Susan A. Maroney

Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation

Significance The generation of thrombin by prothrombinase, a complex composed of activated (a) factors X (FXa) and V (FVa), is a final step in blood coagulation. We demonstrate that tissue factor pathway inhibitor (TFPI) blocks thrombin generation by prothrombinase at physiologically relevant rates and concentrations, but only during the initiation of clot formation. TFPI mediates this inhibitory activity through two high-affinity interactions, one with FXa and one with FVa. This is the first description of an endogenous human protein that inhibits prothrombinase under physiological conditions and may prevent a full thrombotic response to subthreshold coagulant stimuli that otherwise could occlude blood vessels. It provides unique understanding of thrombotic disorders and has important implications for development of anti-TFPI agents to treat hemophilia. Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIβ. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIβ and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIβ in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIβ acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.

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.

Expression of tissue factor pathway inhibitor by endothelial cells and platelets.

Tissue factor pathway inhibitor (TFPI) is a potent anticoagulant protein that abrogates the activity of the tissue factor-factor VIIa catalytic complex that activates blood coagulation in vivo. The importance of TFPI in the regulation of blood coagulation is emphasized by how its activity is modulated in human disease. Decreased TFPI activity contributes to the development of both arterial and venous thrombosis and has been implicated in the thrombotic events occurring in women using oral contraceptives and in patients with paroxysmal nocturnal hemoglobinuria. Both endothelial cells and platelets produce TFPI. Our laboratory is interested in the mechanisms for expression of TFPI on the surface of these cells to better understand how TFPI prevents intravascular thrombosis. Studies of cultured endothelial cells and human placenta have demonstrated that TFPI associates with the cell surface through a glycosyl phosphatidyinositol (GPI)-anchor in a manner that is not dependent on GAGs or altered by heparin. TFPI is not directly bound to the GPI-anchor; instead it appears to bind tightly to a GPI-anchored protein. This GPI-anchored protein appears to be necessary for proper trafficking of TFPI to the cell surface. An alternatively spliced form of TFPI, TFPIbeta, is a truncated form of TFPI that is directly attached to a GPI-anchor. However, it is not clear that human endothelial cells produce TFPIbeta. Platelets produce TFPI but not TFPIbeta. TFPI is expressed on the platelet surface following dual activation with collagen plus thrombin, but not through a GPI-anchor. Studies using mouse models of TFPI deficiency are currently being conducted in our laboratory to determine if distinct physiological functions of endothelial and platelet TFPI exist in vivo.

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.

Alternatively spliced isoforms of tissue factor pathway inhibitor

Tissue factor pathway inhibitor (TFPI) is the major regulator of tissue factor (TF)-induced coagulation. It down regulates coagulation by binding to the TF/fVIIa complex in a fXa dependent manner. It is predominantly produced by microvascular endothelial cells, though it is also found in platelets, monocytes, smooth muscle cells, and plasma. Its physiological importance is demonstrated by the embryonic lethality observed in TFPI knockout mice and by the increase in thrombotic burden that occurs when heterozygous TFPI mice are bred with mice carrying genetic risk factors for thrombotic disease, such as factor V Leiden. Multiple TFPI isoforms, termed TFPIalpha, TFPIbeta, and TFPIdelta in humans and TFPIalpha, TFPIbeta, and TFPIgamma in mice, have been described, which differ in their domain structure and method for cell surface attachment. A significant functional difference between these isoforms has yet to be described in vivo. Both human and mouse tissues produce, on average, approximately 10 times more TFPIalpha message when compared to that of TFPIbeta. Consistent with this finding, several lines of evidence suggest that TFPIalpha is the predominant protein isoform in humans. In contrast, recent work from our laboratory demonstrates that TFPIbeta is the major protein isoform produced in adult mice, suggesting that TFPI isoform production is translationally regulated.

Combined tissue factor pathway inhibitor and thrombomodulin deficiency produces an augmented hypercoagulable state with tissue-specific fibrin deposition

Summary.  Background and Objective: Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are endothelial‐associated anticoagulant proteins thought to control hemostasis in specific vascular beds. Here, we have examined the consequences of TFPI deficiency in the presence of a compounding procoagulant state caused by reduced TM function.

New insights into the biology of tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) dampens the initiation of blood coagulation by inhibiting two potent procoagulant complexes, tissue factor‐factor VIIa (TF–FVIIa) and early forms of prothrombinase. TFPI isoforms, TFPIα and TFPIβ, result from alternative splicing of mRNA, producing distinct C‐terminal ends of the two proteins. Both isoforms inhibit TF–FVIIa, but only TFPIα can inhibit early forms of prothrombinase by binding of its positively charged C‐terminus with high affinity to the acidic B‐domain exosite of FVa, which is generated upon activation by FXa. TFPIα and TFPIβ are produced in cultured human endothelial cells, while platelets contain only TFPIα. Knowledge of the anticoagulant mechanisms and tissue expression patterns of TFPIα and TFPIβ have improved our understanding of the phenotypes observed in different mouse models of TFPI deficiency, the east Texas bleeding disorder, and the development of pharmaceutical agents that block TFPI function to treat hemophilia.