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

Abstract

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.

Fig. 1

Schematic diagrams of mouse (A) TFPIα, (B) TFPIβ and (C) TFPIγ, demonstrating their Kunitz domain structures. The narrow arrows indicate the alternative splice site. The amino acids C-terminal to this site are unique to the isoform. The bold arrow in the C-terminal region of TFPIβ indicates the predicted GPI-anchor attachment site when analyzed using GPI-modification site predictor software (http://mendel.imp.ac.at/gpi/gpi_server.html). The amino acids distal to this site are removed in the ER upon attachment of the GPI-anchor and are not present in mature TFPIβ expressed on the cell surface. (D) Amino acid sequence of K3 from several species. A high amount of amino acid identity exists from primates to fish, demonstrating conservation of K3 structure over 430 Myr of evolution. (E) Amino acid sequences from species with identity to the C-terminal TFPIβ-specific segment.

Fig. 2

TFPIα and TFPIβ have similar tissue mRNA expression patterns, with the majority of expression in endothelial cells. In situ hybridization of mouse (A) lung and (B) heart is shown. No cells specifically staining with either the TFPIα or the TFPIβ probe were identified. Each figure contains a hematoxylin and eosin panel to assess the tissue architecture and various controls as indicated.

Fig. 3

TFPIβ is the predominant protein isoform produced by adult mouse tissues while TFPIα and TFPIβ are produced in mouse placenta and embryo. (A) Western blot analyses demonstrating the deglycosylation patterns of TFPI from mouse heart and mouse placenta with comparison to mouse TFPIα and TFPIβ expressed in CHO cells. Tissues were lysed in CHAPS buffer and precipitated with bovine FXa agarose to concentrate TFPI. Lanes 1 and 5, non-treated; lanes 2 and 6, N-deglycosylated; lanes 3 and 7, O-deglycosylated; lanes 4 and 8, combined N- and O-deglycosylated. The minor ∼35 kDa band in the non-deglycosylated samples of CHO cell TFPIβ has also been observed in Western blots of human TFPIβ produced in CHO cells [26]. (B) Western blot analyses of TFPI from different mouse tissues. The tissue source of TFPI is indicated at the top of the gels. For each tissue an untreated sample is on the left and a combined N- and O-deglycosylated sample is on the right. The embryonic tissue was obtained from an E14.5 fetus. Lung+TFPIα indicates a mixture of lung tissue and TFPIα produced in CHO cells. CHO cells transfected with mouse TFPIα or TFPIβ serve as controls for the migration patterns of the individual isoforms before and after deglycosylation.

Fig. 4

Mice have a minor heparin-releasable pool of TFPIα. (A) TFPI Western blot analysis of combined O- and N-deglycosylated plasma samples from either TFPI^+/− mice (lanes 1 and 2) or wild-type mice (lanes 3 and 4) is shown. Samples from untreated mice are in lanes 1 and 3. Samples obtained 15 min after injection of heparin are in lanes 2 and 4. The arrows indicate the migration of TFPIα and TFPIβ and highlight a minor amount of TFPIα present in the plasma from wild-type mice treated with heparin. Samples from the TFPI^+/− mice are shown to demonstrate that this Western blot analysis is semi-quantitative. (B) TFPI ELISA analysis of mouse plasma before and 15 or 60 min following heparin injection. The white bars are baseline measurements from a sample obtained before injection. The grey bars are from mice injected with saline. The black bars are from mice injected with heparin. The amount of TFPI in plasma following heparin injection is significantly greater than baseline at both 15 and 60 min (P < 0.001), as indicated by the asterisks.