Comparison of cell-surface TFPIα and β

Abstract

Background: Tissue factor pathway inhibitor (TFPI) is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPIα and TFPIβ. A portion of expressed TFPI remains associated with the cell surface through both direct (TFPIβ) and indirect (TFPIα) glycosylphosphatidyl-inositol (GPT)-mediated anchorage. Objective: Compare the structure and properties of TFPIα and TFPIβ. Methods: TFPIα and TFPIβ, 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-1β, LPS, and TNFα for up to 24 hrs and mRNA levels and protein synthesis were determined. Stable clones of ECV304 cells that express reduced levels of TFPIα, TFPIβ 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). Results and conclusions: The deglycosylation studies show that the difference in molecular masses is due to a greater degree of sialylation in O-linked carbohydrate in TFPIβ. The mRNA and protein levels of neither form of TFPI were affected by stimulation of cells with inflammatory stimuli. Although TFPIα comprises 80% of the surface-TFPI, TFPIβ was responsible for the bulk of the cellular FVIIa/TF inhibitory activity, suggesting a potential alternative role for cell surface TFPIα. © 2005 International Society on Thrombosis and Haemostasis.