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Tissue Factor Pathway Inhibitor: Then and Now Paul E. R. Ellery, BSc (Hons), PhD1
Murray J. Adams, BSc (Hons), PhD, MAIMS, FFSc (RCPA)2
1 Blood Research Institute, Blood Center of Wisconsin, Milwaukee,
Wisconsin 2 School of Health Sciences, University of Tasmania, Launceston, Tasmania, Australia
Address for correspondence Murray J. Adams, BSc (Hons), PhD, MAIMS, FFSc (RCPA), Department of Haematology, School of Health Sciences, University of Tasmania, Locked Bag 1322, Launceston, TAS 7250, Australia (e-mail: [email protected]).
Abstract
Keywords
► ► ► ► ►
tissue factor thrombosis protein S hemophilia factor V
Tissue factor pathway inhibitor (TFPI) is the major physiological regulator of tissue factor (TF)-induced blood coagulation. TFPI inhibits the TF-activated factor VII (FVIIa) complex in an activated factor X (FXa)-dependent manner, helping to control thrombin generation and ultimately fibrin formation. The importance of TFPI is demonstrated in models of hemophilia where lower levels of FVIII or FIX are insufficient to overcome its inhibitory effect, resulting in a bleeding phenotype. There are two major isoforms in vivo; TFPIα contains three Kunitz-type inhibitory domains (designated K1, K2, and K3), is secreted by endothelial cells and requires protein S to enhance its anticoagulant activity. In contrast, TFPIβ contains only the K1 and K2 domains, but it is attached to the endothelial surface via a glycosylphosphatidylinositol anchor. This review will initially provide a brief history of the major discoveries related to TFPI, and then discuss new insights into the physiology of TFPI, including updates on its association with protein S and FV, as well as the current understanding of its association with disease.
Tissue factor pathway inhibitor (TFPI) is the major physiological regulator of tissue factor (TF)–induced blood coagulation and is thought to play an important role in modulating the severity of bleeding and clotting disorders.1 Although evidence of an in vivo inhibitor of the trigger of coagulation had been reported nearly 100 years ago, it was not until the 1980s that its “rediscovery,” and thus renewed interest, lead to the purification, cloning, and elucidation of its mechanism of action. The turn of the 21st century has seen continued advances in the understanding of TFPI physiology. For example, it is now known that rather than one, there are two in vivo
isoforms of TFPI (TFPIα and TFPIβ). Furthermore, protein S is now known to be an important cofactor for TFPIα to exert its anticoagulant function. Moreover, recent research is investigating whether inhibitors of TFPI may be exploited for therapeutic advantage, particularly in the treatment of hemophilia. In keeping with the theme of this issue of Seminars in Thrombosis & Hemostasis, this review will provide a brief chronological timeline of the major discoveries related to TFPI and highlight the more important recent advances in knowledge about this important physiological inhibitor.
Brief History
This article is dedicated to Robert (Bob) Oostryck. In 1992, while a Senior Lecturer in Hematology at Curtin University at Curtin University of Technology, Bob asked M.J.A., who was then an undergraduate student interested in research, “Why do hemophiliacs bleed?” The explanation of a lack of factor VIII or factor IX was not only superficially correct, but it also did not take into account the important physiological effects of tissue factor pathway inhibitor (TFPI). Bob was our first mentor, a great teacher, and now friend, who ignited a passion for research and an interest in TFPI that we both still share. Although now retired, Bob retains a keen interest in all things related to coagulation, TFPI, and hemostasis.
published online November 6, 2014
Issue Theme A Short History of Thrombosis and Hemostasis: Part II (40th Year Celebratory Issue); Guest Editor: Emmanuel J. Favaloro, PhD, FFSc (RCPA).
The first evidence for the presence of an endogenous inhibitor of TF was in the early 1920s, with serum reported to inhibit the procoagulant activity of various tissue extracts.2,3 Studies in the 1940s4,5 later confirmed these findings, reporting that the preincubation of crude tissue thromboplastin with serum prevented disseminated intravascular coagulation in animals. Significantly, the effect was shown to be dependent on the presence of Ca2þ, with the inhibitor appearing to bind to thromboplastin.3 Approximately 10 years later, it was
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DOI http://dx.doi.org/ 10.1055/s-0034-1395153. ISSN 0094-6176.
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Semin Thromb Hemost 2014;40:881–886.
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reported that the inhibitor targeted the TF-activated factor VII (FVIIa)–Ca2þ complex, rather than one of these components alone, and suggested that this inhibition was reversible.6 Minimal work was published during the next quarter of a century to characterize the inhibition of TF-FVIIa in human plasma. This was probably due to a focus on the classification of the “intrinsic” and “extrinsic” pathways of coagulation leading to the development of the “waterfall,” and “cascade” theories of blood coagulation.7,8 Delayed and incomplete coagulation following the addition of low concentrations of TF to plasma from patients with hemophilia was reported,9,10 but the connection between this and the inhibition of TF-induced coagulation was not fully understood at this time. In the early 1970s, an inhibitor of activated factor X (FXa) was discovered in the low density lipoprotein plasma fraction,11 and later confirmed to be a fast-acting FXa inhibitor present in all lipoprotein fractions of plasma (low density lipoprotein [LDL] > high density lipoprotein [HDL] > very low density lipoprotein).12,13 A second line of studies that occurred concurrently also suggested the presence of an endogenous inhibitor of TF-FVIIa in both normal and hemophiliac plasma that was dependent on FX or FXa.14–16 Following these findings, there was a significant increase in interest in this inhibitory protein, which was subsequently isolated and purified,17–19 allowing many of its structural and functional features to be extensively investigated. During this period the inhibitor had many names, including lipoprotein-associated coagulation inhibitor,20 TF inhibitor,21 and extrinsic pathway inhibitor,19 before it was formally named TFPI in 1991.22 The discovery of TFPI was subsequently instrumental in redefining the understanding of how clotting occurred in vivo with the formulation of a cell-based model, or “modern hypothesis,” of blood coagulation developed in the mid-1990s (►Fig. 1).
