期刊
JOURNAL OF THROMBOSIS AND HAEMOSTASIS
卷 3, 期 12, 页码 2677-2683出版社
WILEY
DOI: 10.1111/j.1538-7836.2005.01636.x
关键词
coagulation; endothelium-derived factors; inhibitors
资金
- NHLBI NIH HHS [HL60782] Funding Source: Medline
Background: Tissue factor pathway inhibitor (TFPI) is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPI alpha and TFPI beta. A portion of expressed TFPI remains associated with the cell surface through both direct (TFPI beta) and indirect (TFPI alpha) glycosylphosphatidyl-inositol (GPT)-mediated anchorage. Objective: Compare the structure and properties of TFPI alpha and TFPI beta. Methods: TFPI alpha and TFPI beta, 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-l beta, LPS, and TNF alpha for up to 24 hrs and mRNA levels and protein synthesis were determined. Stable clones of ECV304 cells that express reduced levels of TFPI alpha, TFPI beta 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 TFPI beta. The mRNA and protein levels of neither form of TFPI were affected by stimulation of cells with inflammatory stimuli. Although TFPI alpha comprises 80% of the surface-TFPI, TFPI beta was responsible for the bulk of the cellular FVIIa/TF inhibitory activity, suggesting a potential alternative role for cell surface TFPI alpha.
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