CDB15:0001374 SERPINE1 — LRP1

ABSTRACT: Although plasminogen activator inhibitor-1 (PAI-1) is known to stimulate cell migration, little is known about underlying mechanisms. We show that both active and inactive (e.g. cleaved) PAI-1 can activate the Jak/Stat signaling system and stimulate cell migration in chemotaxis, haptotaxis, chemokinesis, and wound healing assays. Moreover, antibodies to the LDL receptor-related protein (LRP) and an LRP antagonist (RAP) blocked these motogenic effects of PAI-1, while a PAI-1 mutant that did not bind to LRP failed to activate the Jak/Stat signaling pathway or to stimulate cell migration. PAI-1 had no chemotactic effect on LRP-deficient cells. These results indicate that LRP is a signaling molecule, that it mediates the migration-promoting activity of PAI-1, and that this activity does not require intact, biologically active PAI-1. Activation of this LRP-dependent signaling pathway by PAI-1 may begin to explain how the inhibitor stimulates cell migration in a variety of normal and pathological processes.

ABSTRACT: It is well-established that complexes of plasminogen-activator inhibitor 1 (PAI-1) with its target enzymes bind tightly to low-density lipoprotein (LDL) receptor-related protein 1 (LRP1), but the molecular details of this interaction are not well-defined. Furthermore, considerable controversy exists in the literature regarding the nature of the interaction of free PAI-1 with LRP1. In this study, we examined the binding of free PAI-1 and complexes of PAI-1 with low-molecular-weight urokinase-type plasminogen activator to LRP1. Our results confirmed that uPA:PAI-1 complexes bind LRP1 with ∼100-fold increased affinity over PAI-1 alone. Chemical modification of PAI-1 confirmed an essential requirement of lysine residues in PAI-1 for the interactions of both PAI-1 and uPA:PAI-1 complexes with LRP1. Results of surface plasmon resonance measurements supported a bivalent binding model in which multiple sites on PAI-1 and uPA:PAI-1 complexes interact with complementary sites on LRP1. An ionic-strength dependence of binding suggested the critical involvement of two charged residues for the interaction of PAI-1 with LRP1 and three charged residues for the interaction of uPA:PAI-1 complexes with LRP1. An enhanced affinity resulting from the interaction of three regions of the uPA:PAI-1 complex with LDLa repeats on LRP1 provided an explanation for the increased affinity of uPA:PAI-1 complexes for LRP1. Mutational analysis revealed an overlap between LRP1 binding and binding of a small-molecule inhibitor of PAI-1, CDE-096, confirming an important role for Lys-207 in the interaction of PAI-1 with LRP1 and of the orientations of Lys-207, -88, and -80 for the interaction of uPA:PAI-1 complexes with LRP1.

ABSTRACT: 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), alpha2-macroglobulin (alpha2-M), protease nexin I, and plasminogen activator inhibitor-1 (PAI-1). ATIII, HCII, and alpha2-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, 125I-thrombin-PAI-1 complexes were most efficiently cleared compared to 125I-thrombin in complex with the serpins ATIII, HCII, alpha1-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-1, since antagonists of receptor function such as LRP-1 and LRP-2 antibodies and the 39-kDa receptor-associated protein blocked 125I-thrombin-PAI-1 endocytosis and degradation. The LRP-mediated clearance of exogenously added 125I-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 125I-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.