Complexes of tissue-type plasminogen activator and its serpin inhibitor plasminogen-activator inhibitor type 1 are internalized by means of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

Abstract

Tissue-type plasmingen activator and urokinase are serine proteases secreted by many cell types that participate in biological processes, such as tissue restructuring, cell migration, and tumor metalst . Clinically, these proteases are used to dissolve coronary fibrin clots that are the proximal causes of acute myocardial infarction. In vivo, the activity of these enzymes is controlled by plasminn-activator inhibitors, members of the serpin family of protease inhibitors. This study shows that tissue-type plsminogen activator-inhibitor complexes bind in solution to low density lipoprotein receptorrelated protein (LRP), a large heterodimeric ubiquitous membrane receptor. In cultured cells, endocytosis and degradation of these complexes is reduced by polyclonal antibodies directed against LRP and inhibited by a Mr 39,000 protein that binds to LRP and inhibits its interaction with previously known ligands, including apolipoprotein E and a2-macroglobulin. We propose a role for LRP in the clearance of plasminogen activatorinhibitor complexes that is analogous to its function in the endocytosis of a2-macroglobulin-protease complexes.Tissue-type plasminogen activator (t-PA) is a blood-borne serine protease of Mr -66,000 that converts the zymogen plasminogen to the highly active fibrinolytic protease plasmin. This activity is exploited clinically to dissolve thrombi that form in coronary arteries and cause acute myocardial infarction. t-PA is secreted as an enzymatically active protein of 527 amino acids. The N-terminal portion of the molecule is organized into four highly conserved structural motifs, whereas the C-terminal portion is a typical serine protease and contains a characteristic catalytic triad (1).Under normal physiological conditions, the concentration of t-PA in plasma is low (:0.1 nM), and much of the endogenous protein circulates as an enzymatically inactive complex composed of t-PA and a powerful inhibitor, the serpin plasminogen-activator inhibitor type 1 (PAI-1) (1, 2). PAl-i reacts rapidly with a series of positively charged residues located near the catalytic site of t-PA (3), forming a heterodimeric complex that is stable to treatment with SDS (4, 5). PAM-acts as a suicide substrate for both t-PA and urokinase, a plasminogen activator with many structural similarities to t-PA (1, 2). These plasminogen activator-inhibitor complexes are cleared from the circulation by the liver (1, 6, 7). In fact, in a perfused liver system t-PA-PAI-1 complex is cleared considerably faster than free t-PA (8). The hepatic receptor(s) responsible for this efficient removal of t-PA-PAI-1 complexes have not been identified, although studies with the human hepatic cell line HepG2 have shown that interaction between t-PA and PAT-i associated with the extracellular matrix is required for recognition and endocytosis (9, 10).The requirement for complex formation of a protease with its inhibitor for efficient receptor-mediated endocytosis is not unprecedented. The protease inhibitor a2-macroglobulin forms a complex with...

Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo

“…10, which is published as supporting information on the PNAS web site), suggesting that the elevated tPA͞uPA expression and activity in these cells may reflect, at least in part, the down-regulation of this protease inhibitor. Indeed, both tPA and uPA are rapidly cleared by cells after complexation with plasminogen activator inhibitor-1 (28,29). Discordance was also observed between the quantity of activity and transcript for the SE target PFK, for which equivalent mRNA levels were detected in parental MDA-MB-231 and 231mfp cells, but activity was observed only in the parental cells (Fig.…”

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor‐related protein/α2‐macroglobulin receptor and transport to endosomes

“…It is believed that the clustering of cysteine-rich type A binding repeats, resembling those found in LDL receptor, is the molecular principle for the ability to bind a variety of ligands so far thought to be unrelated. The physiological importance of recognition of the hitherto identified or proposed ligands -activated c~2-macroglobulin (~2M*), apolipoprotein E (apoE), lipoprotein lipase (LPL), plasminogen activators and complexes with their inhibitor (PA and PA/PAI-1), lipoprotein(a), pseudomonas exotoxin A, human rhinovirus, Lf and the so-called receptor associated protein (RAP) -by a single receptor entity is one crucial question to be answered [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. Within the description of miscellaneous ligands of LRP/~2MR also in vitro binding of Lf was demonstrated [1].…”

α2‐Macroglobulin Receptor Mediates Binding and Cytotoxicity of Plant Ribosome‐Inactivating Proteins

“…a,MR binds ligands other than a,-macroglobulidproteinase complexes, such as a copurifying 40-kDa protein named a,MR-associated protein (a,MRAP; Jensen et al, 1989;Kristensen et al, 1990;Moestrup and Gliemann, 1991), P-migrating very-low-density lipoproteins enriched with apolipoprotein E (Kowal et al, 1990), and lipoprotein lipase (Beisiegel et al, 1991 ;Nykjaer et al, 1993). In addition, a,MR participates in the internalization and degradation of complexes between tissue-type (tPA) or urokinase-type plasminogen activator (uPA) and plasminogen-activator inhibitors (PA1 ; Orth et al, 1992;Herz et al, 1992;Nykjzr et al, 1992bj. Initially, uPA binds to its cell surface receptor (uPARj; uPARbound uPA is then complexed to its inhibitor PAI-1, thus regulating uPA activity.…”