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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: J Thromb Haemost. 2015 Jun;13(0 1):S200–S207. doi: 10.1111/jth.12897

New insights into the biology of tissue factor pathway inhibitor

S A MARONEY *, A E MAST *,†
PMCID: PMC4604745  NIHMSID: NIHMS729139  PMID: 26149025

Summary

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.

Keywords: hemophilia, prothrombinase, TFPI, thrombosis, tissue factor

Introduction

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein produced primarily by the endothelium [1] and megakaryocytes [2,3]. TFPI is produced in at least three alternatively spliced isoforms in humans, TFPIα, TFPIβ, and TFPIδ, and at least three alternatively spliced isoforms in mice, TFPIα, TFPIβ, and TFPIγ [4]. The tissue factor–factor VIIa (TF–FVIIa) catalytic complex is the primary initiator of blood coagulation. TFPI exerts its anticoagulant effects by inhibiting TF–FVIIa in a manner that is dependent on its inhibition of factor Xa (FXa) [5]. All isoforms are capable of inhibiting TF–FVIIa and FXa. Recently, it was recognized that TFPIα is uniquely capable of inhibiting early forms of the prothrombinase complex that assemble before thrombin is generated [6], and evidence is building from a variety of different experimental results that this inhibitory reaction may be physiologically important. These two anticoagulant activities allow TFPI to dampen very early intravascular procoagulant activity and thereby minimize the development of occlusive thrombi and consumptive coagulopathy [7,8]. As such, TFPI impacts a broad range of bleeding and thrombotic disorders [9,10]. This article will review the biochemistry of TFPI anticoagulant function, the tissue expression of the different TFPI isoforms, the relation of TFPI to human thrombotic and bleeding disorders, and the possibility for inhibiting TFPI activity as a treatment for hemophilia.

TFPI biochemistry

TFPI is a multivalent Kunitz-type protease inhibitor with variants that are produced by a single gene transcription of alternatively spliced mRNAs that translate into isoforms that differ in their C-terminal domain structure, their mechanism for cell surface association, and their anticoagulant activity. Protein-level expression of TFPIγ, which is produced only in mice [11], and TFPIδ, which is produced only in humans [4], is uncertain, and details about their structures have been published previously [4]. Here, we focus on TFPIα and TFPIβ, both of which are present in humans and mice. TFPIα contains three tandem Kunitz-type serine protease inhibitor domains, K1, K2, and K3, followed by a basic C-terminal region (Fig. 1) [12], and is thought to be primarily a secreted protein [13]. TFPIβ contains the identical K1 and K2 domains, which are followed by a GPI-anchor attachment sequence that allows direct association with cell surfaces (Fig. 1) [4]. K2 directly binds to and inhibits the active site of FXa [5]. This reaction is enhanced by the basic C-terminal region of TFPIα [14], which directly interacts with FXa [15]. It is also enhanced by protein S [16], which binds to K3 [17] and then localizes TFPIα to phospholipid surfaces where it can more readily interact with FXa [18]. Although soluble forms of TFPI containing only K1 and K2 are relatively poor direct inhibitors of FXa [19], TFPIβ efficiently inhibits FXa when attached to the cell surface via its GPI anchor [20].

Fig. 1.

Fig. 1

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Diagrams of the Kunitz domain structures of TFPIα and TFPIβ with inhibitory functions identified. The C-terminal regions of the two proteins are distinctly different. TFPIα has a third Kunitz domain that binds protein S and a FV homology domain that provides exosite binding during inhibition of early forms of prothrombinase. TFPIβ has a GPI-anchor attachment sequence that allows for its direct association with the endothelial surface.

