Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via ...formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nM). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nM) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nM). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.
Receptor binding of complement C5a leads to proinflammatory activation of many cell types, but the role of receptor-mediated action during arterial remodeling after injury has not been studied. In ...the present study, we examined the contribution of the C5a receptor (C5aR) to neointima formation in apolipoprotein E-deficient mice employing a C5aR antagonist (C5aRA) and a C5aR-blocking monoclonal antibody.
Mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C5aRA and anti-C5aR-blocking monoclonal antibody or vehicle control. Compared with controls, neointima formation was significantly reduced in mice receiving C5aRA or anti-C5aR-blocking monoclonal antibody for 1 week but not for 3 weeks, attributable to an increased content of vascular smooth muscle cells, whereas a marked decrease in monocyte and neutrophil content was associated with reduced vascular cell adhesion molecule-1. As assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and flow cytometry, C5aR was expressed in lesional and cultured vascular smooth muscle cells, upregulated by injury or tumor necrosis factor-alpha, and reduced by C5aRA. Plasma levels and neointimal plasminogen activator inhibitor-1 peaked 1 week after injury and were downregulated in C5aRA-treated mice. In vitro, C5a induced plasminogen activator inhibitor-1 expression in endothelial cells and vascular smooth muscle cells in a C5aRA-dependent manner, possibly accounting for higher vascular smooth muscle cell immigration.
One-week treatment with C5aRA or anti-C5aR-blocking monoclonal antibody limited neointimal hyperplasia and inflammatory cell content and was associated with reduced vascular cell adhesion molecule-1 expression. However, treatment for 3 weeks failed to reduce but rather stabilized plaques, likely by reducing vascular plasminogen activator inhibitor-1 and increasing vascular smooth muscle cell migration.
We report the stepwise transformation of a linear peptide epitope recognized by the anti-transforming growth factor alpha monoclonal antibody Tab2 into peptomers and finally into peptoid analogs. The ...key experiment in this study is the substitution analysis in which each position of the peptide is exchanged by a set of different peptoid building blocks resulting in a peptidomimetic array. After probing the array toward antibody binding, the best binding peptomer spots were selected and subjected to a successive transformation. The best peptoid found in this study has a K(D) of 200 nM when binding to Tab2, which is only 8-fold higher than the starting peptide. Moreover, this approach permits to ask directly questions about the transformation of peptide lead structures into non-peptidic compounds in the context of protein recognition.
Complement activation plays a key role in mediating apoptosis, inflammation, and transplant rejection. In this study, the role of the complement 5a receptor (C5aR) was examined in human renal ...allografts and in an allogenic mouse model of renal transplant rejection. In human kidney transplants with acute rejection, C5aR expression was increased in renal tissue and in cells infiltrating the tubulointerstitium. Similar findings were observed in mice. When recipient mice were treated once daily with a C5aR antagonist before transplantation, long-term renal allograft survival was markedly improved compared with vehicle-treatment (75 versus 0%), and apoptosis was reduced. Furthermore, treatment with a C5aR antagonist significantly attenuated monocyte/macrophage infiltration, perhaps a result of reduced levels of monocyte chemoattractant protein 1 and the intercellular adhesion molecule 1. In vitro, C5aR antagonism inhibited intercellular adhesion molecule 1 upregulation in primary mouse aortic endothelial cells and reduced adhesion of peripheral blood mononuclear cells. Furthermore, C5aR blockade markedly reduced alloreactive T cell priming. These results demonstrate that C5aR plays an important role in mediating acute kidney allograft rejection, suggesting that pharmaceutical targeting of C5aR may have potential in transplantation medicine.
Introduction
Tissue factor pathway inhibitor (TFPI) is a three-Kunitz domain (KD1-3) protease inhibitor that downregulates the extrinsic coagulation pathway. TFPI has a double inhibitory effect; it ...inactivates factor Xa (FXa) by 1:1 binding via its KD2, and it prevents further FX activation by binding the tissue factor (TF) – factor VIIa (FVIIa) complex via its KD1 and the formation of a quaternary complex. Recently, we demonstrated the crystal structure of a linear TFPI inhibitory peptide composed of 20 amino acids, bound to a TFPI protein composed of N-terminus and KD1. On the other hand, a cyclic TFPI inhibitory peptide of 23 amino acids was shown to co-crystallize with TFPI KD1-KD2. Molecular fusion of the linear and cyclic peptide by an optimized linker sequence would thus target two independent epitopes and combine the antagonistic properties of the two peptides.