Fig. 1 Cell-based model of blood coagulation. Cells such as endothelial cells and monocytes express TF following vessel damage and exposure to inflammatory mediators, to trigger the TF pathway of blood coagulation. TF combines with FVII to form the TF-FVIIa complex, which then activates FIX and FX to FIXa and FXa, respectively. Small amounts of thrombin are generated, leading to amplification of coagulation through FXI (and FV and FVIII) activation. A thrombin burst leads to fibrin formation (propagation), which helps to stabilize the platelet plug. TFPI exerts its regulatory effect by direct inhibition of FXa, the FXa-dependent inhibition of the TF-FVIIa complex, and inhibition of “early” forms of prothrombinase. This inhibitory effect is more difficult to overcome in hemophilia A (FVIII deficiency) and B (FIX deficiency), resulting in decreased thrombin generation and a bleeding phenotype. TF, tissue factor; TFPI, tissue factor pathway inhibitor.
Isoforms
determined, but may include temporal and/or spatial control of TFPIβ expression. The endothelium of the microvasculature, but not large vessels, is the primary site of TFPI synthesis.31,32 Endothelial cells produce both TFPIα and TFPIβ mRNA,28,33 with TFPI protein located intracellularly and within caveolae at the endothelial surface.34–36 In vitro, the majority of surface expressed TFPI is removed following phospholipase C treatment,28,36–38 a characteristic of GPI-anchored proteins, with
Two TFPI isoforms, TFPIα and TFPIβ, arise from alternative splicing toward the 3′ end of the TFPI pre-mRNA. TFPIα consists of 276 amino acids,23 is 43 kDa in mass24,25 and comprises a negatively charged N-terminus, three Kunitztype inhibitory domains joined by linker regions, and a highly positively charged C-terminus.23 TFPIβ contains 193 amino acids; however, it is heavily glycosylated and therefore has the same molecular weight as TFPIα.26 It shares homology with TFPIα from the N-terminus through the second Kunitz domain.27 However, instead of a third Kunitz domain and positively charged C-terminus, it contains a glycosylphosphatidylinositol (GPI) anchor signal sequence that allows it to be GPI-anchored at the cell surface (►Fig. 2).28 Exon 2, comprising part of the 5′ untranslated region of TFPI pre-mRNA, is also alternatively spliced.29 This exon was recently demonstrated as a general repressor of translation whose effects are overcome by element(s) within the TFPIα, but not TFPIβ, 3′ UTR.30 As such, the splicing of exon 2 represents a “molecular switch” that controls TFPIβ protein production at the translational level. The physiological relevance of this splicing event remains to be
Fig. 2 Isoforms of TFPI. TFPI exists as two isoforms: TFPIα and TFPIβ. TFPIα contains a negatively charged N-terminus, followed by three Kunitz domains (K1, K2, and K3) and a positively charged C-terminus. K1 and K2 bind to FVIIa and FXa, respectively, to inhibit TF-mediated coagulation. K3 binds to protein S, which is a cofactor for the direct inhibition of FXa, and in the absence of FXa, the direct inhibition of TFFVIIa. TFPIβ lacks K3 and has an altered C-terminus that allows binding to the cell surface via a GPI anchor. Protein S is therefore a cofactor for TFPIα, but not TFPIβ. GPI, glycosylphosphatidylinositol; TF, tissue factor; TFPI, tissue factor pathway inhibitor.
Physiology
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Tissue Factor Pathway Inhibitor
Distribution Plasma contains 10 to 50% of the total in vivo pool of TFPI,44 which is heterogeneous in mass.45,46 TFPIα accounts for 10 to 30% of plasma TFPI, whereas the remainder is variably truncated at its C-terminal end and bound to plasma lipoproteins.45,46 The role of lipoprotein bound TFPI is unclear. Patients with hypo- or abetalipoproteinemia have decreased LDL-bound plasma TFPI but normal plasma TFPI activity and are not prothrombotic.47 Furthermore, treatment of hyperbetalipoproteinemic patients with lipid lowering drugs decreases LDL-bound plasma TFPI without affecting plasma TFPIα or the anticoagulant potential of the plasma.48 A recent study, however, demonstrated that LDLbound TFPI is a more effective inhibitor of FXa than TFPIα, inhibiting the propagation phase of thrombin generation in vitro.49 Further studies are required to determine the functional role of lipoprotein-associated TFPI. Platelets account for 5 to 10% of the total in vivo pool of TFPI50,51; however, unlike endothelial cells, platelets produce only TFPIα mRNA and protein.50–52 Quiescent platelets do not secrete or express cell surface TFPI,51,53 but secrete TFPIα in response to activation with thrombin and/or Ca2þ ionophore.50,51 Furthermore, coated platelets express cell surface TFPIα that is not GPI-anchored.51 In an electrolytic injury thrombosis model, mice lacking hematopoietic (presumably platelet) TFPIα have increased platelet accumulation at the site of vessel injury,54 demonstrating the importance of platelet TFPIα in limiting in vivo thrombus formation.
Association with Disease
correlates with multiple thrombin generation parameters in FVIII-inhibited plasma.58 Furthermore, FVIII-deficient mice lacking platelet TFPIα have decreased blood loss in a tail vein bleeding model.59 Finally, in a small number of patients, it was demonstrated that those with severe hemophilia B, who characteristically have a milder bleeding phenotype compared with those with severe hemophilia A, also have lower plasma TFPIα.60 Larger studies measuring plasma and platelet TFPI are warranted to better define the contribution of each to the bleeding phenotype of people with hemophilia.