K1 binds to and inhibits the active site of FVIIa, thereby inhibiting procoagulant activity produced by the TF–FVIIa catalytic complex [5]. The inhibition of FVIIa is enhanced by the presence of FXa [21]. Thus, TFPI has been described as a FXa-dependent inhibitor of TF–FVIIa with the inhibition of TF–FVIIa by K1 occurring after inhibition of FXa by K2. However, it appears that this is not the true inhibitory mechanism. Instead, K1 and K2 bind the active sites of FVIIa and FXa, respectively, simultaneously and immediately after FX is activated by TF–FVIIa [22].

As TFPIα and TFPIβ each have K1 and K2, both inhibit TF–FVIIa. The GPI anchor of TFPIβ localizes to caveolae on the endothelial cell surface and greatly enhances its anticoagulant activity [23]. Studies using altered forms of TFPI that localize either to the bulk plasma membrane or to caveolae, as well as cells transfected with TFPIβ, found that localization of TFPI to caveolae enhances the inhibition of TF–FVIIa-mediated generation of FXa, while having no effect on the direct inhibition of FXa [24].

In addition to inhibiting FXa and TF–FVIIa, TFPIα efficiently inhibits early forms of prothrombinase [6], the catalytic complex consisting of FVa, FXa, phospholipids, and Ca++ that rapidly converts prothrombin to thrombin. Early studies found that although TFPIα efficiently inhibits prothrombinase from cleaving amidolytic substrates, it poorly inhibits prothrombinase from cleaving prothrombin, its physiological substrate [25]. However, these initial experiments were performed with thrombin-activated FVa in which the entire B-domain of FV is removed. A first clue to identifying the ability of TFPIα to inhibit early forms of prothrombinase was recognizing that a basic 9 amino acid stretch within the TFPIα C-terminal region, LIKTKRKRK, is homologous with a basic region in the FV B-domain (Fig. 2) [6]. This homology is retained in all mammals, as well as some birds and reptiles, which strongly suggested it was physiologically important. Because of this homology, we considered the possibility that TFPIα inhibits prothrombinase assembled with forms of FVa produced early in the procoagulant response that are fully active but retain acidic portions of the B-domain as occurs when FV is activated by FXa and in some forms of platelet FVa, but not in thrombin-activated FV (Fig. 2) [26,27], and identified a potent inhibitory effect [6]. To briefly summarize the inhibitory mechanism, there are distinct basic and acidic regions within the large central B-domain of FV. These domains interact with each other to maintain FV in an inactive conformation [28]. Removal of only the basic region of the B-domain (the region with TFPIα homology) converts FV into a fully active cofactor [28]. When the basic region of the B-domain is removed, the TFPIα basic C-terminal region binds very tightly to the acidic region of the B-domain, providing an exosite interaction for inhibition of prothrombinase that is mediated by binding of K2 to the active site of FXa [6]. Key points about the inhibition of prothrombinase by TFPIα are as follows:

Fig. 2.

Fig. 2

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Diagram demonstrating how the FV B-domain is cleaved sequentially by FXa (Xa) and thrombin (IIa). The B-domain contains a basic region (blue box) with homology to the C-terminus of TFPIα and an acidic region (red box). The basic and acidic regions interact with each other to maintain FV in an inactive conformation. Cleavage of FV by FXa removes the basic region of the B-domain; the acidic region is retained. When FXa-activated FVa assembles into prothrombinase, the acidic region provides a high-affinity exosite for binding of the TFPIα C-terminus that allows for inhibition of prothrombinase by TFPIα. When thrombin is present, it rapidly removes the entire B-domain including the acidic region. Therefore, prothrombinase assembled with thrombin-activated FVa is not readily inhibited by TFPIα.

1 TFPIα inhibits thrombin generation by prothrombinase assembled with FXa-activated FVa with an IC50 value of 0.9 nM, which is 42-fold lower than the IC50 value of 37.5 nM observed for prothrombinase assembled with thrombin-activated FVa [6].

2 TFPIβ is a poor inhibitor of prothrombinase assembled with any form of FVa [6].

3 Monoclonal anti-TFPI antibodies against K2 or K3C, but not K1, block the endogenous TFPIα-mediated prothrombinase inhibitory activity in collagen-activated platelets, indicating this is a TF–FVIIa-independent inhibitory activity [6].

