Comparison of cell-surface TFPIalpha and beta.
BACKGROUND
Tissue factor pathway inhibitor (TFPI) is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPIalpha and TFPIbeta. A portion of expressed TFPI remains associated with the cell surface through both direct (TFPIbeta) and indirect (TFPIalpha) glycosylphosphatidyl-inositol (GPT)-mediated anchorage.


OBJECTIVE
Compare the structure and properties of TFPIalpha and TFPIbeta.


METHODS
TFPIalpha and TFPIbeta, with protein molecular masses of 36 and 28 kDa, respectively, migrate similarly (46 kDa) on SDS-PAGE. Experiments using specific glycosidases were carried out to determine the different glycosylation pattern of the two forms. ECV304 cells, a cell line with some endothelial properties, were stimulated with IL-lbeta, LPS, and TNFalpha for up to 24 hrs and mRNA levels and protein synthesis were determined. Stable clones of ECV304 cells that express reduced levels of TFPIalpha, TFPIbeta or both were produced using a plasmid-based small-interfering RNA technique. Surface TFPI activity was determined by a two-stage chromogenic assay based on the ability of each form to inhibit FXa activation by FVIIa on cells with comparable amount of tissue factor (TF).


CONCLUSIONS
The deglycosylation studies show that the difference in molecular masses is due to a greater degree of sialylation in O-linked carbohydrate in TFPIbeta. The mRNA and protein levels of neither form of TFPI were affected by stimulation of cells with inflammatory stimuli. Although TFPIalpha comprises 80% of the surface-TFPI, TFPIbeta was responsible for the bulk of the cellular FVIIa/TF inhibitory activity, suggesting a potential alternative role for cell surface TFPIalpha.
Piro
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Division of Hematology, Washington University School of Medicine, St Louis, MO, USA.
Broze
G J
GJ
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D020869:Gene Expression Profiling; D006031:Glycosylation; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008074:Lipoproteins / Q000032:analysis* / Q000235:genetics / Q000502:physiology*; D019158:N-Acetylneuraminic Acid; D020033:Protein Isoforms / Q000032:analysis / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000954:Antigens, Surface; D018836:Inflammation Mediators; D007375:Interleukin-1; D008070:Lipopolysaccharides; D008074:Lipoproteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C051928:lipoprotein-associated coagulation inhibitor; D019158:N-Acetylneuraminic Acid