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Keywords:

  • alternative splicing;
  • hemophilia;
  • migration;
  • TFPI;
  • tissue factor

Summary

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.