To investigate the potential role of plasminogen activator inhibitor-1 (PAI-1), which is released from the alpha-granules of activated platelets, in thrombolysis resistance, we employed a model (the "Chandler loop") that mimics the formation of arterial thrombi in vivo and that can be manipulated in terms of rheological parameters and composition of blood cells. Light and electron microscopy revealed that the distribution of blood cells in Chandler thrombi is polarized, as it is in arterial thrombi, resulting in platelet-rich "white heads" and red blood cell-rich "red tails.". Resistance toward tissue-type plasminogen activator (TPA)-mediated thrombolysis parallels the presence of platelets that are fully activated in this system. We demonstrate that the PAI-1 released by the alpha-granules is preferentially retained within the thrombus and that the concentration of PAI-1 antigen is higher in the head than in the tail of the thrombus. The relative thrombolysis resistance of the heads of Chandler thrombi can be largely abolished by inclusion of an anti-PAI-1 monoclonal antibody that blocks that inhibitory activity of PAI-1 toward TPA. We propose that PAI-1, released from activated platelets, plays a key role in thrombolysis resistance and/or reocclusion after thrombolytic therapy. This is due to binding of PAI-1 to polymerized fibrin within the thrombus, followed by inhibition of TPA-mediated fibrinolysis.