Methods
The binding properties of simultaneous interaction of the linear and cyclic peptide with TFPI were studied in Biacore experiments using immobilized human TFPI 1-160 on a CM5 chip. Measurements with the linear or cyclic peptide were done with and without prior saturation of TFPI with the linear peptide and the fusion peptide. The results were confirmed by native-PAGE analysis of peptide/KD1-KD2 mixtures, where the TFPI fragment KD1-KD2 had been incubated with either linear or cyclic peptide or both.
The TFPI inhibitory effect of the linear, cyclic, and fusion peptide was assessed in several TFPI sensitive assays including inhibition of FXa, FX activation by TF/FVIIa, and thrombin generation. Calibrated automated thrombography (CAT) was performed in human hemophilia plasma triggered with low tissue factor. To model a situation of elevated plasma levels of TFPI, the assay was carried out at TFPI concentrations up to 10 nM, which is 40-fold higher than the physiological TFPI plasma concentration.
Results
Biacore binding studies demonstrated that binding kinetics of the cyclic peptide to TFPI 1-160 were not influenced by prior saturation of immobilized TFPI with the linear peptide and vice versa. Prior saturation of immobilized TFPI with the fusion peptide prohibited the linear and cyclic peptide from binding to TFPI, clearly demonstrating the independent binding of the two peptides to different epitopes. By native-PAGE, the linear peptide shifted the KD1-KD2 band completely, whereas the cyclic peptide shifted it only partially. In the presence of both peptides, KD1-KD2 shifted to the highest MW to charge ratio, indicating the formation of a ternary complex consisting of K1-K2, cyclic, and linear peptide.
Although the linear and cyclic peptide inhibited TFPI in functional assays, fusion of the two molecular entities provided the most efficient inhibition of TFPI. This was most evident in assays involving multiple epitopes of TFPI to provide functions such as inhibition of extrinsic FX activation complex and thrombin generation, or at high TFPI concentrations. Thrombin generation assays using of 5- to 40-fold elevated TFPI showed that, separately, the two monomeric peptides are only partial inhibitors, and that a mixture of these peptides led to an improved response. However, molecular fusion of the two entities resulted in the most efficient TFPI neutralization. Thus, a synergistic effect is achieved by linking both peptides. Importantly, thrombin generation compromised by a 40-fold of normal TFPI level is normalized by fusion peptide concentrations as low as 50 nM.
Summary
Based on structural information, we developed a peptide inhibitor composed of two TFPI inhibitory entities. Binding studies support an independent binding mode to non-overlapping binding sites without allosteric cross-talk between binding sites. This introduces synergistic improvement of binding and functional inhibition by bivalent interaction with TFPI. This optimized fusion peptide facilitates efficient TFPI neutralization and resistance to highly increased TFPI levels. Our results further support the use of a fusion peptide in the development of subcutaneous treatment for patients with hemophilia including those with inhibitors.
Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Redl:Baxter Innovations GmbH, Vienna, Austria: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Kammlander:Baxter Innovations GmbH: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Reineke:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Brandstetter:Department of Molecular Biology, University of Salzburg, Salzburg, Austria: Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.
Introduction
Tissue factor pathway inhibitor (TFPI) is an important inhibitor of the extrinsic coagulation pathway as it inhibits factor Xa (FXa) and the tissue factor (TF) – FVIIa complex. ...Inhibition of TFPI with blocking antibodies, aptamers, or peptide inhibitors improves hemostasis and may become an option to treat patients with hemophilia including those with inhibitors. We developed a TFPI-antagonistic fusion peptide (FP) consisting of a linear and a cyclic peptide connected by a linker. The two peptide entities bind to different epitopes on TFPI and together synergistically inhibit TFPI. The FP was further improved by half-life extending (HL) non-covalent albumin binding. HL-FP was characterized for in vitroinhibition of TFPI, pharmacokinetics, and improvement of coagulation in animal models of hemophilia.
Methods
HL-FP was characterized in a set of in vitro assays for binding to and inhibition of TFPI. Interaction with immobilized TFPI was studied by BiaCore. Functional inhibition was analyzed in model assay systems such as inhibition of FXa and FX activation by TF/FVIIa and plasma assays according to the calibrated automated thrombography (CAT) protocol at low TF in hemophilia plasma. Addition of TFPI simulated conditions of potentially elevated TFPI plasma concentrations. In a single dose PK study, mice (n=6 per time point) received 400 nmol/kg of the HL-FP intravenously (i.v.) or subcutaneously (s.c.). Plasma was sampled up to 38 h after dosing and HL-FP level quantified by a compound specific LC-MS protocol. To provide an ex vivo activity measure, FVIII inhibitory antibodies were added to mouse plasma to mimic a hemophilic condition and then analyzed by calibrated automated thrombography (CAT). A 2-week repeated i.v. dose study in mice investigated TFPI accumulation due to HL-FP. HL-FP was dosed at 40, 400, and 2000 nmol/kg and mouse plasma TFPI levels determined by ELISA. The efficacy of the HL-FP was studied in a hemophilia A mouse tail cut model and in a marmoset monkey model of ex vivoimprovement of coagulation. FVIII knockout mice (n=16 per group) were dosed i.v. with 12-400 nmol/kg HL-FP in the presence of a sub-therapeutic level of recombinant FVIII (10 U/kg) and blood loss (mg) was assessed. Marmoset monkeys (N=4) received 400 nmol/kg HL-FP i.v. and plasma samples obtained 1 h after dosing were analyzed by CAT in the presence of FVIII inhibitory antibodies.
