THE LOW-DENSITY-LIPOPROTEIN RECEPTOR-RELATED PROTEIN CAN FUNCTION INDEPENDENTLY FROM HEPARAN-SULFATE PROTEOGLYCANS IN TISSUE FACTOR PATHWAYINHIBITOR ENDOCYTOSIS

Tissue factor pathway inhibitor (TFPI) is a plasma serine protease inh ibitor that directly inhibits coagulation factor Xa and regulates bloo d coagulation via inhibition of factor VIIa-tissue factor enzymatic ac tivity. 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, K-d = 30 nM), and, that following binding, the low densit y lipoprotein receptor-related protein (LRP) mediated TFPI uptake and degradation. We subsequently reported heparan sulfate proteoglycans (H SPGs) constitute a second receptor system involved in TFPI catabolism, In the present study, mouse embryonic fibroblasts heterozygous and ho mozygous-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 I-125-TFPI. LRP heteroz ygous and homozygous-negative cells bind I-125-TFPI similarly, and the 39-kDa protein, an inhibitor of all known ligand interactions with LR P, does not alter I-125-TFPI binding to these cells, TFPI can be cross -linked to LRP on [S-35]cysteine-labeled hepatoma and LRP-heterozygous cells but not LRP-negative cells, When HSPGs are blocked with protami ne, I-125-TFPI binds in a 39-kDa protein-inhibitable manner to 41,000 high affinity sites/ hepatoma cell (K-d = 2.3 nM), Blockade of HSPGs w ith protamine results in significantly more I-125-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, relativ e to the total pool of cross-linked proteins, 5-fold more TFPI is cros s-linked to LRP. Finally, we show TFPI inhibits I-125-alpha(2)-macrogl obulin-methylamine binding to hepatoma cells and that carboxyl-termina l residues 115-319 of the 39-kDa protein inhibit both I-125-TFPI degra dation and binding when binding conditions contain protamine, Together , our results suggest that while the majority of TFPI binds to cell su rface HSPGs, LRP can function independently from HSPGs in the binding and uptake of TFPI.