Regulation of Tissue Factor Signaling by Tissue Factor Pathway Inhibitor.

Tissue factor initiates the coagulation cascade as well as cellular signaling through the activation of cell surface protease activated receptors (PARs), relevant to inflammation, tumor metastasis and angiogenesis. Tissue factor pathway inhibitor-1 (TFPI-1) is the major regulator of TF initiated coagulation, but recombinant TFPI-1 (rTFPI-1) has shown limited efficacy on sepsis-associated inflammatory responses and mortality. In TNF-stimulated human umbilical vein endothelial cells (HUVECs), we demonstrate with specific blocking antibodies that endogenous TFPI effectively controls TF procoagulant activity as well as TF-dependent signaling through PAR1 and PAR2. The effect of rTFPI-1 on coagulation and signaling was tested in TF-transfected CHO-cells or in HUVECs transduced with TF to overcome the inhibitory threshold of endogenous TFPI-1. Under conditions that specifically measure signaling of the ternary TF-VIIa-Xa complex, 2.5–5 nM rTFPI-1 inhibited TF-dependent Xa generation by >80% but the signaling of the ternary complex through PAR1 at these concentrations of rTFPI-1 was only marginally inhibited. Moreover, studies with proteoglycan-deficient CHO cells provided genetic evidence that PG binding facilitates the inhibitory activities of rTFPI-1 on coagulation and signaling. In HUVECs that co-expressed PAR2 with TF, three different signaling responses of the ternary TF-VIIa-Xa complex were inhibited by ~ 50% at 1.2–2.5 nM rTFPI-1, but the inhibitory potency of rTFPI-1 to block Xa generation was consistently higher relative to its effect on signaling. These data demonstrate that coagulation initiation phase signaling can occur in the absence of the activation of coagulation. The results also establish that endogenously expressed TFPI-1, known to be entirely GPI-anchored in endothelial cells, is a potent, negative regulator of ternary complex-mediated PAR signaling. The efficacy of rTPFI-1 appears to be influenced by the presence of PAR2, further emphasizing the close reciprocal signaling linkage of PAR2 and TF demonstrated in angiogenesis and cellular signaling targeting the TF cytoplasmic domain by phosphorylation.