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Background— The endothelium is a major source of tissue factor pathway inhibitor (TFPI), the endogenous regulator of TF-induced coagulation, and a significant proportion of the expressed TFPI remains associated with the endothelial surface. Methods and Results— Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment reduced TFPI at the surface of cultured endothelial cells by ≈80%, and at least a portion of the TFPI released by PI-PLC contained an intrinsic glycosylphosphatidylinositol (GPI) anchor that is recognized by anti-crossreactive determinant antibodies. Endothelial cells express both of the alternatively spliced forms of TFPI mRNA at a ratio of TFPIβ/TFPIα mRNA of ≈0.1 to 0.2. In Chinese hamster ovary (CHO) cells, TFPIα is predominantly secreted, whereas TFPIβ is a GPI-anchored membrane protein. Like TFPIβ, the small proportion of the TFPIα expressed by CHO cells that remains surface associated is also released by PI-PLC treatment, suggesting that it is bound to a separate GPI-anchored protein(s) at the surface of the cells. Conclusions— Both direct (TFPIβ) and indirect (TFPIα) GPI-mediated membrane anchorage is involved in localizing TFPI to the surface of cells.

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