A Potential Treatment for Hemophilia? It has long been hypothesized that TFPI is a viable target for the treatment of hemophilia. Inhibition of TFPI would allow coagulation to proceed through TF-FVIIa and FXa, circumventing the need for coagulation amplification through the factor(s) deficient in patients with hemophilia (►Fig. 1). Indeed, in a rabbit model of hemophilia, administration of anti-TFPI antibody significantly decreased bleeding after injury.61 Several TFPI inhibitory compounds have been, or are now under development for the treatment of hemophilia, including the monoclonal antibody mAb 2021,62 AV513,63 small peptides,64 and the aptamer ARC19499.65 mAb 2021, which binds K2 and prevents TFPI inhibition of FXa, decreases bleeding in a rabbit model of hemophilia.62 Importantly, a single bolus remains efficacious for 1 week when administered intravenously and it can be administered subcutaneously.62 This represents a marked improvement compared with factor replacement therapy, which is currently used for the treatment of hemophilia. ARC19499 increased thrombin generation and improved clot formation in hemophilic plasma in vitro and decreased bleeding times in a nonhuman primate model of hemophilia A.65 However, human clinical trials were terminated because the aptamer was found to promote bleeding via release of TFPIα from the endothelium, and increase the circulating half-life.66,67 Developing TFPI inhibitory compounds that avoid these issues is critical for their transition to clinical use.
Thrombophilia There is no clear evidence that associates low circulating plasma levels of TFPI with increased thrombotic risk, as is the case with other natural inhibitors (i.e., protein C, protein S, and antithrombin). Plasma levels of TFPI have been reported in a wide range of diseases with thrombotic phenotype, displaying a wide degree of variability, and generally similar to, or higher than, levels in healthy individuals.55 Probable reasons include the heterogeneous in vivo distribution of TFPI, variable anticoagulant activities of the different forms of the molecule, and poor correlation between different assay methods.56,57
Hemophilia As an inhibitor of TF-FVIIa and FXa, TFPI plays an important role in hemophilia because it forces amplification of coagulation through FVIII and FIX. Therefore, it is reasonable to hypothesize that differences in TFPI concentration would affect the bleeding phenotype of these patients. Indeed, it has recently been demonstrated that plasma TFPIα strongly
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Recent Advances Tissue Factor Pathway Inhibitor and Protein S Almost a decade ago, it was demonstrated that protein S acts as a cofactor for the direct inhibition of FXa by TFPI.68 It was subsequently established that protein S accelerates formation of the initial K2-FXa encounter complex, but not isomerization of the final tight complex.69 In the absence of FXa, protein S is also a cofactor in the direct inhibition of TF-FVIIa by TFPI.70 Binding of TFPI to protein S requires K3, with Arg199 and Glu226 of K3 identified as critical for this inhibition.71,72 As such, protein S is a cofactor for TFPIα, but not TFPIβ,73 and is hypothesized to localize TFPIα to membrane surfaces73 to allow optimal interaction with FXa.74 The laminin G-type 1 subunit within the sex-hormone-binding globulin–like domain of protein S was recently identified as critical for binding to TFPIα.74 TFPI and free protein S circulate in a complex in vivo.75 Plasma TFPIα is therefore correlated with plasma levels of free Seminars in Thrombosis & Hemostasis
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recent evidence suggesting that this is TFPIβ.39 It remains unknown if TFPIα is present at the endothelial surface, and if so, binding to a putative GPI-anchored coreceptor28,33,37,40 or glycosaminoglycans33,36,37,41 is unlikely. Instead, evidence suggests that TFPIα is constitutively secreted from endothelial cells and is present in an intracellular granule, from which it is released by heparin.28,42,43
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and total protein S.76 Patients with hereditary or acquired protein S deficiency tend to have reduced plasma TFPIα that is thought to contribute to their procoagulant state.75
Tissue Factor Pathway Inhibitor and Factor V FV circulates in vivo in an inactive pro-cofactor state, in which it is maintained via association of two highly evolutionary conserved regions of amino acids within the FV B-domain, one predominantly basic and the other acidic, that together comprise the procofactor regulatory region (PRR).77 Proteolytic cleavage at multiple sites within the B-domain by thrombin removes the entire PRR, transforming FV to an active cofactor.78–80 Conversely, FXa activates FV by removing only the basic region of the PRR, whereas the acidic region is retained by the active FV protein.81–83 Forms of FV resembling FXa-activated FV (FVa) are also found stored within platelets.84,85 The evolutionary conserved, highly basic region within the TFPIα C-terminus and the basic region of the FV PRR share significant homology,86 suggesting an interaction between TFPIα and FV. Indeed, recent studies demonstrate that the TFPIα C-terminus can bind to the acidic region of the FV PRR with similar affinity to that of the FV PRR basic region.86,87 In accordance with this, it has been demonstrated that FVimmunodepleted plasma and that from FV-deficient patients have markedly reduced levels of TFPIα, suggesting that TFPIα and FV circulate as a complex in vivo.75,88 It is hypothesized that the reduced plasma TFPIα accounts for, at least in part, the relatively mild bleeding phenotype observed in FV-deficient patients.88 Association of TFPIα with FV has recently been described as the cause of the moderately severe bleeding diathesis observed in patients with FV East Texas.89 These patients have a mutation in exon 13 of the FV gene, which encodes the B-domain. This mutation produces a novel, preferentially used splice donor site that results in a mature protein containing a greatly shortened B-domain missing the basic region, but retaining the acidic region. This form of FV, termed FV short, quickly associates with plasma TFPIα in vivo and is thought to enhance its circulating half-life. As such, these patients have a plasma TFPIα concentration that is approximately 10 fold greater than normal, resulting in a bleeding phenotype probably due to the direct inhibition of TF-FVIIa that can occur with increased TFPIα concentrations and/or inhibition of early forms of prothrombinase (see the next section).
