PLASMINOGEN-ACTIVATOR INHIBITOR-1 AND VITRONECTIN PROMOTE THE CELLULAR CLEARANCE OF THROMBIN BY LOW-DENSITY-LIPOPROTEIN RECEPTOR-RELATED PROTEIN-1 AND PROTEIN-2

Thrombin is a multifunctional protein that has both proteinase and gro wth factor-like activities. Its regulation is largely mediated by inte raction with a host of inhibitors including antithrombin III (ATIII), heparin cofactor II (HCII), alpha(2)-macroglobulin (alpha(2)-M), prote ase nexin I, and plasminogen activator inhibitor-1 (PAI-1). ATIII, HCI I, and alpha(2)-M are all abundant in blood and can inactivate blood-b orne thrombin leading to rapid hepatic clearance of the thrombin-inhib itor complex. PAI-1 alone, a poor solution phase inhibitor of thrombin , can efficiently inhibit thrombin in the presence of native vitronect in (VN), In this study, active thrombin was found to be efficiently en docytosed and degraded by cultured pre-type II pneumocyte cells, and b oth processes could be blocked by polyclonal antibodies to PAI-1. When the relative efficiency of cellular endocytosis of thrombin in comple x with a number of inhibitors was examined, I-125-thrombin-PAI-1 compl exes were most efficiently cleared compared to I-125-thrombin in compl ex with the serpins ATIII, HCII, alpha(1)-proteinase inhibitor, or D-p henylalanyl-L-prolyl-L-arginine chloromethyl ketone. Low density lipop rotein receptor-related proteins 1 (LRP) and 2 (gp330/megalin) mediate the endocytosis of thrombin-PAI-l, since antagonists of receptor func tion such as LRP-1 and LRP-2 antibodies and the 39-kDa receptor-associ ated protein blocked I-125-thrombin-PAI-1 endocytosis and degradation. The LRP-mediated clearance of exogenously added I-125-thrombin by cul tured 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-throm bin clearance was found to occur only in the presence of native VN and not with its conformationally altered form. The results highlight a n ovel mechanism for cellular clearance of thrombin involving native VN promoting the interaction of thrombin and PAI-1 and the subsequent end ocytosis of the complex by LRP-1 or LRP-2. This pathway is potentially important for the regulation of the potent biological activities of t hrombin, particularly at sites of vascular injury.