Full Length ArticleInterplay between alternatively spliced Tissue Factor and full length Tissue Factor in modulating coagulant activity of endothelial cells

- Interplay between asTF and flTF in endothelial cells is unknown.
- Concomitant expression of asTF and flTF does not alter the coagulant potential of cells and/or MVs.
- The two TF protein isoforms do not co-localize in endothelial cells.
- High levels of asTF decrease flTF expression, mainly in non-lipid raft plasma membrane fractions.
- High levels of asTF increase ER stress.

BackgroundFull length Tissue factor (flTF) is a key player in hemostasis and also likely contributes to venous thromboembolism (VTE), the third most common cardiovascular disease. flTF and its minimally coagulant isoform, alternatively spliced TF (asTF), have been detected in thrombi, suggesting participation of both isoforms in thrombogenesis, but data on participation of asTF in hemostasis is lacking. Therefore, we assessed the role of asTF in flTF cofactor activity modulation, using a co-expression system.ObjectiveTo investigate the interplay between flTF and asTF in hemostasis on endothelial cell surface.MethodsImmortalized endothelial (ECRF) cells were adenovirally transduced to express asTF and flTF, after which flTF cofactor activity was measured on cells and microvesicles (MVs). To study co-localization of flTF/asTF proteins, confocal microscopy was performed. Finally, intracellular distribution of flTF was studied in the presence or absence of heightened asTF levels.ResultsLevels of flTF antigen and cofactor activity were not affected by asTF co-expression. asTF and flTF were found to localize in distinct subcellular compartments. Only upon heightened overexpression of asTF, lower flTF protein levels and cofactor activity were observed. Heightened asTF levels also induced a shift of flTF from non-raft to lipid raft plasma membrane fractions, and triggered the expression of ER stress marker BiP. Proteasome inhibition resulted in increased asTF - but not flTF - protein expression.ConclusionAt moderate levels, asTF appears to have negligible impact on flTF cofactor activity on endothelial cells and MVs; however, at supra-physiological levels, asTF is able to reduce the levels of flTF protein and cofactor activity.