Discordant expression of tissue factor and its activity in polarized epithelial cells. Asymmetry in anionic phospholipid availability as a possible explanation

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 inv estigation was performed to elucidate possible reasons for the discordant e xpression of TF and its activity on the surface of polarized epithelial cel ls using a human intestinal epithelial cell line, Caco-5 and Madin-Darby ca nine kidney epithelial cells, type II (MDCK-II). Functional activity of coa gulation factor VIIa (VIIa) in complex with TF was 6- to 7-fold higher on t he apical than the,basolateral surface in polarized Caco-5 cells. In contra st, no significant difference was found in the formation of TF/VIIa complex es between the apical and basolateral surface. Confocal microscopy of Caco- 5 cells showed TF expression on both the apical and the basolateral surface domains. Studies with MDCK-II cells showed that the specific functional ac tivity of TF expressed on the apical cell surface was 5-fold higher than on the basolateral surface. To test whether differential expression of TF pat hway inhibitor (TFPI) on the apical and basolateral surface could account f or differences in TF/VIIa functional activity, we measured cell-surface-bou nd TFPI activity in Caco-5 cells. Small but similar amounts of TFPI were fo und 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 t he cell membrane could regulate TF/VIIa functional activity, we measured th e distribution of anionic phospholipids on the apical and basolateral surfa ce by annexin V binding and thrombin generation. The results showed that th e anionic phospholipid content on the basolateral surface, compared with th e 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 bas olateral surface without affecting the number of TF/VIIa complexes formed o n the surface. Overall, our data suggest that an uneven expression of TF/VI Ia activity between the apical and basolateral surface of polarized epithel ial cells is caused by differences in anionic phospholipid content between the two surface domains and not from a polar distribution of TFPI. (C) 1999 by The American Society of Hematology.