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

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an alternatively spliced protein with two isoforms, TFPIα and TFPIß, 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 TFPIß 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, TFPIß was a slightly better inhibitor than TFPIα, which was approximately three-fold better than TFPI-160, a soluble, altered form of TFPI similar to TFPIß. In direct FXa inhibitory assays, TFPIß had an IC50 2.5-fold lower than that of TFPIα and 56-fold lower than that of TFPI-160. TFPIß inhibited TF-mediated CHO cell migration though Matrigel, whereas TFPIα and TFPI-160 were poor inhibitors, demonstrating that TFPIß effectively blocks TF-initiated signaling events during cellular migration through matrices that are not permeable to soluble forms of TFPI. Furthermore, TFPIß inhibited TF-dependent CHO cell infiltration into lung tissue following tail vein injection into SCID mice, and blocked the development of consumptive coagulopathy. CONCLUSIONS: TFPIß is a slightly better inhibitor of TF procoagulant activity than TFPIα. As a surface-associated protein, TFPIß 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.

Temporal expression of alternatively spliced forms of tissue factor pathway inhibitor in mice.

BACKGROUND: Mouse tissue factor pathway inhibitor (TFPI) is produced in three alternatively spliced isoforms that differ in domain structure and mechanism for cell surface binding. Tissue expression of TFPI isoforms in mice was characterized as an initial step for identification of their physiological functions. METHODS AND RESULTS: Sequence homology demonstrates that TFPIalpha existed over 430 Ma while TFPIbeta and TFPIgamma evolved more recently. In situ hybridization studies of heart and lung did not reveal any cells exclusively expressing a single isoform. Although our previous studies have demonstrated that TFPIalpha mRNA is more prevalent than TFPIbeta or TFPIgamma mRNA in mouse tissues, western blot studies demonstrated that TFPIbeta is the primary protein isoform produced in adult tissues, while TFPIalpha is expressed during embryonic development and in placenta. Consistent with TFPIbeta as the primary isoform produced within adult vascular beds, the TFPI isoform in mouse plasma migrates like TFPIbeta in SDS-PAGE and mice have a much smaller heparin-releasable pool of plasma TFPIalpha than humans. CONCLUSIONS: The data demonstrate that alternatively spliced isoforms of TFPI are temporally expressed in mouse tissues at the level of protein production. TFPIalpha and TFPIbeta are produced in embryonic tissues and in placenta while adult tissues produce almost exclusively TFPIbeta.

Tissue factor pathway inhibitor-gamma is an active alternatively spliced form of tissue factor pathway inhibitor present in mice but not in humans.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor procoagulant activity produced as two alternatively spliced isoforms, TFPIalpha and TFPIbeta, which differ in domain structure and mechanism for cell surface association. 3' Rapid amplification of cDNA ends was used to search for new TFPI isoforms. TFPIgamma, a new alternatively spliced form of TFPI, was identified and characterized. METHODS: The tissue expression, cell surface association and anticoagulant activity of TFPIgamma were characterized and compared to those of TFPIalpha and TFPIbeta through studies of mouse and human tissues and expression of recombinant proteins in Chinese hamster ovary (CHO) cells. RESULTS: TFPIgamma is produced by alternative splicing using the same 5'-splice donor site as TFPIbeta and a 3'-splice acceptor site 276 nucleotides beyond the stop codon of TFPIbeta in exon 8. The resulting protein has the first two Kunitz domains connected to an 18 amino acid C-terminal region specific to TFPIgamma. TFPIgamma mRNA is differentially produced in mouse tissues but is not encoded within the human TFPI gene. When expressed in CHO cells, TFPIgamma is secreted into conditioned media and effectively inhibits tissue factor procoagulant activity. CONCLUSIONS: TFPIgamma is a third alternatively spliced form of TFPI that is widely expressed in mouse tissues but not made by human tissues. It contains the first two Kunitz domains and is a secreted, rather than a cell surface-associated, protein. It is a functional anticoagulant and may partially explain the resistance of mice to coagulopathy in tissue factor-mediated models of disease.