| Popis: |
Recent studies have shown a discrepancy between the level of tissue factor (TF) expression and the level of TF procoagulant activity on the apical and basolateral surface domains of polarized epithelial cells. The present investigation was performed to elucidate possible reasons for the discordant expression of TF and its activity on the surface of polarized epithelial cells using a human intestinal epithelial cell line, Caco-2 and Madin-Darby canine kidney epithelial cells, type II (MDCK-II). Functional activity of coagulation factor VIIa (VIIa) in complex with TF was 6- to 7-fold higher on the apical than the basolateral surface in polarized Caco-2 cells. In contrast, no significant difference was found in the formation of TF/VIIa complexes between the apical and basolateral surface. Confocal microscopy of Caco-2 cells showed TF expression on both the apical and the basolateral surface domains. Studies with MDCK-II cells showed that the specific functional activity of TF expressed on the apical cell surface was 5-fold higher than on the basolateral surface. To test whether differential expression of TF pathway inhibitor (TFPI) on the apical and basolateral surface could account for differences in TF/VIIa functional activity, we measured cell-surface-bound TFPI activity in Caco-2 cells. Small but similar amounts of TFPI were found on both surfaces. Further, addition of inhibitory anti-TFPI antibodies induced a similar enhancement of TF/VIIa activity on both surface domains. Because the availability of anionic phospholipids on the outer leaflet of the cell membrane could regulate TF/VIIa functional activity, we measured the distribution of anionic phospholipids on the apical and basolateral surface by annexin V binding and thrombin generation. The results showed that the anionic phospholipid content on the basolateral surface, compared with the apical surface, was 3- to 4-fold lower. Mild acid treatment of polarized Caco-2 cells, which markedly increased the anionic phospholipid content on the basolateral surface membrane, increased the TF/VIIa activity on the basolateral surface without affecting the number of TF/VIIa complexes formed on the surface. Overall, our data suggest that an uneven expression of TF/VIIa activity between the apical and basolateral surface of polarized epithelial cells is caused by differences in anionic phospholipid content between the two surface domains and not from a polar distribution of TFPI. |
