Josephine P. Ferrel

A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression.

Summary.  Background: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)‐anchored co‐receptor. Objectives/methods: Cellular trafficking of TFPI within aerolysin‐resistant ECV304 and EA.hy926 cells, which do not express GPI‐anchored proteins on their surface, was compared with their wild‐type counterparts. Results and conclusions: Although aerolysin‐resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild‐type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin‐resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin‐resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI‐anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co‐receptor in the ER/Golgi. The co‐receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild‐type cells or to lysosomes of aerolysin‐resistant cells. TFPI that escapes co‐receptor binding is secreted through the same pathway in both wild‐type and aerolysin‐resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.

Absence of hematopoietic tissue factor pathway inhibitor mitigates bleeding in mice with hemophilia

Tissue factor pathway inhibitor (TFPI) blocks thrombin generation via the extrinsic blood coagulation pathway. Because the severe bleeding in patients with hemophilia occurs from deficiency of intrinsic blood coagulation pathway factor VIII or IX, pharmacological agents that inactivate TFPI and, therefore, restore thrombin generation via the extrinsic pathway, are being developed for treatment of hemophilia. Murine models of combined TFPI and factor VIII deficiency were used to examine the impact of TFPI deficiency on bleeding and clotting in hemophilia. In breeding studies, Factor VIII null (F8−/−) did not rescue the embryonic death of TFPI null (Tfpi−/−) mice. Tfpi+/− did not alter the bleeding phenotype of F8−/− mice. However, total inhibition of intravascular TFPI through injection of anti-TFPI antibody mitigated tail vein bleeding. Interestingly, tail blood loss progressively decreased at doses greater than needed to totally inhibit plasma TFPI, suggesting that inhibition of a sequestered pool of TFPI released at the injury site mitigates bleeding. Because TFPI is sequestered within platelets and released following their activation, the function of platelet TFPI was examined in F8−/− mice lacking hematopoietic cell TFPI that was generated by fetal liver transplantation. Blood loss after tail transection significantly decreased in Tfpi+/−;F8−/− mice with hematopoietic Tfpi−/− cells compared with Tfpi+/−;F8−/− mice with Tfpi+/+ hematopoietic cells. Additionally, following femoral vein injury, Tfpi+/−;F8−/− mice with Tfpi−/− hematopoietic cells had increased fibrin deposition compared with identical-genotype mice with Tfpi+/+ hematopoietic cells. These findings implicate platelet TFPI as a primary physiological regulator of bleeding in hemophilia.

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

Summary.  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 TFPIα existed over 430 Ma while TFPIβ and TFPIγ 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 TFPIα mRNA is more prevalent than TFPIβ or TFPIγ mRNA in mouse tissues, western blot studies demonstrated that TFPIβ is the primary protein isoform produced in adult tissues, while TFPIα is expressed during embryonic development and in placenta. Consistent with TFPIβ as the primary isoform produced within adult vascular beds, the TFPI isoform in mouse plasma migrates like TFPIβ in SDS‐PAGE and mice have a much smaller heparin‐releasable pool of plasma TFPIα than humans. Conclusions: The data demonstrate that alternatively spliced isoforms of TFPI are temporally expressed in mouse tissues at the level of protein production. TFPIα and TFPIβ are produced in embryonic tissues and in placenta while adult tissues produce almost exclusively TFPIβ.

Murine Hematopoietic Cell Tissue Factor Pathway Inhibitor Limits Thrombus Growth

Objective—Tissue factor (TF)–factor VIIa initiates blood coagulation and is found on microparticles that accumulate within intravascular thrombi. Tissue factor pathway inhibitor (TFPI), a factor Xa (fXa)–dependent inhibitor of TF–factor VIIa, is produced by megakaryocytes and is present in platelets. We sought to determine the role of platelet TFPI in regulation of thrombus growth. Methods and Results—Western blot analyses demonstrated that murine platelets produce TFPI&agr;, the most evolutionarily conserved alternatively spliced isoform of TFPI. A mouse model of hematopoietic cell TFPI deficiency was developed by transplanting irradiated TFPI+/− mice with TFPI−/− fetal liver cells. Platelets from transplanted mice totally lack TFPI inhibitory activity. An electrolytic vascular injury model was used to assess thrombus growth in the femoral vein and carotid artery. Mice lacking hematopoietic TFPI developed larger femoral vein and carotid artery thrombi than TFPI+/− mice transplanted with TFPI+/+ hematopoietic cells, as evidenced by increased platelet accumulation. Conclusion—Hematopoietic TFPI limits thrombus growth following vascular injury. Because platelets are the primary hematopoietic cell accumulating within a growing thrombus, these findings suggest that TFPI present within platelets functions to limit intravascular thrombus growth, likely through inhibition of the procoagulant activity of blood borne TF.

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

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.

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

Summary.  Background: Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor procoagulant activity produced as two alternatively spliced isoforms, TFPIα and TFPIβ, 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. TFPIγ, a new alternatively spliced form of TFPI, was identified and characterized. Methods: The tissue expression, cell surface association and anticoagulant activity of TFPIγ were characterized and compared to those of TFPIα and TFPIβ through studies of mouse and human tissues and expression of recombinant proteins in Chinese hamster ovary (CHO) cells. Results: TFPIγ is produced by alternative splicing using the same 5′‐splice donor site as TFPIβ and a 3′‐splice acceptor site 276 nucleotides beyond the stop codon of TFPIβ in exon 8. The resulting protein has the first two Kunitz domains connected to an 18 amino acid C‐terminal region specific to TFPIγ. TFPIγ mRNA is differentially produced in mouse tissues but is not encoded within the human TFPI gene. When expressed in CHO cells, TFPIγ is secreted into conditioned media and effectively inhibits tissue factor procoagulant activity. Conclusions: TFPIγ 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.

Caveolae optimize tissue factor-Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor

TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)-fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI-GPI (TFPI-glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI-TM (TFPI-transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent-insoluble membrane fractions, whereas TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-fVIIa than TFPI-TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIβ and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.