inhibit these “early” forms of the prothrombinase complex.86 As expected, inhibition is dependent on K2 and the TFPIα Cterminus.86 Furthermore, this inhibition can be prevented by negatively charged molecules such as heparin or polyphosphate, demonstrating the importance of the interaction between the TFPIα C-terminus and acidic region of the FVa PRR in this inhibition.86
Conclusion and Future Directions Although there were reports in the early part of the 20th century that a physiological inhibitor of TF-induced coagulation was present in blood, it was not until the 1980s that this entity, TFPI, was identified. Following its discovery, the structure, function, and other physiological features of TFPI were further investigated and characterized through the 1990s and into the 21st century. The most recent literature continues to explore the characteristics of the TFPIα and TFPIβ isoforms of the molecule, as well as improving understanding of its interactions with protein S, and with FV in the prothrombinase complex. The next chapter of the evolving history of TFPI will include the identification of further individuals affected by FV East Texas and other bleeding diatheses caused by markedly elevated TFPI, and the development of TFPI inhibitors that may be used to treat bleeding disorders such as hemophilia.
Conflict of Interest The authors have no conflict of interest to declare.
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Tissue Factor Pathway Inhibitor and the Inhibition of Prothrombinase Initial studies, performed using prothrombinase complexes formed with thrombin-activated FV, demonstrated that TFPI is a poor inhibitor of FXa within the prothrombinase complex.90 However, prothrombinase formed during the early stages of coagulation contains FXa-activated FVa that retains the acidic region of the PRR.91 A portion of FV within platelets that is able to assemble into prothrombinase has a similar structure.84,85 This form of prothrombinase is critical for the initiation of coagulation.91 A recent study demonstrated that TFPIα, at physiologically relevant concentrations, can Seminars in Thrombosis & Hemostasis
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lipoprotein associated coagulation inhibitor. Blood 1988;72(6): 2020–2025 Maroney SA, Haberichter SL, Friese P, et al. Active tissue factor pathway inhibitor is expressed on the surface of coated platelets. Blood 2007;109(5):1931–1937 Jayachandran M, Sanzo A, Owen WG, Miller VM. Estrogenic regulation of tissue factor and tissue factor pathway inhibitor in platelets. Am J Physiol Heart Circ Physiol 2005;289(5): H1908–H1916 Novotny W, Day K, Wun T-C, Nieces G. Platelet LACI is present in novel granules. Blood 1990;76(Suppl 1):469 Maroney SA, Cooley BC, Ferrel JP, Bonesho CE, Mast AE. Murine hematopoietic cell tissue factor pathway inhibitor limits thrombus growth. Arterioscler Thromb Vasc Biol 2011;31(4):821–826 Winckers K, ten Cate H, Hackeng TM. The role of tissue factor pathway inhibitor in atherosclerosis and arterial thrombosis. Blood Rev 2013;27(3):119–132 Adams M. Tissue factor pathway inhibitor: new insights into an old inhibitor. Semin Thromb Hemost 2012;38(2):129–134 Adams MJ, Oostryck R. A comparative study of functional assays for tissue factor pathway inhibitor using normal plasma and clinical samples. Blood Coagul Fibrinolysis 2000;11(4):327–333 Knappe S, Gorczyca ME, Jilma B, et al. Plasmatic tissue factor pathway inhibitor is a major determinant of clotting in factor VIII inhibited plasma or blood. Thromb Haemost 2013;109(3): 450–457 Maroney SA, Cooley BC, Ferrel JP, et al. Absence of hematopoietic tissue factor pathway inhibitor mitigates bleeding in mice with hemophilia. Proc Natl Acad Sci U S A 2012;109(10):3927–3931 Tardy-Poncet B, Piot M, Chapelle C, Berger C, Tardy B. Difference in TFPI levels between haemophilia A and B patients. Haemophilia 2011;17(2):312–313 Erhardtsen E, Ezban M, Madsen MT, et al. Blocking of tissue factor pathway inhibitor (TFPI) shortens the bleeding time in rabbits with antibody induced haemophilia A. Blood Coagul Fibrinolysis 1995;6(5):388–394 Hilden I, Lauritzen B, Sørensen BB, et al. Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model. Blood 2012;119(24): 5871–5878 Prasad S, Lillicrap D, Labelle A, et al. Efficacy and safety of a newclass hemostatic drug candidate, AV513, in dogs with hemophilia A. Blood 2008;111(2):672–679 Dockal M, Hartmann R, Fries M, et al. Small peptides blocking inhibition of factor Xa and tissue factor-factor VIIa by tissue factor pathway inhibitor (TFPI). J Biol Chem 2014;289(3): 1732–1741 Waters EK, Genga RM, Schwartz MC, et al. Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor. Blood 2011;117(20):5514–5522 Dockal M, Hartmann R, Knappe S, et al. Effect of increased tissue factor pathway inhibitor (TFPI) levels on factor Xa inhibition and global hemostasis in the presence of TFPI-antagonistic aptamer BAX 499. Blood 2012:120;2207 Dockal M, Pachlinger R, Hartmann R, et al. Biological explanation of clinically observed elevation of TFPI plasma levels after treatment with TFPI-antagonistic aptamer BAX 499 [Abstract]. Blood 2012;120:1104 Hackeng TM, Seré KM, Tans G, Rosing J. Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor. Proc Natl Acad Sci U S A 2006;103(9):3106–3111 Peraramelli S, Suylen DP, Rosing J, Hackeng TM. The Kunitz 1 and Kunitz 3 domains of tissue factor pathway inhibitor are required for efficient inhibition of factor Xa. Thromb Haemost 2012;108(2): 266–276 Peraramelli S, Thomassen S, Heinzmann A, et al. Direct inhibition of factor VIIa by TFPI and TFPI constructs. J Thromb Haemost 2013; 11(4):704–714
