Quantification and Characterization of Human Endothelial Cell–Derived Tissue Factor Pathway Inhibitor-2

From the Department of Pathology (M.I., W.K.), University of New Mexico School of Medicine, Albuquerque, NM, and ZymoGenetics, Inc (D.C.F.), Seattle, Wash.

Correspondence to Dr Walter Kisiel, Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131-5301.

Abstract—Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a serine protease inhibitor consisting of three tandemly-arranged Kunitz-type protease inhibitor domains. While TFPI-2 is a potent inhibitor of trypsin, plasmin, kallikrein, and factor XIa in the test tube, the function of this inhibitor in vivo remains unclear. In the present study, we investigated the synthesis and secretion of TFPI-2 by cultured endothelial cells derived from human umbilical vein, aorta, saphenous vein, and dermal microvessels to gain insight into its biological function. While all endothelial cells examined synthesized and secreted TFPI-2, dermal microvascular endothelial cells synthesized threefold to sevenfold higher levels of TFPI-2. Approximately 60% to 90% of the TFPI-2 secreted by endothelial cells was directed to the subendothelial extracellular matrix (ECM). When cultured human umbilical vein endothelial cells were stimulated with inflammatory mediators such as phorbol 12-myristate,13-acetate; endotoxin; and tumor necrosis factor-, TFPI-2 synthesis by these cells increased twofold to 14-fold. Recombinant TFPI-2 bound to dermal microvascular endothelial cell monolayers and its ECM in a specific, dose-dependent, and saturable manner with K_d values of 21 and 24 nmol/L, respectively. TFPI-2 interacted with 4.5x10¹⁰ sites/cm² (3x10⁵ sites/cell) and 2.3x10¹¹ sites/cm² on endothelial cells and ECM, respectively. In the presence of rabbit anti–TFPI-2 IgG, but not preimmune IgG, endothelial cells dissociated from the culture flask in a time- and IgG concentration–dependent manner. Our findings provide evidence that endothelial cell–derived TFPI-2 is primarily secreted into the abluminal space and presumably plays an important role in maintaining the integrity of the ECM essential for cell attachment.

Key Words: tissue factor pathway inhibitor-2 • endothelial cell • extracellular matrix • proteoglycan

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