed that D-RGDW reduced the quantity of platelet-bound fibrin, and accelerated lysis near the platelet surface with both native rt-PA and the PAI-resistant variant. These observations suggest that the dense network of fibrin bound to the platelet surface is protected from fibrinolysis by tissue-type plasminogen activators. Further, uncoupling fibrin from its platelet receptors uniquely hastens fibrinolysis at the cell/fibrin interface.