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alpha is required for protein S-dependent enhancement of factor Xa inhibition. Blood 2010;116(8):1344–1351 Ahnström J, Andersson HM, Hockey V, et al. Identification of functionally important residues in TFPI Kunitz domain 3 required for the enhancement of its activity by protein S. Blood 2012; 120(25):5059–5062 Wood JP, Ellery PE, Maroney SA, Mast AE. Protein S is a cofactor for platelet and endothelial tissue factor pathway inhibitor-α but not for cell surface-associated tissue factor pathway inhibitor. Arterioscler Thromb Vasc Biol 2014;34(1):169–176 Reglińska-Matveyev N, Andersson HM, Rezende SM, et al. TFPI cofactor function of protein S: essential role of the protein S SHBGlike domain. Blood 2014;123(25):3979–3987 Castoldi E, Simioni P, Tormene D, Rosing J, Hackeng TM. Hereditary and acquired protein S deficiencies are associated with low TFPI levels in plasma. J Thromb Haemost 2010;8(2): 294–300 Dahm AE, Sandset PM, Rosendaal FR. The association between protein S levels and anticoagulant activity of tissue factor pathway inhibitor type 1. J Thromb Haemost 2008;6(2):393–395 Bos MH, Camire RM. A bipartite autoinhibitory region within the B-domain suppresses function in factor V. J Biol Chem 2012; 287(31):26342–26351 Kane WH, Davie EW. Cloning of a cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplasmin. Proc Natl Acad Sci U S A 1986;83(18): 6800–6804 Kane WH, Ichinose A, Hagen FS, Davie EW. Cloning of cDNAs coding for the heavy chain region and connecting region of human factor V, a blood coagulation factor with four types of internal repeats. Biochemistry 1987;26(20):6508–6514 Jenny RJ, Pittman DD, Toole JJ, et al. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci U S A 1987;84(14):4846–4850 Smith CM, Hanahan DJ. The activation of factor V by factor Xa or alpha-chymotrypsin and comparison with thrombin and RVV-V action. An improved factor V isolation procedure. Biochemistry 1976;15(9):1830–1838 Foster WB, Nesheim ME, Mann KG. The factor Xa-catalyzed activation of factor V. J Biol Chem 1983;258(22):13970–13977 Odegaard B, Mann K. Proteolysis of factor Va by factor Xa and activated protein C. J Biol Chem 1987;262(23):11233–11238 Viskup RW, Tracy PB, Mann KG. The isolation of human platelet factor V. Blood 1987;69(4):1188–1195 Monković DD, Tracy PB. Functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin. J Biol Chem 1990;265(28):17132–17140 Wood JP, Bunce MW, Maroney SA, Tracy PB, Camire RM, Mast AE. Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation. Proc Natl Acad Sci U S A 2013;110(44):17838–17843 Ndonwi M, Girard TJ, Broze GJ Jr. The C-terminus of tissue factor pathway inhibitor α is required for its interaction with factors V and Va. J Thromb Haemost 2012;10(9):1944–1946 Duckers C, Simioni P, Spiezia L, et al. Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency. Blood 2008;112(9):3615–3623 Vincent LM, Tran S, Livaja R, Bensend TA, Milewicz DM, Dahlbäck B. Coagulation factor V(A2440G) causes east Texas bleeding disorder via TFPIα. J Clin Invest 2013;123(9):3777–3787 Mast AE, Broze GJ Jr. Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase. Blood 1996; 87(5):1845–1850 Schuijt TJ, Bakhtiari K, Daffre S, et al. Factor Xa activation of factor V is of paramount importance in initiating the coagulation system: lessons from a tick salivary protein. Circulation 2013;128(3): 254–266
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Tissue Factor Pathway Inhibitor: Then and Now Paul E. R. Ellery, BSc (Hons), PhD1
Murray J. Adams, BSc (Hons), PhD, MAIMS, FFSc (RCPA)2
1 Blood Research Institute, Blood Center of Wisconsin, Milwaukee,
Wisconsin 2 School of Health Sciences, University of Tasmania, Launceston, Tasmania, Australia
Address for correspondence Murray J. Adams, BSc (Hons), PhD, MAIMS, FFSc (RCPA), Department of Haematology, School of Health Sciences, University of Tasmania, Locked Bag 1322, Launceston, TAS 7250, Australia (e-mail: [email protected]).
Abstract
Keywords
► ► ► ► ►
tissue factor thrombosis protein S hemophilia factor V
Tissue factor pathway inhibitor (TFPI) is the major physiological regulator of tissue factor (TF)-induced blood coagulation. TFPI inhibits the TF-activated factor VII (FVIIa) complex in an activated factor X (FXa)-dependent manner, helping to control thrombin generation and ultimately fibrin formation. The importance of TFPI is demonstrated in models of hemophilia where lower levels of FVIII or FIX are insufficient to overcome its inhibitory effect, resulting in a bleeding phenotype. There are two major isoforms in vivo; TFPIα contains three Kunitz-type inhibitory domains (designated K1, K2, and K3), is secreted by endothelial cells and requires protein S to enhance its anticoagulant activity. In contrast, TFPIβ contains only the K1 and K2 domains, but it is attached to the endothelial surface via a glycosylphosphatidylinositol anchor. This review will initially provide a brief history of the major discoveries related to TFPI, and then discuss new insights into the physiology of TFPI, including updates on its association with protein S and FV, as well as the current understanding of its association with disease.
