Endogenous tissue factor pathway inhibitor has a limited effect on host defence in murine pneumococcal pneumonia

SummaryStreptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Coagulation and inflammation interact in the host response to infection. Tissue factor pathway inhibitor (TFPI) is a natural anticoagulant protein that inhibits tissue factor (TF), the main activator of inflammation-induced coagulation. It was the objective of this study to investigate the effect of endogenous TFPI levels on coagulation, inflammation and bacterial growth during S. pneumoniae pneumonia in mice. The effect of low endogenous TFPI levels was studied by administration of a neutralising anti-TFPI antibody to wild-type mice, and by using genetically modified mice expressing low levels of TFPI, due to a genetic deletion of the first Kunitz domain of TFPI (TFPIK1(-/-)) rescued with a human TFPI transgene. Pneumonia was induced by intranasal inoculation with S. pneumoniae and samples were obtained at 6, 24 and 48 hours after infection. Anti-TFPI reduced TFPI activity by ~50 %. Homozygous lowTFPI mice and heterozygous controls had ~10 % and ~50 % of normal TFPI activity, respectively. TFPI levels did not influence bacterial growth or dissemination. Whereas lung pathology was unaffected in all groups, mice with ~10 % (but not with ~50 %) of TFPI levels displayed elevated lung cytokine and chemokine concentrations 24 hours after infection. None of the groups with low TFPI levels showed an altered procoagulant response in lungs or plasma during pneumonia. These data argue against an important role for endogenous TFPI in the antibacterial, inflammatory and procoagulant response during pneumococcal pneumonia.

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Factor Xa Enhances the Binding of Tissue Factor Pathway Inhibitor to Acidic Phospholipids

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650369 ◽

1996 ◽

Vol 75 (05) ◽

pp. 796-800 ◽

Cited By ~ 10

Author(s):

Sanne Valentin ◽

Inger Schousboe

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Xa ◽

Microtitre Plate ◽

C Terminus ◽

Microtitre Plate Assay ◽

Kunitz Domain ◽

Tissue Factor Pathway ◽

Acidic Phospholipids

SummaryIn the present study, the interaction between tissue factor pathway inhibitor (TFPI) and phospholipids has been characterized using a microtitre plate assay. TFPI was shown to bind calcium-independently to an acidic phospholipid surface composed of phosphatidylserine, but not a surface composed of the neutral phosphatidylcholine. The interaction was demonstrated to be dependent on the presence of the TFPI C-terminus. The presence of heparin (1 U/ml, unfractionated) was able to significantly reduce the binding of TFPI to phospholipid. The interaction of TFPI with phosphatidylserine was significantly decreased in the presence of calcium, but this was counteracted, and even enhanced, following complex formation of TFPI with factor Xa prior to incubation with the phospholipid surface. Moreover, a TFPI variant, not containing the third Kunitz domain and the C-terminus, was unable to bind to phospholipid. However, following the formation of a TFPI/factor Xa-complex this TFPI variant was capable of interacting with the phospholipid surface. This indicates that the role of factor Xa as a TFPI cofactor, at least in part, is to mediate the binding of TFPI to the phospholipid surface.

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Recombinant Tissue Factor Pathway Inhibitor in Severe Community-acquired Pneumonia

American Journal of Respiratory and Critical Care Medicine ◽

10.1164/rccm.201007-1167oc ◽

2011 ◽

Vol 183 (11) ◽

pp. 1561-1568 ◽

Cited By ~ 71

Author(s):

Richard G. Wunderink ◽

Pierre-François Laterre ◽

Bruno Francois ◽

Dominique Perrotin ◽

Antonio Artigas ◽

...

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Community Acquired Pneumonia ◽

Tissue Factor Pathway ◽

Severe Community Acquired Pneumonia

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Recombinant human tissue factor pathway inhibitor exerts anticoagulant, anti-inflammatory and antibacterial effects in murine pneumococcal pneumonia

Critical Care ◽

10.1186/cc8083 ◽

2009 ◽

Vol 13 (Suppl 4) ◽

pp. P27

Author(s):

F Boogaard ◽

X Brands ◽

M Schultz ◽

M Levi ◽

J Roelofs ◽

...

