The Efficient Catabolism of Thrombin-Protease Nexin 1 Complexes Is a Synergistic Mechanism That Requires Both the LDL Receptor-related Protein and Cell Surface Heparins

Abstract

Protease nexin 1 (PN1) is a serine protease inhibitor (SERPIN) that acts as a suicide substrate for thrombin (Th) and urokinase-type plasminogen activator (uPA). PN1 forms 1:1 stoichiometric complexes with these proteases, which are then rapidly bound, internalized, and degraded. The low density lipoprotein receptor-related protein (LRP) is the receptor responsible for the internalization of protease-PN1 complexes. However, we found that the LRP is not significantly involved in the initial cell surface binding of thrombin-PN1, leading us to investigate what cellular component was responsible for this initial interaction. Since Th-PN1 complexes retain a high-affinity for heparin after complex formation, unlike several of the other SERPINs, we tested the possibility that cell surface heparins were involved in initial complex binding. Soluble heparin was found to be a potent inhibitor of the binding of Th-PN1 to the cell surface and greatly facilitated the dissociation of Th-PN1 complexes pre-bound in the absence of soluble heparin. To ascertain the role of cell surface heparins, further studies were done using complexes of thrombin and PN1(K7E), a variant of PN1 in which the heparin binding site was rendered non-functional. When added at equal initial concentrations of complexes, Th-PN1(K7E) was catabolized 5-to 10-fold less efficiently than Th-PN1, a direct result of the greatly diminished initial binding of the Th-PN1(K7E) complexes. These data demonstrate the sizable contribution of cell surface heparins to Thrombin-PN1 complex binding and support a model in which these heparins act to concentrate the complexes at the cell surface facilitating their subsequent LRP-dependent endocytosis. Protease nexin 1 (PN1)1 is a 43-kDa serine protease inhibitor that is a member of the SERPIN superfamily (1). It is an important physiological regulator of thrombin in tissues, and like its plasma counter part, antithrombin III, it is activated by heparin (1, 2). Like other members of the SERPIN family, PN1 is proteolytically attacked by its target protease but arrests proteolytic cleavage resulting in the formation of a 1:1 stoichiometric complex with that protease (1, 2). Only PN1 that is in complex with a protease is internalized and degraded by cells at a significant rate (3). This is presumably due to the formation or unmasking of a receptor binding site in PN1 when in complex with a protease, which is absent in the free SERPIN. One candidate site within the PN1 sequence for this function has recently been identified (4). Mechanistically, this ensures that SERPINs will remain extracellular until they have become part of an inhibitory complex. Once they have formed an inhibitory complex with a protease, the rapid removal of protease-SERPIN complexes is important for two reasons. First, since there is evidence to suggest that protease-SERPIN complexes may not be covalently linked (5), the uninternalized complexes represent a potential source of active protease formed when the complexes dissociate. Second, in the case of one S...

Control of thrombin signaling through PI3K is a mechanism underlying plasticity between hair follicle dermal sheath and papilla cells

“…3C). However, PN-1 internalization or signaling through LRP (Knauer et al, 1997) was not required. Blockade of PN-1 binding to the LRP receptor by the antagonistic receptor-associated protein RAP, used at a concentration interfering with proliferation (Vaillant et al, 2007), did not affect cell volume increase (Fig.…”

“…1F). This overload probably reflects a failure in the clearance of the protease by the low density lipoprotein receptor-related protein-1 (LRP-1), which controls internalization of PN-1-thrombin complexes (Knauer et al, 1997).…”

Protease nexin 1 and its receptor LRP modulate SHH signalling during cerebellar development

“…Free or complexed extracellular PN-1 binds to LRP1, which mediates its internalization (Knauer et al, 1997b). LRP1 was detected in the cell body and along neurites of all CGNPs (Fig.…”

Protease nexin 1 inhibits hedgehog signaling in prostate adenocarcinoma

“…The activity of PN1 is controlled by 3 principle regions of interest: heparin interaction, the reactive center loop (RCL), and the LDL receptor-related protein binding site (LRP) (38,39). The RCL, located in the C terminus of PN1, is required for the inhibitory activity of PN1 and is homologous to the RCL of other serpins (40).…”