Tissue factor pathway inhibitor (TFPI) is the major physiological regulator of tissue factor (TF)–induced blood coagulation and is thought to play an important role in modulating the severity of bleeding and clotting disorders.1 Although evidence of an in vivo inhibitor of the trigger of coagulation had been reported nearly 100 years ago, it was not until the 1980s that its “rediscovery,” and thus renewed interest, lead to the purification, cloning, and elucidation of its mechanism of action. The turn of the 21st century has seen continued advances in the understanding of TFPI physiology. For example, it is now known that rather than one, there are two in vivo
isoforms of TFPI (TFPIα and TFPIβ). Furthermore, protein S is now known to be an important cofactor for TFPIα to exert its anticoagulant function. Moreover, recent research is investigating whether inhibitors of TFPI may be exploited for therapeutic advantage, particularly in the treatment of hemophilia. In keeping with the theme of this issue of Seminars in Thrombosis & Hemostasis, this review will provide a brief chronological timeline of the major discoveries related to TFPI and highlight the more important recent advances in knowledge about this important physiological inhibitor.
Brief History
This article is dedicated to Robert (Bob) Oostryck. In 1992, while a Senior Lecturer in Hematology at Curtin University at Curtin University of Technology, Bob asked M.J.A., who was then an undergraduate student interested in research, “Why do hemophiliacs bleed?” The explanation of a lack of factor VIII or factor IX was not only superficially correct, but it also did not take into account the important physiological effects of tissue factor pathway inhibitor (TFPI). Bob was our first mentor, a great teacher, and now friend, who ignited a passion for research and an interest in TFPI that we both still share. Although now retired, Bob retains a keen interest in all things related to coagulation, TFPI, and hemostasis.
published online November 6, 2014
Issue Theme A Short History of Thrombosis and Hemostasis: Part II (40th Year Celebratory Issue); Guest Editor: Emmanuel J. Favaloro, PhD, FFSc (RCPA).
The first evidence for the presence of an endogenous inhibitor of TF was in the early 1920s, with serum reported to inhibit the procoagulant activity of various tissue extracts.2,3 Studies in the 1940s4,5 later confirmed these findings, reporting that the preincubation of crude tissue thromboplastin with serum prevented disseminated intravascular coagulation in animals. Significantly, the effect was shown to be dependent on the presence of Ca2þ, with the inhibitor appearing to bind to thromboplastin.3 Approximately 10 years later, it was
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DOI http://dx.doi.org/ 10.1055/s-0034-1395153. ISSN 0094-6176.
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Semin Thromb Hemost 2014;40:881–886.
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reported that the inhibitor targeted the TF-activated factor VII (FVIIa)–Ca2þ complex, rather than one of these components alone, and suggested that this inhibition was reversible.6 Minimal work was published during the next quarter of a century to characterize the inhibition of TF-FVIIa in human plasma. This was probably due to a focus on the classification of the “intrinsic” and “extrinsic” pathways of coagulation leading to the development of the “waterfall,” and “cascade” theories of blood coagulation.7,8 Delayed and incomplete coagulation following the addition of low concentrations of TF to plasma from patients with hemophilia was reported,9,10 but the connection between this and the inhibition of TF-induced coagulation was not fully understood at this time. In the early 1970s, an inhibitor of activated factor X (FXa) was discovered in the low density lipoprotein plasma fraction,11 and later confirmed to be a fast-acting FXa inhibitor present in all lipoprotein fractions of plasma (low density lipoprotein [LDL] > high density lipoprotein [HDL] > very low density lipoprotein).12,13 A second line of studies that occurred concurrently also suggested the presence of an endogenous inhibitor of TF-FVIIa in both normal and hemophiliac plasma that was dependent on FX or FXa.14–16 Following these findings, there was a significant increase in interest in this inhibitory protein, which was subsequently isolated and purified,17–19 allowing many of its structural and functional features to be extensively investigated. During this period the inhibitor had many names, including lipoprotein-associated coagulation inhibitor,20 TF inhibitor,21 and extrinsic pathway inhibitor,19 before it was formally named TFPI in 1991.22 The discovery of TFPI was subsequently instrumental in redefining the understanding of how clotting occurred in vivo with the formulation of a cell-based model, or “modern hypothesis,” of blood coagulation developed in the mid-1990s (►Fig. 1).
Fig. 1 Cell-based model of blood coagulation. Cells such as endothelial cells and monocytes express TF following vessel damage and exposure to inflammatory mediators, to trigger the TF pathway of blood coagulation. TF combines with FVII to form the TF-FVIIa complex, which then activates FIX and FX to FIXa and FXa, respectively. Small amounts of thrombin are generated, leading to amplification of coagulation through FXI (and FV and FVIII) activation. A thrombin burst leads to fibrin formation (propagation), which helps to stabilize the platelet plug. TFPI exerts its regulatory effect by direct inhibition of FXa, the FXa-dependent inhibition of the TF-FVIIa complex, and inhibition of “early” forms of prothrombinase. This inhibitory effect is more difficult to overcome in hemophilia A (FVIII deficiency) and B (FIX deficiency), resulting in decreased thrombin generation and a bleeding phenotype. TF, tissue factor; TFPI, tissue factor pathway inhibitor.
