Comparison of the inhibitory activities of human tissue factor pathway inhibitor (TFPI)α and TFPIβ

Abstract

Background: Tissue factor pathway inhibitor (TFPI) is an alternatively spliced protein with two isoforms, TFPIα and TFPIβ, which differ in their C-terminal structure and cellular localization. Detailed characterization of their inhibitory activity is needed to define potentially unique inhibitory roles in tissue factor (TF)-mediated thrombotic and inflammatory disease, and to understand how pharmaceuticals targeted to different structural regions of the TFPI isoforms alter hemostasis in hemophilia patients.

Methods: The TF inhibitory activity of TFPIβ localized to the surface of CHO cells was compared with that of soluble TFPIα by the use of in vitro and in vivo assays.

Results: In TF-factor VIIa-mediated FXa generation assays, TFPIβ was a slightly better inhibitor than TFPIα, which was approximately three-fold better than TFPI-160, a soluble, altered form of TFPI similar to TFPIβ. In direct FXa inhibitory assays, TFPIβ had an IC50 2.5-fold lower than that of TFPIα and 56-fold lower than that of TFPI-160. TFPIβ inhibited TF-mediated CHO cell migration though Matrigel, whereas TFPIα and TFPI-160 were poor inhibitors, demonstrating that TFPIβ effectively blocks TF-initiated signaling events during cellular migration through matrices that are not permeable to soluble forms of TFPI. Furthermore, TFPIβ inhibited TF-dependent CHO cell infiltration into lung tissue following tail vein injection into SCID mice, and blocked the development of consumptive coagulopathy.

Conclusions: TFPIβ is a slightly better inhibitor of TF procoagulant activity than TFPIα. As a surface-associated protein, TFPIβ is a much better inhibitor of TF-mediated cellular migration than soluble TFPIα, and may specifically act in the inhibition of TF-mediated signaling events on inflamed endothelium and/or monocytes.

Fig. 1

CHO-TF and CHO-TF/TFPIβ cells have similar TF expression and activity. (A) Flow cytometry for TF on CHO-TF (filled dark grey) and CHO-TF/TFPIβ cells (solid line) and for TFPI on CHO-TF/TFPIβ cells (dotted line). The isotype control is shaded light grey. The data are representative of 3 experiments. (B) TF-FVIIa-mediated FXa generation on CHO-TF cells and CHO-TF/TFPIβ cells. TFPIβ decreases the amount of FXa generated (p<0.001) and can be totally reversed by anti-TFPI polyclonal antibody or by treatment of the cells with PIPLC (mean ± SEM, n=3).

Fig. 2

TFPIβ is an effective inhibitor TF-FVIIa and FXa. (A) TF-FVIIa-mediated FXa generation (mean ± SEM, n=3) and (B) FXa activity (mean ± SEM, n=3) were measured in the presence of glycosylated TFPIα (□), non-glycosylated TFPIα (■), TFPI-160 (▲) or CHO-TF/TFPIβ cells (●). Cellular TF was constant in all experiments. Data are shown as percent activity compared to the “no TFPI” control in (A) or as uninhibited FXa activity in (B).

Fig. 3

TFPIβ blocks CHO-TF cell migration through Matrigel by preventing thrombin generation and activation of PAR-1. Cellular migration was standardized to that of CHO-TF cells. (A) Migration of CHO-TF/TFPIβ cells compared to CHO cells, CHO-TF cells and CHO-TF cells in the presence of 100nM/well recombinant TFPI-160, glycosylated TFPIα or non-glycosylated TFPIα (mean ± SEM, n=3). (B) Migration of CHO-TF cells in the presence of SCH79797 (PAR-1 antagonist), rNAP-C2 (TF-FVIIa inhibitor), rTAP (FXa inhibitor), or argatroban (thrombin inhibitor), all at 10 μM (*p<0.001, †p<0.01) (mean ± SEM, n=3).

Fig. 4

TFPIβ reduces TF-mediated tissue infiltration and consumptive coagulopathy. Histology of the lungs from SCID mice ten days following injection with (A) CHO; (B) CHO-TF; or (C) CHO-TF/TFPIβ cells. (A) Arrowhead indicates a small cluster of CHO cells within the lung. (B) Arrow indicates a large intravascular thrombus. (D) Histopathological grading (1⁺–4⁺) of lungs ten days following injection cells. (E) Platelet counts ten days following injection of cells (*p<0.001, † p<0.05).