Keyword(s):

Tissue Factor ◽

Human Tissue ◽

Pneumococcal Pneumonia ◽

Tissue Factor Pathway Inhibitor ◽

Antibacterial Effects ◽

Tissue Factor Pathway ◽

Anti Inflammatory

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Engineering Kunitz Domain of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis: An Antifibrinolytic Agent with Potential to Replace Aprotinin

Blood ◽

10.1182/blood.v112.11.24.24 ◽

2008 ◽

Vol 112 (11) ◽

pp. 24-24

Author(s):

Madhu S Bajaj ◽

Godwin I Ogueli ◽

Amy E Schmidt ◽

Sreejesh Shanker ◽

S. Paul Bajaj

Keyword(s):

Blood Loss ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Viia ◽

Total Blood Loss ◽

Total Blood ◽

Antifibrinolytic Agent ◽

Kunitz Domain ◽

Factor Xia ◽

Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin and therefore it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this report, structure based investigations and analysis of the serine proteases profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ (nomenclature of Schechter and Berger, BBRC, 27:157–162, 1967) position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu17 (bovine pancreatic trypsin inhibitor/aprotinin numbering) in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with the wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in E. Coli, folded and purified to homogeneity. Amino-terminal sequences and mass spectra revealed proper folding of the KD1-WT and KD1-L17R. As compared to KD1-WT, the KD1-L17R neither prolonged the activated partial thromboplastin time of normal plasma nor it inhibited factor XIa, plasma kallikrein or factor VIIa/tissue factor. Further, KD1-L17R inhibited plasmin with ~4-fold increased affinity. In a mouse liver laceration model of bleeding from small vessels, KD1-L17R reduced total blood loss by 84% compared with KD1-WT, which reduced total blood loss by 10%. Moreover, in this bleeding model, KD1-L17R was more effective than aprotinin (70% reduction), which has been used as an antifibrinolytic agent to decrease blood loss during major surgery. In this model, KD1-L17R was also more effective than the lysine analogue tranexamic acid (52% reduction). In additional studies, in a tail transection model of bleeding from a large vessel, KD1-L17R reduced total blood loss by 70% and was more effective than KD1-WT (46% reduction), aprotinin (43% reduction) and tranexamic acid (67% reduction). Notably, as compared to aprotinin, renal toxicity manifesting as multifocal tubular necrosis by histopathology was not observed with KD1-L17R or KD1-WT. In conclusion, KD1-L17R is a specific inhibitor of plasmin without anticoagulant properties and is more effective in reducing blood loss compared with known antifibrinolytic agents in clinical use.

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Peptides Binding to Kunitz Domain 1 of Tissue Factor Pathway Inhibitor (TFPI) Inhibit All Functions of TFPI and Improve Thrombin Generation of Hemophilia Plasma

Blood ◽

10.1182/blood.v118.21.2245.2245 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2245-2245

Author(s):

Michael Dockal ◽

Johannes Brandstetter ◽

Martin Ludwiczek ◽

Georg Kontaxis ◽

Markus Fries ◽

...

Keyword(s):

