title: The low density lipoprotein receptor-related protein can function independently from heparan sulfate proteoglycans in tissue factor pathway inhibitor endocytosis.
abstract: Tissue factor pathway inhibitor (TFPI) is a plasma serine protease inhibitor that directly inhibits coagulation factor Xa and regulates blood coagulation via inhibition of factor VIIa-tissue factor enzymatic activity. We previously demonstrated that >90% of TFPI bound to a single population of low affinity binding sites on hepatoma cells (2 x 10(6) sites/cell, Kd = 30 nM), and, that following binding, the low density lipoprotein receptor-related protein (LRP) mediated TFPI uptake and degradation. We subsequently reported heparan sulfate proteoglycans (HSPGs) constitute a second receptor system involved in TFPI catabolism. In the present study, mouse embryonic fibroblasts heterozygous and homozygous-negative for disruption of the LRP gene were used to further examine the roles of LRP and HSPGs in TFPI endocytosis. We demonstrate that LRP is absolutely required for degrading 125I-TFPI. LRP heterozygous and homozygous-negative cells bind 125I-TFPI similarly, and the 39-kDa protein, an inhibitor of all known ligand interactions with LRP, does not alter 125I-TFPI binding to these cells. TFPI can be cross-linked to LRP on [35S]cysteine-labeled hepatoma and LRP-heterozygous cells but not LRP-negative cells. When HSPGs are blocked with protamine, 125I-TFPI binds in a 39-kDa protein-inhibitable manner to 41,000 high affinity sites/hepatoma cell (Kd = 2.3 nM). Blockade of HSPGs with protamine results in significantly more 125I-TFPI degradation by LRP-positive cells. TFPI can be cross-linked to LRP in the absence and presence of protamine. However, in the presence of protamine, relative to the total pool of cross-linked proteins, 5-fold more TFPI is cross-linked to LRP. Finally, we show TFPI inhibits 125I-alpha2-macroglobulin-methylamine binding to hepatoma cells and that carboxyl-terminal residues 115-319 of the 39-kDa protein inhibit both 125I-TFPI degradation and binding when binding conditions contain protamine. Together, our results suggest that while the majority of TFPI binds to cell surface HSPGs, LRP can function independently from HSPGs in the binding and uptake of TFPI.
authors.affiliation: Department of Pediatrics, Jewish Hospital at Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: I
authors.initials: I
authors.lastname: Warshawsky
authors.forename: J
authors.initials: J
authors.lastname: Herz
authors.forename: G J
authors.initials: GJ
authors.lastname: Broze
authors.forename: A L
authors.initials: AL
authors.lastname: Schwartz
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D004705:Endocytosis; D006023:Glycoproteins / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism*; D007700:Kinetics; D026563:LDL-Receptor Related Protein-Associated Protein; D008074:Lipoproteins / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D051379:Mice; D011479:Protamines / Q000494:pharmacology; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats; D011971:Receptors, Immunologic / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D003545:Cysteine; D006023:Glycoproteins; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D026563:LDL-Receptor Related Protein-Associated Protein; D008074:Lipoproteins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011479:Protamines; D011509:Proteoglycans; D011971:Receptors, Immunologic; C051928:lipoprotein-associated coagulation inhibitor