Results
HL-FP bound to and efficiently inhibited TFPI as demonstrated in several in vitro test systems. Binding affinity of < 1nM correlated well with functional inhibition of TFPI in model assays, resulting in IC50s of ~0.7nM. The HL-fusion peptide (HL-FP) efficiently inhibited plasma TFPI, which resulted in an improvement of all thrombin generation parameters in plasma of hemophilia A and B patients, with EC50s ranging from 6 to 20nM. HL-FP increased peak thrombin levels of hemophilia plasma to or slightly above a range established for individual normal plasma. Non-covalent binding to albumin substantially increased the half-life to ~4 h with ~ 50% s.c. bioavailability in mice. The ex vivo procoagulant activity determined by CAT correlated well with HL-FP plasma concentrations. In the repeated dose study, the HL-FP was well tolerated and did not accumulate TFPI, which strongly indicates that HL-FP did not interfere with TFPI clearance receptor interactions. HL-FP significantly reduced bleeding in the hemophilia mouse tail cut bleeding model at a dose as low as 40 nmol/kg. In marmoset monkeys, HL-FP efficiently improved ex vivo plasma thrombin generation, even at low peptide plasma concentrations (25- 55 nM).
Summary
We developed a TFPI inhibitor composed of two TFPI antagonistic peptides that completely inhibits TFPI. Introduction of an entity non-covalently bound to albumin provides intermediate half-life extension and s.c. bioavailability. This HL-FP improved coagulation and hemostasis in animal models of hemophilia and did not interfere with TFPI clearance receptor interactions. TFPI-antagonistic peptides with a prolonged half-life, resistance to elevated TFPI, and minimal interference with TFPI clearance. Our HL-FP appears to be useful in preventing bleeding in hemophilia and provides a FVIII and FIX independent approach for non-i.v. treatment.
Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Kammlander:Baxter Innovations GmbH, Vienna, Austria: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Reineke:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Schiviz:Baxter Innovations GmbH, Vienna, Austria: Employment. Hoellriegl:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.
Renal fibrosis is the final common pathway of most progressive renal diseases. C5 was recently identified as a risk factor for liver fibrosis. This study investigated the role of C5 in the ...development of renal tubulointerstitial fibrosis by (1) induction of renal fibrosis in wild-type and C5(-/-) mice by unilateral ureteral ligation (UUO) and (2) investigation of the effects of a C5a receptor antagonist (C5aRA) in UUO. In C5(-/-) mice, when compared with wild-type controls, markers of renal fibrosis (Sirius Red, type I collagen, fibronectin, alpha-smooth muscle actin, vimentin, and infiltrating macrophages) were significantly reduced on day 5 of UUO. On day 10, fibronectin mRNA and protein expression were still reduced in the C5(-/-) mice. Cortical mRNA of all PDGF isoforms and of TGF-beta(1) (i.e., central mediators of renal disease) were significantly reduced in C5(-/-) mice when compared with controls. Renal tubular cell expression of the C5aR was sparse in normal cortex but markedly upregulated after UUO. Treatment of wild-type UUO mice with C5aRA also led to a significant reduction of cortical Sirius Red staining, fibronectin protein expression, and PDGF-B mRNA expression on day 5. Neither genetic C5 deficiency nor C5aRA treatment caused any histologic changes in the nonobstructed kidneys. In cultured murine cortical tubular cells, C5a stimulated production of TGF-beta(1), and this was inhibited by C5aRA. Using a combined genetic and pharmacologic approach, C5, in particular C5a, is identified as a novel profibrotic factor in renal disease and as a potential new therapeutic target.