Amino Acid ◽

Tissue Factor ◽

Active Site ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Peptide Bond ◽

Model System ◽

Kunitz Domain ◽

Tissue Factor Pathway ◽

Antagonistic Peptide

Abstract Abstract 2245 Blood coagulation is initiated by the tissue factor-factor VIIa (TF-FVIIa) complex which cleaves and activates coagulation factor X to Xa (FXa). Tissue factor pathway inhibitor (TFPI) controls this key process and thus plays a crucial role in maintaining the delicate balance of pro- and anticoagulant processes. Inhibition of TFPI in hemophilia plasma and in a rabbit model of hemophilia has been shown to improve coagulation and hemostasis (Nordfang et al., Thromb Haemost. 1991;66:464; Erhardsen et al., Blood Coagulation and Fibrinolysis 1995;6:388). TFPI is a Kunitz-type protease inhibitor that inhibits FXa and TF-FVIIa. TFPI is a slow, tight-binding FXa inhibitor which rapidly forms a loose FXa-TFPI complex that slowly isomerises to a tight FXa-TFPI* complex. The FXa-TFPI* complex inhibits TF-FVIIa by formation of a quaternary FXa-TFPI-TF-FVIIa complex. Using a library approach, we selected a peptide which binds and inhibits TFPI. We located the binding site of the antagonistic peptide on TFPI by NMR spectroscopy. Residues of TFPI undergoing the strongest chemical shift changes were exclusively found on the Kunitz domain 1 (KD1). NMR data were confirmed by solving the crystal structure of KD1 in complex with the antagonistic peptide at 2.55 Å resolution. Like in related Kunitz domains, the robustness of this approximately 60-amino-acid long folding module largely depends on stabilization by the three disulfides bonds and a hydrophobic cluster of three phenylalanines. The disulfide bridging of the P2 residue induces conformational constraints on the reactive centre loop (RCL), thereby establishing an extended RCL conformation; consequently, the amino acid side chains flanking the “scissile” peptide bond are exposed to the solvent. This RCL geometry also explains why the distorted, improperly activated scissile peptide bond is hardly cleaved. Whereas Cys-Lys/Arg is a rather conserved P2-P1 motif, reflecting the topological restraints in Kunitz protease inhibitors, proline at position P3 induces an additional conformational constraint on the RCL, which would not be possible in the narrow active site of FXa. Proline at the P3 and to a lesser extent Lys rather than Arg at P1 thus represent two major specificity determinants of KD1 towards FVIIa over FXa. The structure of the 20-mer peptide can be segmented into (i) an N-terminal anchor; (ii) an Ω-shaped loop; (iii) an intermediate segment; (iv) a tight glycine loop; and (v) a C-terminal α-helix that is anchored to KD1 at its RCL and two-strand β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots but the major driving force of complex formation is steric surface complementarity. One of the optimized peptides, which binds to KD1 of TFPI, had an affinity for TFPI of <1 nM. In a model system, the peptide blocked both FXa inhibition by TFPI (IC50=5 nM) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (IC50=5.7 nM). In FVIII-depleted plasma, the peptide enhanced thrombin generation 9-fold (EC50=4 nM). Detailed kinetic analysis in a model system showed that the peptide almost fully inhibited TFPI and prevented the transition from the loose to the tight FXa-TFPI* complex, but did not affect formation of the loose FXa-TFPI complex. Since KD1 binds to the active site of FVIIa and KD2 to the active site of FXa our kinetic data with the KD1-binding peptide show that KD1 is not only important for FVIIa inhibition but is also required for FXa inhibition, i.e. for the transition from the loose to the tight FXa-TFPI* complex. In line with this mechanism, the peptide did not affect FXa inhibition by the isolated KD2. The peptide was also able to dissociate preformed FXa-TFPI* and FXa-TFPI-TF-FVIIa complexes and liberate active FXa and TF-FVIIa. In summary, we developed a peptide that binds to KD1 of TFPI, that prevents FXa-TFPI and FXa-TFPI-TF-FVIIa complex formation and that enhances coagulation under hemophilia conditions. Disclosures: Dockal: Baxter Innovations GmbH: Employment. Brandstetter:University of Salzburg: Employment. Ludwiczek:Baxter Innovations GmbH: Employment. Kontaxis:University of Vienna: Employment. Fries:Baxter Innovations GmbH: Employment. Thomassen:Maastricht University: Employment. Heinzmann:Maastricht University: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Prohaska:Baxter Innovations GmbH: Employment. Hartmann:Baxter Innovations GmbH: Employment. Rosing:Maastricht University: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

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Tissue factor pathway inhibitor (TFPI) in disseminated intravascular coagulation: low levels of the activated factor X–TFPI complex

Blood Coagulation & Fibrinolysis ◽

10.1097/00001721-200010000-00002 ◽

2000 ◽

Vol 11 (7) ◽

pp. 591-598 ◽

Cited By ~ 11

Author(s):

N. Iversen ◽

F. G. Strekerud ◽

U. Abildgaard

Keyword(s):

Tissue Factor ◽

Disseminated Intravascular Coagulation ◽

Tissue Factor Pathway Inhibitor ◽

Factor X ◽

Intravascular Coagulation ◽

Tissue Factor Pathway ◽

Low Levels

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Structural and functional characterization of tissue factor pathway inhibitor following degradation by matrix metalloproteinase-8

Biochemical Journal ◽

10.1042/bj20020696 ◽

2002 ◽

Vol 367 (2) ◽

pp. 451-458 ◽

Cited By ~ 30

Author(s):

Anna C. CUNNINGHAM ◽

Karen A. HASTY ◽

Jan J. ENGHILD ◽

Alan E. MAST

Keyword(s):

Matrix Metalloproteinase ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Proteolytic Enzymes ◽

Functional Characterization ◽

Factor Xa ◽

Terminal Region ◽

The Third ◽

Kunitz Domain ◽

Tissue Factor Pathway

Vascular injury results in the activation of coagulation and the release of proteolytic enzymes from neutrophils and connective- tissue cells. High concentrations of these inflammatory proteinases may destroy blood coagulation proteins, contributing to coagulation and bleeding disorders associated with severe inflammation. Matrix metalloproteinase-8 (MMP-8) is released from neutrophils at sites of inflammation and vascular disease. We have investigated the effect of MMP-8 degradation on the anticoagulant function of tissue factor pathway inhibitor (TFPI) as a potential pathological mechanism contributing to coagulation disorders. MMP-8 cleaves TFPI following Ser174 within the connecting region between the second and third Kunitz domains (kcat/Km75M-1·s-1) as well as following Lys20 within the NH2-terminal region. MMP-8 cleavage of TFPI decreases the anticoagulant activity of TFPI in factor Xa initiated clotting assays as well as the ability of TFPI to inhibit factor Xa in amidolytic assays. Yet, MMP-8 cleavage does not alter the ability of TFPI to inhibit trypsin. Since the inhibition of both factor Xa and trypsin is mediated by binding to the second Kunitz domain, these results suggest that regions of TFPI other than the second Kunitz domain may directly interact with factor Xa. 125I-factor Xa ligand blots of TFPI fragments generated following MMP-8 degradation were used for probing binding interactions between factor Xa and regions of TFPI, other than the second Kunitz domain. In experiments performed under reducing conditions that disrupt the Kunitz domain structure, 125I-factor Xa binds to the C-terminal fragment of MMP-8-degraded TFPI. This fragment contains portions of TFPI distal to Ser174, which include the third Kunitz domain and the basic C-terminal region. An altered form of TFPI lacking the third Kunitz domain, but containing the C-terminal region, was used to demonstrate that the C-terminal region directly interacts with factor Xa.