4 Protein S does not enhance inhibition of early forms of prothrombinase by TFPIα [18].

This inhibition occurs at physiologically relevant rates and protein concentrations in biochemical assays and is the first human protein described to inhibit prothrombinase from activating prothrombin to thrombin. No other endogenous inhibitor has been found to block this stage of the clotting cascade under physiological conditions, including the antithrombin/heparin [29] and protein Z/protein Z-dependent protease inhibitor complexes [30]. In addition, FVa, when assembled in prothrombinase, is protected from degradation by the activated protein C/protein S complex [31].

TFPI expression

Polymorphisms present within the TFPI promoter have been reported to alter the plasma concentration of human TFPI [32]. However, many of these initial reports were not reproduced when examined in larger populations [33]. Thus, promoter regulation of TFPI mRNA production is unclear. However, it is known that the protein production of TFPIα verses TFPIβ is under translational control. The human TFPI 5′ untranslated region undergoes alternative splicing of exon 2. Exon 2 is a repressor that prevents translation of TFPIβ, but not TFPIα [34]. Thus, exon 2 splicing is a molecular switch that alters the translation of different TFPI isoforms produced by 3′ splicing events. The differential expression of exon 2 represents a means to provide temporal and tissue-specific expression of TFPIβ anticoagulant activity. Exon 2 splicing does not occur in mice, suggesting that translational control of TFPI isoform expression differs between humans and mice.

Endothelial TFPI

TFPIα and TFPIβ both exhibit anticoagulant effects via FXa-dependent inhibition of TF–FVIIa [20]. However, TFPIα is uniquely capable of inhibition of early forms of prothrombinase assembled with forms of FVa that retain the acidic region of the B-domain [6]. Thus, detailed knowledge of cellular TFPI isoform expression within vascular beds is important for understanding how TFPI modulates intravascular blood coagulation under normal conditions and in patients with bleeding and thrombotic disorders. In situ hybridization studies of murine tissues revealed that TFPIα and TFPIβ mRNA have identical cellular expression patterns with the vascular endothelium as the major site of synthesis [35]. Quantitative PCR analyses have shown that TFPIα mRNA is about 10-fold more abundant than TFPIβ mRNA in human and mouse tissues and cultured human endothelial cells [11,36]. However, consistent with the translational control mechanisms for TFPI described above, the protein expression patterns of the TFPI isoforms do not match the mRNA expression patterns [35]. The understanding of TFPI cell surface association has undergone changes, as experimental systems evolved to study the protein. It was originally thought that TFPIα and TFPIβ were indirectly and directly, respectively, attached to the endothelial cell surface [37]. Current evidence suggests that TFPIβ is the only isoform on the endothelial surface in mouse tissues [35], as well as on the surface of cultured human endothelial cells and human placental microsomes [13]. Growth of cultured human endothelial cells in the presence of heparin increases the amount of TFPIα secreted into conditioned media in a manner that is dose and time dependent [38]. It has been estimated that cultured human endothelial cells produce 10–50 times more TFPIα than TFPIβ in a 24-h period [13]. These data suggest that heparin may induce secretion of TFPIα from an uncharacterized human endothelial cell granule which is distinct from the Weibel-Palade body [39]. Complete characterization of TFPI isoform processing by endothelial cells and how the processing is modified by biological compounds will better define how endothelial TFPI is stored and secreted into plasma in vivo.

Plasma TFPI

Human plasma TFPI consists of about 10–30% TFPIα with the remainder variably C-terminally truncated and associated with lipoproteins [40]. TFPIα increases 2- to 4-fold promptly following heparin infusion and is rapidly reversed by protamine infusion [41]. This suggests human TFPIα binds to glycosaminoglycans on endothelium in vivo through its basic C-terminus. This is distinctly different from mice that do not have a heparin releasable pool of plasma TFPIα [35].