TFPI is an important inhibitor of the extrinsic coagulation pathway. It efficiently inhibits TF-FVIIa and FXa by quaternary complex formation. Plasma contains various truncated forms of TFPI which ...are poor inhibitors, and full length (fl)TFPI (0.3 – 0.5 nM) which is the most active TFPI in plasma. flTFPI is released from platelets upon activation, and increases flTFPI concentrations locally up to 30-fold. Most intravascular TFPI (∼80%) is associated with endothelial cells. Both endothelial forms, TFPIa and TFPIb, are similarily effective inhibitors of FX activation on the endothelial cell surface. Inhibition of TFPI in hemophilia models with blocking antibodies, aptamers or peptide inhibitors improves hemostasis and may become an option to treat hemophilia. Recently, we presented peptide inhibitors of TFPI that enhance coagulation in hemophilia models. Two optimized peptides, JBT-A7 and JBT-B5, efficiently blocked inhibitory activity of TFPI and bound to distinct binding sites.
We demonstrated the crystal structure of JBT-A7, a linear TFPI inhibitory peptide composed of 20 amino acids, bound to NtermK1 (TFPI 1-83). JBT-B5, a cyclic TFPI inhibitory peptide of 23 amino acids, co-crystallized with TFPI KD1-KD2 (TFPI 22-150). Overlaying the KD1 structure in the KD1-KD2/JBT-B5 and the NTermK1/JBT-A7 complex provided atomic details for linking the two peptide entities. Binding of peptides to TFPI and TFPI fragments was studied by BioCore. The TFPI inhibitory potential of the resulting fusion peptide was tested in model systems (FXa inhibition and TF-FVIIa catalyzed FX activation) and global hemostatic assays (TF-triggered thrombin generation) using hemophilia plasma. To model situations of increased TFPI concentration, both model and plasma assays were carried out at TFPI concentrations up to 10 nM, which is 40-50-fold higher than the physiological flTFPI plasma concentration. To characterize the inhibition of platelet TFPI, we used platelets isolated from blood samples and platelet rich plasma from different donors. Binding of a biotinylated fusion peptide on living HUVE cells was assessed by fluorescence activated cell sorting (FACS) and fluorescence microscopy. Inhibition of cell surface TFPI was analyzed on cultivated HUVECs stimulated with TNFa for TF expression. We monitored FXa generation by the TFPI-dependent cell surface FX activation complex by conversion of an FXa-specific fluorogenic substrate.
The overlay of the crystal structures of KD1-KD2/JBT-B5 and the NTermKD1/JBT-A7 complexes revealed non-overlapping epitopes and close proximity of the termini of both peptides. The distance could be bridged by an approximately ten amino acid linker. A fusion peptide with a 10-serine-linker was synthesized and showed highly improved dissociation in Biacore experiments and most efficiently inhibited TFPI activity in the model assays. In contrast, single peptides only partially inhibit TFPI especially at high TFPI concentrations. In thrombin generation assays using hemophilia plasma, the fusion peptide showed a substantially higher ability than the single peptides to increase the thrombin peak even at elevated TFPI. The fusion peptide efficiently inhibited TFPI released from platelets and improved thrombin generation in TFPI deficient plasma reconstituted with platelets as the only source of TFPI released upon platelet activation. The fusion peptide was also shown to bind TFPI on the surface of living HUVECs. This is consistent with its binding epitopes on KD1 and KD2 which result in inhibition of cell surface TFPI in a cell based FX activation assay. Thus, we demonstrate that a molecular fusion peptide most efficiently inhibits all physiologic forms of TFPI.
X-ray structures of binary and ternary peptide TFPI complexes provided atomic details for linking two single peptides to generate a fusion peptide that most efficiently blocks TFPI in plasma, released from platelets and associated with endothelial cells. It most efficiently neutralizes TFPI even at substantially elevated concentrations occurring at sites of platelet activation. Our observations support the notion that targeting TFPI with TFPI inhibitors is a promising novel strategy to mitigate the bleeding risk in hemophilia patients.
Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals, Berlin, Germany: Employment. Brandstetter:Baxter Innovations GmbH, Vienna, Austria: Research Funding. Kammlander:Baxter Innovations GmbH, Vienna, Austria: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Redl:Baxter Innovations GmbH, Vienna, Austria: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.
TFPI is a Kunitz-type protease inhibitor which efficiently regulates the extrinsic coagulation pathway. It is composed of three flexible linked Kunitz-type domains (KD) where KD1 and KD2 are involved ...in efficient inhibition of TF/FVIIa and FXa. TFPI inhibition has been shown to improve coagulation and hemostasis in hemophilia models in vitro and in vivo.
