Tissue-type plasminogen activator (t-PA), a serine protease that catalyzes the initial and rate-limiting step in the fibrinolytic cascade, is cleared rapidly in vivo by the liver. Using chemical crosslinking, we have recently identified a plasminogen-activator inhibitor type 1 (PAI-1)-independent t-PA clearance receptor on rat hepatoma MH1C1 cells with a relative molecular mass of %-500 kDa. Another recently identified membrane receptor, low density lipoprotein receptorrelated protein/a2-macroglobulin receptor (LRP/a2MR), was also detected on MH1Cj hepatoma cells by using immunoprecipitation with anti-LRP/a2MR antibody. When analyzed by SDS/PAGE, we found the t-PA receptor identified on MH1Cj cells comigrated with the large subunit of LRP/a2MR.The t-PA receptor was immunoprecipitated by an anti-LRP/a2MR antibody after chemical crosslinking of specifically bound 12'I-labeled t-PA to its receptor. Through chemical crosslinking studies, we found that t-PA and methylamineactivated a2-macroglobulin could bind to LRP/a2MR simultaneously without competing with one another for binding, suggesting that the two ligands bound to two independent sites on the LRP/a2MR molecule. Furthermore, a 39-kDa protein, which modulates ligand binding to LRP/a2MR, was also found to inhibit t-PA binding to its receptor. These data thus show that the t-PA clearance receptor identified on MH1Cj hepatoma cells is LRP/a2MR.Tissue-type plasminogen activator (t-PA) plays an essential role in the fibrinolytic process by catalyzing the conversion of the zymogen plasminogen to plasmin (1,2). The plasmin thus activated can proteolytically degrade the fibrin network associated with blood clots. The role of t-PA as an initiator in thrombolysis has been exploited as a thrombolytic agent (3-5). However, the half-life of t-PA in plasma is very short, ranging from 1-4 min in rodents (6-11) to 5-10 min in humans (12, 13).In vivo studies have shown that the liver is the major organ responsible for t-PA clearance (6-11). Kinetic studies have suggested that specific receptors exhibiting characteristics of receptor-mediated endocytosis are involved in the clearance process. At least two classes of clearance mechanisms exist: a noncarbohydrate-mediated pathway via hepatocytes and a carbohydrate-mediated pathway via endothelial cells (14). In hepatocytes, detailed studies of the clearance mechanism have revealed two types of clearance receptor: plasminogenactivator inhibitor type 1 (PAI-1)-dependent (15)(16)(17)(18)(19)(20) and -independent (11,21) receptors. Recent studies from our laboratory have identified a PAI-1-independent t-PA receptor on rat hepatoma MH1C1 cells (22). Binding of t-PA to this receptor occurs predominantly via the free form and requires neither the protease active site nor the presence of bioactive PAI-1. This situation contrasts with the PAI-1-dependent t-PA receptor we have previously characterized on human hepatoma HepG2 cells, for which the predominant specificbinding species is the t-PA-PAI-1 complex (16)(17)(18)(19)22). Simi...