TISSUE FACTOR PATHWAY INHIBITOR IN ENDOTHELIAL-CELLS COLOCALIZES WITHGLYCOLIPID MICRODOMAINS CAVEOLAE - REGULATORY MECHANISM(S) OF THE ANTICOAGULANT PROPERTIES OF THE ENDOTHELIUM/

Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters unif ormly distributed both on the cell surface and intracellularly. We her e demonstrate by immunofluorescence that TFPI colocalizes in EC with c aveolin, urokinase-type plasminogen activator receptor, and glycosphin golipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveol in. After ultracentrifugation of rat lung or EC homogenates through de nsity gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-1 00-insoluble extracts of EC. TFPI incorporates [1-H-3]ethanolamine and is cleaved from the cell surface by phosphatidylinositol-phospholipas e C, indicating a specific glycosylphosphatidylinositol-anchorage mech anism for TFPI in the plasma membrane. Clustering of TFPI and its loca lization in caveolae are dependent on the presence of cholesterol in t he membrane. Agonist-induced stimulation of EC caused marked changes o f distribution for both TFPI and caveolin at subcellular level, with s ubsequent increase of the cell surface-associated inhibitory activity toward tissue factor.factor VIIa. Our findings suggest that, beside th eir function in transcytosis, potocytosis, cell surface proteolysis, a nd regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.