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Down-regulation of monocyte tissue factor mediated by tissue factor pathway inhibitor and the low density lipoprotein
1Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
Abstract:
Inflammatory mediators like bacterial lipopolysaccharide induce monocytes to express tissue factor (TF), the cell-surface protein that triggers the blood clotting cascade in hemostasis and thrombotic disease. The physiologic ligand for TF is the serine protease, factor VIIa (FVIIa), and the resulting bimolecular enzyme, TF/FVIIa, can be reversibly inhibited by tissue factor pathway inhibitor (TFPI). Culturing monocytic cells in the presence of both FVIIa and TFPI caused down-regulation of TF expression via reducing its half-life. To exert this effect, FVIIa had to be competent to bind both TF and TFPI, and TFPI had to contain the C-terminal domain required for binding to other cell-surface receptors, including the low density lipoprotein receptor-related protein (LRP). TF down-regulation by FVIIa plus TFPI was abrogated by the 39-kDa receptor-associated protein, which blocks binding of all known ligands to LRP. Furthermore, treatment with FVIIa plus TFPI caused monocyte TF to colocalize with alpha-adaptin, a component of clathrin-coated pits. Thus, in addition to reversibly inhibiting TF/FVIIa catalytic activity, TFPI also mediates the permanent down-regulation of cell-surface TF in monocytic cells via LRP-dependent internalization and degradation. This represents an unusual mechanism for receptor internalization, requiring ligand-dependent bridging of one cell-surface receptor (TF) to a second cell-surface receptor (LRP), the latter being capable of clathrin-mediated internalization.
Insights
Tissue factor pathway inhibitor (TFPI) and factor VIIa (FVIIa) reduce tissue factor (TF) expression in monocytes. This involves TFPI binding to low density lipoprotein receptor-related protein (LRP) for TF internalization and degradation.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Monocytes express tissue factor (TF), a key initiator of blood coagulation and thrombosis, upon inflammatory stimulation.
- TF/FVIIa complex activity is regulated by tissue factor pathway inhibitor (TFPI), a reversible inhibitor.
- The role of TFPI in regulating TF expression beyond reversible inhibition is not fully understood.
Purpose of the Study:
- To investigate the mechanism by which TFPI influences TF expression in monocytic cells.
- To determine the role of FVIIa binding and TFPI's C-terminal domain in TF regulation.
- To elucidate the cellular pathway involved in TF down-regulation mediated by TFPI.
Main Methods:
- Monocytic cells were cultured with FVIIa and TFPI.
- TF expression levels and half-life were measured.
- The role of FVIIa binding competence, TFPI C-terminal domain, and LRP was assessed using blocking agents.
- TF colocalization with clathrin-coated pit components was examined.
Main Results:
- FVIIa and TFPI co-culture led to TF down-regulation by reducing TF half-life.
- FVIIa binding to TF and TFPI, and TFPI's C-terminal binding to LRP, were essential for this effect.
- Receptor-associated protein blocked TF down-regulation, confirming LRP involvement.
- TF was observed to colocalize with alpha-adaptin in clathrin-coated pits.
Conclusions:
- TFPI, in conjunction with FVIIa, mediates permanent down-regulation of cell-surface TF in monocytes.
- This process involves LRP-dependent internalization and degradation of TF.
- It represents an unusual mechanism of receptor internalization via ligand-dependent bridging to LRP and clathrin-mediated endocytosis.