Abstract
BackgroundTissue factor (TF) pathway inhibitor (TFPI) exists in two isoforms; TFPIα and TFPIβ. Both isoforms are cell surface attached mainly through glycosylphosphatidylinositol (GPI) anchors. TFPIα has also been proposed to bind other surface molecules, like glycosaminoglycans (GAGs). Cell surface TFPIβ has been shown to exert higher anticoagulant activity than TFPIα, suggesting alternative functions for TFPIα. Further characterization and search for novel TFPI binding partners is crucial to completely understand the biological functions of cell associated TFPI.Methods and ResultsPotential association of TFPI to heparan sulphate (HS) proteoglycans in the syndecan family were evaluated by knock down studies and flow cytometry analysis. Cell surface colocalization was assessed by confocal microscopy, and native PAGE or immunoprecipitation followed by Western blotting was used to test for protein interaction. Heparanase was used to enzymatically degrade cell surface HS GAGs. Anticoagulant potential was evaluated using a factor Xa (FXa) activity assay. Knock down of syndecan-3 in endothelial,- smooth muscle- and breast cancer cells reduced the TFPI surface levels by 20-50%, and an association of TFPIα to syndecan-3 on the cell surface was demonstrated. Western blotting indicated that TFPIα was found in complex with syndecan-3. The TFPI bound to syndecan-3 did not inhibit the FXa generation. Removal of HS GAGs did not release TFPI antigen from the cells.ConclusionsWe demonstrated an association between TFPIα and syndecan-3 in vascular cells and in cancer cells, which did not appear to depend on HS GAGs. No anticoagulant activity was detected for the TFPI associated with syndecan-3, which may indicate coagulation independent functions for this cell associated TFPI pool. This will, however, require further investigation.
Highlights
Tissue factor (TF) pathway inhibitor (TFPI) is the endogenous inhibitor of TF-induced blood coagulation, and it exists in two isoforms; TFPIα and TFPIβ
Potential association of TFPI to heparan sulphate (HS) proteoglycans in the syndecan family were evaluated by knock down studies and flow cytometry analysis
We demonstrated an association between TFPIα and syndecan-3 in vascular cells and in cancer cells, which did not appear to depend on heparan sulfate (HS) GAGs
Summary
Tissue factor (TF) pathway inhibitor (TFPI) is the endogenous inhibitor of TF-induced blood coagulation, and it exists in two isoforms; TFPIα and TFPIβ. TFPIα consists of three tandem Kunitz inhibitory domains and a highly positively charged C-terminal end [8], whereas TFPIβ contains the first two Kunitz domains followed by a different C-terminus encoding a glycosylphosphatidylinositol (GPI) attachment signal peptide, directing it to the cell surface [1, 9]. Due to these dissimilarities, TFPIβ is exclusively bound to the cell membrane, while TFPIα can either be secreted to the extracellular environment, or be bound to the cell surface through an as yet unidentified GPI linked molecule, and to some extent to heparan sulfate (HS) proteoglycans (HSPGs) [9,10,11,12].
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