Plasminogen activator inhibitor-1 and vitronectin promote the cellular clearance of thrombin by low density lipoprotein receptor-related proteins 1 and 2

Thrombin is a multifunctional protein that has both proteinase and growth factor-like activities. Its regulation is largely mediated by interaction with a host of inhibitors including antithrombin III (ATIII), heparin cofactor II (HCII), alpha(2)-macroglobulin (alpha(2)-M), protease nexin I, and plasminogen activator inhibitor-1 (PAI-1). ATIII, HCII, and alpha(2)-M are all abundant in blood and can inactivate blood-borne thrombin leading to rapid hepatic clearance of the thrombin-inhibitor complex. PAI-1 alone, a poor solution phase inhibitor of thrombin, can efficiently inhibit thrombin in the presence of native vitronectin (VN), In this study, active thrombin was found to be efficiently endocytosed and degraded by cultured pre-type II pneumocyte cells, and both processes could be blocked by polyclonal antibodies to PAI-1. When the relative efficiency of cellular endocytosis of thrombin in complex with a number of inhibitors was examined, I-125-thrombin-PAI-1 complexes were most efficiently cleared compared to I-125-thrombin in complex with the serpins ATIII, HCII, alpha(1)-proteinase inhibitor, or D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone. Low density lipoprotein receptor-related proteins 1 (LRP) and 2 (gp330/megalin) mediate the endocytosis of thrombin-PAI-l, since antagonists of receptor function such as LRP-1 and LRP-2 antibodies and the 39-kDa receptor-associated protein blocked I-125-thrombin-PAI-1 endocytosis and degradation. The LRP-mediated clearance of exogenously added I-125-thrombin by cultured cells was found to be enhanced 5-fold by inclusion of wild-type PAI-1 but by only 2-fold when a mutant form of PAI-1 that is unable to bind VN was included. This wild-type PAI-1 enhancement of I-125-thrombin clearance was found to occur only in the presence of native VN and not with its conformationally altered form. The results highlight a novel mechanism for cellular clearance of thrombin involving native VN promoting the interaction of thrombin and PAI-1 and the subsequent endocytosis of the complex by LRP-1 or LRP-2. This pathway is potentially important for the regulation of the potent biological activities of thrombin, particularly at sites of vascular injury.