Caveolin-1 Expression Regulates Cell Surface Exposure and Anticoagulant Function of Tissue Factor Pathway Inhibitor

Abstract number: OR078

Lupu C, Hu X, Lupu F

Tissue factor pathway inhibitor (TFPI) blocks tissue factor-factor VIIa (TF-FVIIa)-activation of factors X and IX through formation of TF-FVIIa-FXa-TFPI complex. Most TFPI in vivo associates with caveolae in endothelial cells (EC). The mechanism of this association and the anticoagulant role of the caveolar TFPI are not yet known. We studied how the expression of caveolin-1 (Cav-1) affects the distribution and function of TFPI. We expressed Cav-1 and EGFP-tagged TFPI in HEK293, a cell line that is deficient of TFPI and Cav-1. Immunofluorescence and live-cell microscopy revealed that considerably more TFPI is exposed on the cell surface in the presence of Cav-1, and with much decreased membrane lateral mobility. As reflected by inhibition of FX activation, TFPI has significantly higher inhibitory activity against both endogenously expressed TF and exogenously added TF-FVIIa. Confocal microscopy suggests that the caveolae-associated TFPI is responsible for inducing the co-localization of the quaternary complex with caveolae. The significance of these observations was investigated in EC by using RNA interference to deplete them of Cav-1. We found a highly significant positive correlation between the exposure of TFPI on the cell surface and the levels of Cav-1, for both HUVEC and EA.hy 926 cells. On molar basis, cell surface TFPI in normal EC was able to prevent up to seven-fold more FXa generation than in the cells with silenced Cav-1 expression. Fluorescence microscopy confirmed that the assembly of the quaternary complex was impaired in Cav-1 depleted cells, apparently due to the inability of TFPI to support the localization of the complex in caveolae. Our results suggest that caveolae could concentrate, through TFPI, the interacting molecules and facilitate the formation of the quaternary complex. The process identifies Cav-1 and/or caveolae as a key factor in the regulation of TFPI-dependent inhibition of TF-driven coagulation in EC.