title: Syndecan-3 and TFPI colocalize on the surface of endothelial-, smooth muscle-, and cancer cells.
abstract: BACKGROUND
  Tissue 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.
  
  
  RESULTS
  Potential 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway; Department of Haematology and Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
authors.forename: Mari
authors.initials: M
authors.lastname: Tinholt
authors.affiliation: Department of Haematology and Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway.
authors.forename: Benedicte
authors.initials: B
authors.lastname: Stavik
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway.
authors.forename: William
authors.initials: W
authors.lastname: Louch
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway.
authors.forename: Cathrine Rein
authors.initials: CR
authors.lastname: Carlson
authors.affiliation: Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway.
authors.forename: Marit
authors.initials: M
authors.lastname: Sletten
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America.
authors.forename: Wolfram
authors.initials: W
authors.lastname: Ruf
authors.affiliation: Department of Haematology and Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway.
authors.forename: Grethe
authors.initials: G
authors.lastname: Skretting
authors.affiliation: Department of Haematology and Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
authors.forename: Per Morten
authors.initials: PM
authors.lastname: Sandset
authors.affiliation: Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway.
authors.forename: Nina
authors.initials: N
authors.lastname: Iversen
mesh_terms: D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D008074:Lipoproteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053670:Syndecan-3 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D008074:Lipoproteins; D012333:RNA, Messenger; C508301:SDC3 protein, human; D053670:Syndecan-3; C051928:lipoprotein-associated coagulation inhibitor; D015942:Factor VIIa