Isoforms
determined, but may include temporal and/or spatial control of TFPIβ expression. The endothelium of the microvasculature, but not large vessels, is the primary site of TFPI synthesis.31,32 Endothelial cells produce both TFPIα and TFPIβ mRNA,28,33 with TFPI protein located intracellularly and within caveolae at the endothelial surface.34–36 In vitro, the majority of surface expressed TFPI is removed following phospholipase C treatment,28,36–38 a characteristic of GPI-anchored proteins, with
Two TFPI isoforms, TFPIα and TFPIβ, arise from alternative splicing toward the 3′ end of the TFPI pre-mRNA. TFPIα consists of 276 amino acids,23 is 43 kDa in mass24,25 and comprises a negatively charged N-terminus, three Kunitztype inhibitory domains joined by linker regions, and a highly positively charged C-terminus.23 TFPIβ contains 193 amino acids; however, it is heavily glycosylated and therefore has the same molecular weight as TFPIα.26 It shares homology with TFPIα from the N-terminus through the second Kunitz domain.27 However, instead of a third Kunitz domain and positively charged C-terminus, it contains a glycosylphosphatidylinositol (GPI) anchor signal sequence that allows it to be GPI-anchored at the cell surface (►Fig. 2).28 Exon 2, comprising part of the 5′ untranslated region of TFPI pre-mRNA, is also alternatively spliced.29 This exon was recently demonstrated as a general repressor of translation whose effects are overcome by element(s) within the TFPIα, but not TFPIβ, 3′ UTR.30 As such, the splicing of exon 2 represents a “molecular switch” that controls TFPIβ protein production at the translational level. The physiological relevance of this splicing event remains to be
Fig. 2 Isoforms of TFPI. TFPI exists as two isoforms: TFPIα and TFPIβ. TFPIα contains a negatively charged N-terminus, followed by three Kunitz domains (K1, K2, and K3) and a positively charged C-terminus. K1 and K2 bind to FVIIa and FXa, respectively, to inhibit TF-mediated coagulation. K3 binds to protein S, which is a cofactor for the direct inhibition of FXa, and in the absence of FXa, the direct inhibition of TFFVIIa. TFPIβ lacks K3 and has an altered C-terminus that allows binding to the cell surface via a GPI anchor. Protein S is therefore a cofactor for TFPIα, but not TFPIβ. GPI, glycosylphosphatidylinositol; TF, tissue factor; TFPI, tissue factor pathway inhibitor.
Physiology
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Tissue Factor Pathway Inhibitor
Distribution Plasma contains 10 to 50% of the total in vivo pool of TFPI,44 which is heterogeneous in mass.45,46 TFPIα accounts for 10 to 30% of plasma TFPI, whereas the remainder is variably truncated at its C-terminal end and bound to plasma lipoproteins.45,46 The role of lipoprotein bound TFPI is unclear. Patients with hypo- or abetalipoproteinemia have decreased LDL-bound plasma TFPI but normal plasma TFPI activity and are not prothrombotic.47 Furthermore, treatment of hyperbetalipoproteinemic patients with lipid lowering drugs decreases LDL-bound plasma TFPI without affecting plasma TFPIα or the anticoagulant potential of the plasma.48 A recent study, however, demonstrated that LDLbound TFPI is a more effective inhibitor of FXa than TFPIα, inhibiting the propagation phase of thrombin generation in vitro.49 Further studies are required to determine the functional role of lipoprotein-associated TFPI. Platelets account for 5 to 10% of the total in vivo pool of TFPI50,51; however, unlike endothelial cells, platelets produce only TFPIα mRNA and protein.50–52 Quiescent platelets do not secrete or express cell surface TFPI,51,53 but secrete TFPIα in response to activation with thrombin and/or Ca2þ ionophore.50,51 Furthermore, coated platelets express cell surface TFPIα that is not GPI-anchored.51 In an electrolytic injury thrombosis model, mice lacking hematopoietic (presumably platelet) TFPIα have increased platelet accumulation at the site of vessel injury,54 demonstrating the importance of platelet TFPIα in limiting in vivo thrombus formation.
Association with Disease
correlates with multiple thrombin generation parameters in FVIII-inhibited plasma.58 Furthermore, FVIII-deficient mice lacking platelet TFPIα have decreased blood loss in a tail vein bleeding model.59 Finally, in a small number of patients, it was demonstrated that those with severe hemophilia B, who characteristically have a milder bleeding phenotype compared with those with severe hemophilia A, also have lower plasma TFPIα.60 Larger studies measuring plasma and platelet TFPI are warranted to better define the contribution of each to the bleeding phenotype of people with hemophilia.
A Potential Treatment for Hemophilia? It has long been hypothesized that TFPI is a viable target for the treatment of hemophilia. Inhibition of TFPI would allow coagulation to proceed through TF-FVIIa and FXa, circumventing the need for coagulation amplification through the factor(s) deficient in patients with hemophilia (►Fig. 1). Indeed, in a rabbit model of hemophilia, administration of anti-TFPI antibody significantly decreased bleeding after injury.61 Several TFPI inhibitory compounds have been, or are now under development for the treatment of hemophilia, including the monoclonal antibody mAb 2021,62 AV513,63 small peptides,64 and the aptamer ARC19499.65 mAb 2021, which binds K2 and prevents TFPI inhibition of FXa, decreases bleeding in a rabbit model of hemophilia.62 Importantly, a single bolus remains efficacious for 1 week when administered intravenously and it can be administered subcutaneously.62 This represents a marked improvement compared with factor replacement therapy, which is currently used for the treatment of hemophilia. ARC19499 increased thrombin generation and improved clot formation in hemophilic plasma in vitro and decreased bleeding times in a nonhuman primate model of hemophilia A.65 However, human clinical trials were terminated because the aptamer was found to promote bleeding via release of TFPIα from the endothelium, and increase the circulating half-life.66,67 Developing TFPI inhibitory compounds that avoid these issues is critical for their transition to clinical use.