Platelet TFPI

While human endothelial cells produce both TFPIα and TFPIβ, megakaryocytes produce only TFPIα which localizes within quiescent platelets, but not on the platelet surface [3]. Similar to humans, murine platelets also contain only TFPIα [7]. The location of TFPIα within platelets is uncertain, but it is not in α-granules [3]. TFPIα is secreted from platelets upon activation with thrombin [2,3]. When platelets are dual activated with thrombin and convulxin, TFPIα is secreted and also localizes to the platelet surface [3]. As such, platelet TFPIα represents an anticoagulant protein that is secreted by a procoagulant cell. The biological function of platelet TFPIα was investigated by producing mice lacking hematopoietic cell TFPIα by fetal liver transplantation into irradiated adult heterozygous TFPI mice. The transplanted mice had unaltered plasma TFPI concentration, indicating that platelets do not contribute to the plasma TFPI pool [7]. This finding is consistent with the lack of TFPIα in murine plasma. When subjected to an electrolytic injury model of thrombus growth in the femoral vein or carotid artery, and when compared to mice transplanted with wild-type fetal liver cells, those lacking hematopoietic cell TFPI developed larger thrombi characterized by increased platelet accumulation [7]. Thus, platelet TFPIα appears to function to limit the growth of intravascular thrombi and acts independently of the presence of plasma or endothelium pools of TFPI.

Studies of TFPI deficiency in mice

A number of mouse models have demonstrated physiological synergies between TFPI deficiency and procoagulant proteins such as TF, FVIIa, FV Leiden, and thrombomodulin. These studies reveal rescue of embryonic lethal phenotypes and severe postnatal coagulopathies.

Rescue of embryonic lethality associated with the lack of TFPI

Genetically altered mice lacking K1 (TFPItm1Gjb; Tfpi−/−) die during embryogenesis. Between 40 and 70% of the embryos die between E9.5 and E11.5 from yolk sac hemorrhage, and the remainder die before birth from a consumptive coagulopathy. The older embryos display fibrin deposition in the liver and brain, and some have short tails [8]. The TFPI null mice can be rescued from embryonic lethality by breeding with mice expressing low levels of TF (F3tm1Dco/Tg[F3]1Nmk). During pregnancy, the lack of TFPI decreased formation of placental blood pools present in the TFlow mice, and the low TF decreased the placental vascular defects present in the TFPI null mice. Similarly in adult mice, the lack of TFPI decreased the lung hemorrhage in the TFlow mice, suggesting that TFPI and TF counterbalance each other to maintain hemostasis in the lung. However, in pregnant TFlow/TFPI null mice, the lack of TFPI did not prevent death from uterine hemorrhage, suggesting that larger amounts of TF are required to maintain hemostasis during childbirth regardless of the presence or absence of TFPI [42]. TFPI null mice were also rescued to birth by breeding into mice that lack FVII (F7tm1Edr) but succumbed within days after birth with intracranial and intra-abdominal bleeding. Interestingly, the embryonic lethality of TFPI null mice is also rescued by a 50% decrease in FVII. However, these mice die at or immediately after birth. While their heart, lungs, kidneys, and liver develop normally, they have evidence of both brain hemorrhage and fibrin deposition throughout the brain [43].

Interaction of heterozygous TFPI deficiency with other prothrombotic risk factors

Mice with heterozygous TFPI (Tfpi+/−) deficiency are born at the expected frequency [8]. They do not exhibit signs of a procoagulant state under standard husbandry conditions but do have augmented thrombus volume following electrolytic vascular injury [44]. The underlying propensity of Tfpi−/− mice to thrombosis is exposed by breeding into mice with other procoagulant mutations. Mice homozygous for FV Leiden (F5tm2Dgi) also display only a mild prothrombotic phenotype. However, in the presence of heterozygous TFPI deficiency, the pups die of severe perinatal thrombosis in the liver, lungs, and kidneys [45]. Similarly, mice with decreased thrombomodulin function (Thbdtm1Wlr) have only a mild prothrombotic state when on a C57Bl6 background. However, in the presence of heterozygous TFPI deficiency, the mice exhibit partial embryonic lethality, while surviving adult mice have a generalized prothrombotic state with tissue selective fibrin deposition in the liver and brain [44].

