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 4.5ϫ10 10 sites/cm 2 (3ϫ10 5 sites/cell) and 2.3ϫ10 11 sites/cm 2 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. (Arterioscler Thromb Vasc Biol. 1998;18:40-46.) Key Words: tissue factor pathway inhibitor-2 Ⅲ endothelial cell Ⅲ extracellular matrix Ⅲ proteoglycan H uman TFPI-2 is a 32-kD serine protease inhibitor with an overall Kunitz inhibitory domain organization similar to TFPI, a major factor Xa-and factor VIIa-tissue factor inhibitor found in human plasma.1,2 Recombinant human TFPI-2 has recently been shown to be a strong inhibitor of trypsin, plasmin, plasma kallikrein, and factor XIa amidolytic activity. 3 TFPI-2 also weakly inhibited the amidolytic and proteolytic activities of chymotrypsin, factor VIIa-tissue factor, factor IXa-polylysine, and cathepsin G, but failed to significantly inhibit the amidolytic activities of glandular kallikrein, urinary plasminogen activator, tissue plasminogen activator, activated protein C, factor Xa, thrombin, and leukocyte elastase. Recent studies revealed that TFPI-2 is identical to an inhibitory protein isolated from human placenta designated as placental protein 5, or PP5.4,5 PP5 is synthesized in endothelial cells 6 and circulates in blood of normal men and nonpregnant women in extremely low concentrations (0.43 to 0.49 ng/mL). 7 The level of PP5 increases 40-fold t...