Thrombophilia There is no clear evidence that associates low circulating plasma levels of TFPI with increased thrombotic risk, as is the case with other natural inhibitors (i.e., protein C, protein S, and antithrombin). Plasma levels of TFPI have been reported in a wide range of diseases with thrombotic phenotype, displaying a wide degree of variability, and generally similar to, or higher than, levels in healthy individuals.55 Probable reasons include the heterogeneous in vivo distribution of TFPI, variable anticoagulant activities of the different forms of the molecule, and poor correlation between different assay methods.56,57
Hemophilia As an inhibitor of TF-FVIIa and FXa, TFPI plays an important role in hemophilia because it forces amplification of coagulation through FVIII and FIX. Therefore, it is reasonable to hypothesize that differences in TFPI concentration would affect the bleeding phenotype of these patients. Indeed, it has recently been demonstrated that plasma TFPIα strongly
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Recent Advances Tissue Factor Pathway Inhibitor and Protein S Almost a decade ago, it was demonstrated that protein S acts as a cofactor for the direct inhibition of FXa by TFPI.68 It was subsequently established that protein S accelerates formation of the initial K2-FXa encounter complex, but not isomerization of the final tight complex.69 In the absence of FXa, protein S is also a cofactor in the direct inhibition of TF-FVIIa by TFPI.70 Binding of TFPI to protein S requires K3, with Arg199 and Glu226 of K3 identified as critical for this inhibition.71,72 As such, protein S is a cofactor for TFPIα, but not TFPIβ,73 and is hypothesized to localize TFPIα to membrane surfaces73 to allow optimal interaction with FXa.74 The laminin G-type 1 subunit within the sex-hormone-binding globulin–like domain of protein S was recently identified as critical for binding to TFPIα.74 TFPI and free protein S circulate in a complex in vivo.75 Plasma TFPIα is therefore correlated with plasma levels of free Seminars in Thrombosis & Hemostasis
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recent evidence suggesting that this is TFPIβ.39 It remains unknown if TFPIα is present at the endothelial surface, and if so, binding to a putative GPI-anchored coreceptor28,33,37,40 or glycosaminoglycans33,36,37,41 is unlikely. Instead, evidence suggests that TFPIα is constitutively secreted from endothelial cells and is present in an intracellular granule, from which it is released by heparin.28,42,43
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and total protein S.76 Patients with hereditary or acquired protein S deficiency tend to have reduced plasma TFPIα that is thought to contribute to their procoagulant state.75
Tissue Factor Pathway Inhibitor and Factor V FV circulates in vivo in an inactive pro-cofactor state, in which it is maintained via association of two highly evolutionary conserved regions of amino acids within the FV B-domain, one predominantly basic and the other acidic, that together comprise the procofactor regulatory region (PRR).77 Proteolytic cleavage at multiple sites within the B-domain by thrombin removes the entire PRR, transforming FV to an active cofactor.78–80 Conversely, FXa activates FV by removing only the basic region of the PRR, whereas the acidic region is retained by the active FV protein.81–83 Forms of FV resembling FXa-activated FV (FVa) are also found stored within platelets.84,85 The evolutionary conserved, highly basic region within the TFPIα C-terminus and the basic region of the FV PRR share significant homology,86 suggesting an interaction between TFPIα and FV. Indeed, recent studies demonstrate that the TFPIα C-terminus can bind to the acidic region of the FV PRR with similar affinity to that of the FV PRR basic region.86,87 In accordance with this, it has been demonstrated that FVimmunodepleted plasma and that from FV-deficient patients have markedly reduced levels of TFPIα, suggesting that TFPIα and FV circulate as a complex in vivo.75,88 It is hypothesized that the reduced plasma TFPIα accounts for, at least in part, the relatively mild bleeding phenotype observed in FV-deficient patients.88 Association of TFPIα with FV has recently been described as the cause of the moderately severe bleeding diathesis observed in patients with FV East Texas.89 These patients have a mutation in exon 13 of the FV gene, which encodes the B-domain. This mutation produces a novel, preferentially used splice donor site that results in a mature protein containing a greatly shortened B-domain missing the basic region, but retaining the acidic region. This form of FV, termed FV short, quickly associates with plasma TFPIα in vivo and is thought to enhance its circulating half-life. As such, these patients have a plasma TFPIα concentration that is approximately 10 fold greater than normal, resulting in a bleeding phenotype probably due to the direct inhibition of TF-FVIIa that can occur with increased TFPIα concentrations and/or inhibition of early forms of prothrombinase (see the next section).
inhibit these “early” forms of the prothrombinase complex.86 As expected, inhibition is dependent on K2 and the TFPIα Cterminus.86 Furthermore, this inhibition can be prevented by negatively charged molecules such as heparin or polyphosphate, demonstrating the importance of the interaction between the TFPIα C-terminus and acidic region of the FVa PRR in this inhibition.86
Conclusion and Future Directions Although there were reports in the early part of the 20th century that a physiological inhibitor of TF-induced coagulation was present in blood, it was not until the 1980s that this entity, TFPI, was identified. Following its discovery, the structure, function, and other physiological features of TFPI were further investigated and characterized through the 1990s and into the 21st century. The most recent literature continues to explore the characteristics of the TFPIα and TFPIβ isoforms of the molecule, as well as improving understanding of its interactions with protein S, and with FV in the prothrombinase complex. The next chapter of the evolving history of TFPI will include the identification of further individuals affected by FV East Texas and other bleeding diatheses caused by markedly elevated TFPI, and the development of TFPI inhibitors that may be used to treat bleeding disorders such as hemophilia.
Conflict of Interest The authors have no conflict of interest to declare.
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Tissue Factor Pathway Inhibitor and the Inhibition of Prothrombinase Initial studies, performed using prothrombinase complexes formed with thrombin-activated FV, demonstrated that TFPI is a poor inhibitor of FXa within the prothrombinase complex.90 However, prothrombinase formed during the early stages of coagulation contains FXa-activated FVa that retains the acidic region of the PRR.91 A portion of FV within platelets that is able to assemble into prothrombinase has a similar structure.84,85 This form of prothrombinase is critical for the initiation of coagulation.91 A recent study demonstrated that TFPIα, at physiologically relevant concentrations, can Seminars in Thrombosis & Hemostasis
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