TFPI and thrombosis

Total TFPI deficiency has not been observed clinically, suggesting that it is required for human embryonic development, as it is in mice. While low plasma TFPI is associated with venous and arterial thrombotic disease, in most studies an increased risk is only observed in individuals with plasma TFPIα levels at or below the 10th percentile of the normal reference range [9]. Of particular interest is that oral estrogen therapies decrease the total plasma TFPI concentration and activity by about 25% [46]. The severe perinatal thrombosis observed in mice with heterozygous TFPI deficiency and FV Leiden suggests that these two procoagulant risk factors synergize to produce severe thrombotic disease. Women with FV Leiden increase their risk for thrombotic disease five-fold when they use oral contraceptives [47]. It is tempting to speculate that this occurs when the oral contraceptive decreases TFPI levels, which then synergize with FV Leiden to greatly reduce endogenous anticoagulant activity and cause thrombosis.

TFPI and bleeding

As discussed above, TFPIα rapidly inhibits prothrombinase assembled with FXa-activated FVa in vitro [6]. Because only trace amounts of thrombin are needed to rapidly remove the entire B-domain [26], there was some question whether the forms of FVa that retain the acidic region of the B-domain are physiologically important. However, a key role for these forms of FVa was identified in studies examining the anticoagulant properties of a tick saliva anticoagulant protein (TIX-5), which found that it exerts anticoagulant activity by binding the B-domain of FV and specifically blocking FXa-mediated activation of FV. Further, this study found that activation of FV by FXa, occurring before thrombin is produced, is an essential step in the initiation of coagulation [48]. This is a newly recognized control point in the procoagulant response that may be blocked endogenously by TFPIα.

Further study of the inhibition of prothrombinase by TFPIα may advance knowledge of the biochemical mechanisms underlying a wide range of bleeding and thrombotic disorders. For example, patients with the east Texas bleeding disorder produce an altered form of FV(a) that lacks the basic region of the B-domain and therefore is similar to FXa-activated FVa. Plasma TFPIα is elevated approximately 10-fold in these patients. It is likely that TFPIα tightly binds to the acidic region of the altered FV east Texas B-domain. This stabilizes TFPIα by protecting it from clearance or degradation and produces the bleeding symptoms [10]. The bleeding symptoms in these patients may be caused by inhibition of prothrombinase by the circulating TFPIα/FV east Texas complex rather than TF-FVIIa inhibition, providing an important example of how TFPIα exerts physiological anticoagulant activity via specific, high-affinity interactions with FVa and FXa in humans [6].

There are other examples of how inhibition of prothrombinase by TFPIα may have direct clinical relevance. TFPIα-mediated prothrombinase inhibition is blocked by physiologically important polyanions that prevent the exosite interaction between the basic region of TFPIα and the acidic region of the FV B-domain [6]. These data provide biochemical explanations for why heparin is procoagulant in the absence of antithrombin [25,49] and why polyphosphate drives thrombin generation in platelet-rich plasma [PRP] stimulated with histones [50].

Treatment for hemophilia

Hemophilia A and B are caused by deficiency of FVIII or FIX, respectively. These bleeding disorders are treated with plasma-derived or recombinant proteins for the missing clotting factor. Patients may develop inhibitory antibodies that inactivate the replaced factor making bleeding events difficult to control. These patients can be treated with bypass agents such as recombinant FVIIa or plasma-derived activated prothrombin complex concentrate, but additional options to treat these patients are needed [51]. TFPI inhibits thrombin generation through the extrinsic coagulation pathway by inhibiting the TF/FVIIa complex [21] and the common coagulation pathway by inhibiting early forms of prothrombinase [6]. These biochemical mechanisms for inhibition of clotting by TFPI suggest that severe bleeding in patients with hemophilia not only requires the absence of FVIII or FIX, but also the presence of TFPI, which forces amplification of a clotting response to proceed via activation of FIX by TF–FVIIa.

