Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor

Abstract

Tissue factor pathway inhibitor (TFPI) is a primary regulator of the initiation of blood coagulation. TFPI is internalized and degraded by HepG2 cells through the low-density-lipoprotein receptor-related protein (LRP) but also binds another molecule present on the cell surface at approx. 10-fold the abundance of LRP [Warshawsky, Broze and Schwartz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668]. When HepG2 cells are washed with heparin or dextran sulphate, a substance that binds TFPI is removed from the cell surface and can be detected in a slot-blot assay. Preincubation with trypsin destroys the reactivity of the TFPI-binding component in the slot-blot assay, suggesting that it is a protein. In addition, when the sulphation of glycosaminoglycans (GAGs) is prevented by growing the HepG2 cells in the presence of 30 mM sodium chlorate, TFPI binding is unaffected, whereas the binding of bovine lipoprotein lipase, a protein known to associate with cell-surface GAGs, falls to 50% of control levels. Dextran sulphate washes of HepG2 cells grown in sodium chlorate have an equal reactivity in slot-blot experiments to that of non-treated cells, suggesting that GAGs are not totally responsible for the binding activity observed. By using the slot blot to follow binding activity and conventional protein purification techniques, a protein species that migrates at 40 kDa after reduction was identified in the HepG2 cell wash. The binding of this protein to TFPI was confirmed with immobilized TFPI. Amino acid sequence analysis identified this protein species as a proteolytic fragment of glypican-3 (also called OCI-5), a member of the glypican family of glycosylphosphatidylinositol-anchored proteoglycans.

Reversible Regulation of Tissue Factor–Induced Coagulation by Glycosyl Phosphatidylinositol–Anchored Tissue Factor Pathway Inhibitor

“…Proteoglycan receptors that are known to bind TFPI-1 are the transmembrane-anchored ryudocan/syndecan 4 14 and the glycosyl phosphatidylinositol (GPI)-anchored glypican 3. 15 In a previous study, we found evidence that the primary anchoring of endogenous TFPI-1 on the surface of ECV304 cells is through a GPI linkage, 16 and similar conclusions were drawn from studies using primary human umbilical vein endothelial cells in culture. 17 The type of membrane anchoring influences the localization of cell surface receptors to specific areas of the cell membrane.…”

“…ECV304 cells express members of the GPI-anchored glypican family of heparan-sulfated proteoglycans that likely associate with the detergent-insoluble membrane domains as a result of their specific membrane attachment. A previous study has demonstrated that the glypican 3 core protein binds specifically to immobilized TFPI-1, 15 and we find no significant displacement of the endogenous inhibitor from ECV304 cells on incubation with heparin. Clusters of negatively charged residues in glypican 1 and 3 are highly conserved 32 and may contribute to protein-protein interactions with basic regions of TFPI, resulting in high-affinity binding that is only Figure 3.…”

“…By reverse-transcriptase PCR, ECV304 cells expressed mRNA (data not shown) for 2 of the known GPI-anchored heparan-sulfated proteoglycan receptors, glypican 1 and 3, the latter of which binds TFPI-1. 15 In pooled low-density fractions from large quantities of cells (from ten 150-cm 2 flasks) that were treated with heparinase, the glypican 1-reactive antibody S1 detected a 68-kDa band (data not shown) that had a molecular mass corresponding to the published size of the glypican 1 core protein. 27 These data are consistent with localization of GPI-anchored proteoglycan receptors in the glycosphingolipid-rich microdomains.…”

Regulation of tissue factor–induced signaling by endogenous and recombinant tissue factor pathway inhibitor 1

“…However, endogenous, GPI-anchored TFPI-1 controls TF procoagulant activity in these cell types. [18][19][20][21][22][23] TNF-␣-stimulated HUVECs show increased ERK1/2 phosphorylation when triggered with a stabilized ternary TF-VIIa-Xa complex using the nematode inhibitor NAPc2. 9 We had also used mRNA expression levels of the TR3 orphan receptor gene, which has been demonstrated in endothelial cells of atherosclerotic lesions in vivo, 37 as an additional, potentially relevant readout for TFdependent signaling.…”

“…12,14,15 Endogenous TFPI-1 is directly or indirectly anchored by glycosylphosphatidylinositol (GPI) on cell surfaces. [17][18][19][20][21][22][23] Lipoprotein receptor-related protein (LRP) and heparan sulfate proteoglycan (HSPG) have been implicated in the binding and internalization of recombinant forms of TFPI-1, depending on their C-terminal polybasic portions. 24,25 Endothelial cells, which are normally deficient in LRP, may bind TFPI-1 via HSPG and the very-low-density lipoprotein receptor.…”