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Plasmatic tissue factor pathway inhibitor is a major determinant of clotting in factor VIII inhibited plasma or blood

Thrombosis and Haemostasis ◽

10.1160/th12-07-0529 ◽

2013 ◽

Vol 109 (03) ◽

pp. 450-457 ◽

Cited By ~ 16

Author(s):

Sabine Knappe ◽

Bernd Jilma ◽

Ulla Derhaschnig ◽

Rudolf Hartmann ◽

Michael Palige ◽

...

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Ex Vivo ◽

Major Determinant ◽

Prospective Clinical Study ◽

Acquired Haemophilia ◽

C Terminus ◽

Kunitz Domain ◽

Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a major inhibitor of coagulation. We therefore hypothesised that high plasmatic TFPI levels are associated with impaired ex vivo clotting in a model of acquired haemophilia. Blood samples were collected in a prospective clinical study from 30 healthy volunteers. Coagulation in normal or factor VIII (FVIII)-inhibited human blood or plasma was measured by the calibrated automated thrombogram (CAT) and rotational thromboelastometry (ROTEM). Both methods are global haemostatic assays that provide insight into the whole coagulation process. Monoclonal mouse antibodies raised against either the C-terminus or the Kunitz domain 2 of TFPI were used to determine full-length (fl-) and total TFPI by an enzyme-immunoassay. Clotting times and parameters of thrombin generation correlated with TFPI levels. Subjects with low fl-TFPI levels had significantly shorter clotting times and a higher endogenous thrombin potential (ETP) compared to those with high fl-TFPI levels (p≤0.005 for all). An even stronger effect was seen in FVIII-inhibited blood/plasma: ROTEM clotting time was 26% shorter (p=0.01) and the ETP assessed by CAT was >2-fold higher in subjects with low fl-TFPI levels (p≤0.0001). Plasmatic TFPI is a major determinant of coagulation in global haemostatic tests particularly when FVIII is missing. Thus, inhibition of TFPI might be a promising novel treatment approach, especially in haemophilia patients with FVIII inhibitors.

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Low levels of tissue factor pathway inhibitor (TFPI) increase the risk of venous thrombosis

Blood ◽

10.1182/blood-2002-10-3188 ◽

2003 ◽

Vol 101 (11) ◽

pp. 4387-4392 ◽

Cited By ~ 151

Author(s):

Anders Dahm ◽

Astrid van Hylckama Vlieg ◽

Bjorn Bendz ◽

Frits Rosendaal ◽

Rogier M. Bertina ◽

...

Keyword(s):

Risk Factor ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Large Population ◽

Population Based ◽

Vein Thrombosis ◽

Tissue Factor Pathway ◽

Low Levels ◽

Free Antigen

Abstract There is now strong experimental evidence that tissue factor pathway inhibitor (TFPI) is a critical inhibitor to modulate tissue factor–induced coagulation, but the role of TFPI as a risk factor for thrombosis is yet to be to be determined. This study investigated the role of low TFPI levels for the development of deep-vein thrombosis (DVT). We determined TFPI activity and TFPI-free and total antigen levels in the subjects enrolled in the Leiden Thrombophilia Study, which is a large population-based case-control study of 474 patients and 474 controls. The odds ratio (OR) for DVT in subjects who had TFPI-free antigen levels below the 10th percentile, as compared with those who had TFPI-free antigen levels above this cutoff, was 1.7 (95% confidence interval [CI], 1.1-2.6). The ORs for low TFPI activity and low total antigen were also mildly increased. When the 5th percentile was used as a cutoff, the ORs were 2.1 (95% CI, 1.1-4.1) for both TFPI-free antigen and TFPI total antigen. Exogenous female hormones had a profound lowering effect on TFPI levels, with lower levels in oral contraceptive users than in premenopausal nonusers, who had lower levels than men and postmenopausal women. These results indicate that low levels of TFPI, especially low TFPI-free and total antigen in plasma, constitute a risk factor for DVT.

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