As inhibition of TFPI can restore clotting through the extrinsic and common pathways bypassing the need for clotting to proceed through FVIII and FIX, inhibitors of TFPI are being developed as pharmaceutical agents to treat patients with hemophilia. These agents have several potential advantages over current therapies including efficacy in both hemophilia A and B, efficacy in patients with inhibitors, and the potential for subcutaneous prophylactic therapy that could be administered on a weekly or even monthly basis.

The strategy of inhibiting TFPI to treat hemophilia bleeding was initially validated in vivo using a rabbit model of hemophilia in which infusion of anti-TFPI antibodies shortened the cuticle bleeding time from 26 to 11 min [52]. Antibodies that block TFPI activity have continued to be developed. A high-affinity antibody directed against the active site of K2 [Concizumab] greatly improves thrombin generation in TF-initiated assays of FVIII-deficient plasma and shortened cuticle bleeding time in a rabbit model of hemophilia even at 7 days following a single subcutaneous dose [53]. Concizumab has entered Phase I clinical trials [54]. Peptides with high affinity for TFPI are also being developed. Interestingly, one of the peptides with high affinity for only K1 binding blocked not only TF–FVIIa activation of FX, but also direct inhibition of FXa by K2, demonstrating that K1 enhances inhibition of FXa by K2 [55]. Fucoidans are sulfated polysaccharides found in brown algae and echinoderms. Fucoidans can be procoagulant or anticoagulant with the bioactivity influenced by their structure, molecular weight, degree of sulfation, and monosaccharide composition [56]. Non-anticoagulant fucoidans may produce procoagulant activity by preventing TFPIα from inhibiting early forms of prothrombinase by blocking the charge-dependent interaction between the C-terminus of TFPIα and the acidic region of FVa B-domain [6]. Their procoagulant activity was demonstrated in vivo through restoration of hemostasis in a dog hemophilia A model [57]. An aptamer [BAX499] with high-affinity binding to TFPI through interactions with K1, K3, and the C-terminus showed efficacy in animal models of hemophilia [58]. However, it failed in clinical trials because it paradoxically produced an increase in bleeding episodes among patients with hemophilia. Characterization of plasma from patients enrolled in this trial revealed that the aptamer induced a large increase in plasma TFPIα, possibly by stabilizing it and slowing plasma clearance. Although the aptamer dampened TFPI activity, it did not totally inactivate it, and the large increase in plasma TFPIα produced bleeding [54].

Improved agents for blocking TFPI to treat hemophilia may be produced as knowledge of human TFPI biology increases. All current strategies under development for blocking TFPI activity are designed to inhibit both TFPIα and TFPIβ activities. However, deficiency of only hematopoietic cell TFPI, which is primarily platelet TFPIα, restores hemostasis in a murine hemophilia model [59]. Therefore, development of new agents that block only TFPIα may be therapeutically beneficial when compared to a general TFPI blocking agent because it would restore hemostasis at the site of a bleed without producing a generalized procoagulant state that could result from inhibition of endothelial TFPIβ.

Footnotes

Disclosure of Conflict of Interests

A. E. Mast reports grants from Novo Nordisk, during the conduct of the study and personal fees from Siemens, Biogen Idec, PREVENT, and Sysmex, outside the submitted work. In addition, A. E. Mast has a patent pending on the ‘Method of screening for novel compounds’ that inhibit early forms of prothrombinase or that block the inhibition of early forms of prothrombinase by tissue factor pathway inhibitor.

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