Recombinant KD1-KD2 (residues 22-150) produced by E. coli and complexed to JBT-B5, a cyclic peptide composed of 23 amino acids, was co-crystallized in 20% w/v PEG6000 and 50mM imidazole, pH8.0. JBT-B5 binding to TFPI was verified by BiaCore experiments with TFPI immobilized on a chip surface. Functional inhibition of TFPI by JBT-B5 was tested in model assays including TFPI inhibition of FXa, FX activation by TF/FVIIa, inhibition of TFPI released upon platelet activation, and by global hemostatic assays including calibrated automated thrombography in FVIII-inhibited plasma and rotational thomboelastometry (ROTEM) using FVIII-inhibited whole blood. Inhibition of cell surface TFPI was analyzed in an FX activation assay performed on HUVECs.
TFPI KD1-KD2 bound to JBT-B5 formed a crystal containing two independent complexes in the asymmetric unit. The complexes belong to the orthorhombic spacegroup P212121 and diffracted to a maximum resolution of 1.95 Å. To our knowledge, this is the first TFPI structure consisting of KD1, KD2 and their linker. The KD1-KD2 structure is fully defined in the electron density. Both domains show a Kunitz-type structure, where only ∼1/3 of the structure is engaged in secondary structure elements. These are two short α-helical elements at Ser24-Ala27(KD1)/Asp95-Phe98(KD2) (α1/α3) and Leu69-Met75/Leu140-Glu148 (α2/α4) and a two-stranded β sheet comprising Met39-Asn45/Ile110-Asn116 (β1/β3) and Arg49-Ile55/Lys120-Lys126 (β2/β4). These elements form the topological framework that is stabilized by the three canonical disulfide bonds involving Cys26-Cys76, Cys35-Cys59, and Cys51-Cys72 in KD1 and Cys95-Cys147, Cys106-Cys130, and Cys122-Cys143 in KD2. The 23mer TFPI inhibitor, JBT-B5, is sandwiched between the two Kunitz domains of TFPI and assumes a β-hairpin-like structure. It can be segmented into (i) a two-stranded β sheet comprising Tyr2-Ala8 and Thr17-Phe23; (ii) and a long β-turn loop comprising Met9-Met16. The β-sheet is stabilized by a disulfide bridge (Cys7 and Cys18) and a hydrophobic zipper comprising the side chains of Tyr3, Trp5 and Trp20. Strikingly, JBT-B5 locks KD1-KD2 in a distinct conformational state in which both Kunitz-domains are related via a two-fold symmetry and reactive center loops (RCL) from each KD are forced to opposite sides. The interactions between KD1-KD2 and JBT-B5 are extensive, as are the intramolecular interactions within JBT-B5. Analyzing the interactions between KD1-KD2 and JBT-B5 with the PISA server resulted in a total interaction surface of 1340Å2. More than 2/3 of the interaction surface consists of a hydrophobic anchor in JBT-B5 which interact with residues spread all over TFPI including KD1, KD2 and their linker. In addition, several polar interactions stabilize the KD1-KD2/JBT-B5 complex, explaining JBT-B5´s exlusive binding to human TFPI. Interaction studies revealed high affinity binding to TFPI (KD: 0.5nM). The highly complex and extensive interaction of JBT-B5 with TFPI translates to a highly efficient inhibition of recombinant TFPI and;TFPI released from activated platelets which is demonstrated in model assays on FXa (EC50: 1.3nM) and FXa generation by TF/FVIIa (EC50: 0.2nM). HUVE cell-based FX activation and global hemostasis assays such as thrombin generation in hemophilia plasma (EC50: 4nM) and ROTEM in FVIII-inhibited whole blood confirm the inhibition of all physiologic TFPI forms.
For the first time, a TFPI structure comprising KD1, KD2 and their linker in complex with a TFPI- inhibitory cyclic peptide was solved. This structure provides atomic details explaining the inhibitory mode of action of this efficient TFPI antagonist, and will guide the design of efficient inhibitors for use in hemophilia treatment.
Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Fries:Baxter Innovations GmbH, Vienna, Austria: Employment. Prohaska: Baxter Innovations GmbH, Vienna, Austria: Employment. Pachlinger:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals, Berlin, Germany: Employment. Brandstetter: Baxter Innovations GmbH, Vienna, Austria: Research Funding. Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.
Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via ...formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nm). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nm) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nm). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.
Background: Tissue factor pathway inhibitor (TFPI) inhibits coagulation factors Xa and VIIa.
Results: A de novo synthesized 20-mer peptide that binds to TFPI was structurally and functionally characterized.
Conclusion: The peptide binds to the Kunitz domain 1 of TFPI and blocks inhibition of factor Xa and factor VIIa by TFPI.
Significance: The peptide can potentially prevent bleeding in hemophilia patients.
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