scholarly journals Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction

2015 ◽  
Vol 113 (05) ◽  
pp. 976-987 ◽  
Author(s):  
Sofia Somajo ◽  
Josefin Ahnström ◽  
Juan Fernandez-Recio ◽  
Magdalena Gierula ◽  
Bruno O. Villoutreix ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Tissue Factor ◽  
In Silico ◽  
Tissue Factor Pathway Inhibitor ◽  
Protein S ◽  
Protein Docking ◽  
Amino Acid Residues ◽  
Interaction Sites ◽  
Tissue Factor Pathway

SummaryProtein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50–60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LGdomains.

Cloning, Expression, and Characterization of Mouse Tissue Factor Pathway Inhibitor (TFPI)

1998 ◽  
Vol 79 (02) ◽  
pp. 306-309 ◽  
Author(s):  
Dougald Monroe ◽  
Julie Oliver ◽  
Darla Liles ◽  
Harold Roberts ◽  
Jen-Yea Chang
Keyword(s):  
Amino Acid ◽  
Tissue Factor ◽  
Signal Peptide ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Protein Sequences ◽  
Cloning And Expression ◽  
Mouse Tissue ◽  
Amino Acid Residues ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/ TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

scholarly journals Imbalance in Coagulation/Fibrinolysis Inhibitors Resulting in Extravascular Thrombin Generation in Gliomas of Varying Levels of Malignancy

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 663
Author(s):  
Marek Z. Wojtukiewicz ◽  
Marta Mysliwiec ◽  
Elwira Matuszewska ◽  
Stanislaw Sulkowski ◽  
Lech Zimnoch ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Tissue Factor Pathway Inhibitor ◽  
Protein S ◽  
Systemic Circulation ◽  
Coagulation System ◽  
Lower Grade ◽  
Increased Risk ◽  
Tissue Factor Pathway ◽  
Pai 1

Neoplastic processes are integrally related to disturbances in the mechanisms regulating hemostatic processes. Brain tumors, including gliomas, are neoplasms associated with a significantly increased risk of thromboembolic complications, affecting 20–30% of patients. As gliomas proliferate, they cause damage to the brain tissue and vascular structures, which leads to the release of procoagulant factors into the systemic circulation, and hence systemic activation of the blood coagulation system. Hypercoagulability in cancer patients may be, at least in part, a result of the inadequate activity of coagulation inhibitors. The aim of the study was to evaluate the expression of the inhibitors of the coagulation and fibrinolysis systems (tissue factor pathway inhibitor, TFPI; tissue factor pathway inhibitor-2 TFPI-2; protein C, PC; protein S, PS, thrombomodulin, TM; plasminogen activators inhibitor, PAI-1) in gliomas of varying degrees of malignancy. Immunohistochemical studies were performed on 40 gliomas, namely on 13 lower-grade (G2) gliomas (8 astrocytomas, 5 oligodendrogliomas) and 27 high-grade gliomas (G3–12 anaplastic astrocytomas, 4 anaplastic oligodendrogliomas; G4–11 glioblastomas). A strong expression of TFPI-2, PS, TM, PAI-1 was observed in lower-grade gliomas, while an intensive color immunohistochemical (IHC) reaction for the presence of TFPI antigens was detected in higher-grade gliomas. The presence of PC antigens was found in all gliomas. Prothrombin fragment 1+2 was observed in lower- and higher-grade gliomas reflecting local activation of blood coagulation. Differences in the expression of coagulation/fibrinolysis inhibitors in the tissues of gliomas with varying degrees of malignancy may be indicative of their altered role in gliomas, going beyond that of their functions in the hemostatic system.

scholarly journals Different effects of oral contraceptives containing different progestogens on protein S and tissue factor pathway inhibitor

2008 ◽  
Vol 6 (2) ◽  
pp. 346-351 ◽  
Author(s):  
H. A. A. M. VAN VLIET ◽  
R. M. BERTINA ◽  
A. E. A. DAHM ◽  
F. R. ROSENDAAL ◽  
J. ROSING ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Oral Contraceptives ◽  
Tissue Factor Pathway Inhibitor ◽  
Protein S ◽  
Tissue Factor Pathway

scholarly journals Complementary DNA sequencing of canine tissue factor pathway inhibitor reveals a unique nanomeric repetitive sequence between the second and third Kunitz domains

1994 ◽  
Vol 303 (3) ◽  
pp. 923-928 ◽  
Author(s):  
T J Girard ◽  
D Gailani ◽  
G J Broze
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Peptide Sequence ◽  
Tissue Factor Pathway ◽  
Kunitz Type ◽  
Canine Plasma

Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent inhibitor of the factor VIIa-tissue factor complex of blood coagulation. The primary amino acid sequence of canine TFPI has been deduced from cDNA sequences obtained using the techniques of reverse transcription followed by amplification using PCR and conventional screening of a canine endothelial cell cDNA library. The open reading frame for canine TFPI encodes a signal peptide of 28 amino acids followed by a 40.7 kDa protein of 368 amino acids. Similar to human, rat and rabbit TFPI, canine TFPI contains a negatively-charged cluster of amino acids at its mature amino-terminus, followed by three Kunitz-type proteinase inhibitory domains and a cluster of positively-charged amino acids near its carboxy-terminus. In contrast to other TFPIs, following its second Kunitz-type proteinase inhibitory domain canine TFPI contains an additional amino acid insert which includes a nanomeric peptide-sequence repeated six times. Recombinant canine TFPI was expressed in both bacterial- and insect cell-expression systems for functional analysis and the generation of antibodies. The recombinant canine TFPI inhibits tissue factor-induced coagulation in an in vitro canine system. Immunoprecipitation of TFPI from canine plasma, followed by Western-blot analysis, tentatively identifies canine TFPI as an 80,000 kDa protein. Anti-peptide antibodies raised to the nanomeric peptide repeat immunoprecipitate an identical, cross-reactive, 80,000 kDa protein.

Protein S is a cofactor for tissue factor pathway inhibitor

2008 ◽  
Vol 122 ◽  
pp. S60-S63 ◽  
Author(s):  
J. Rosing ◽  
L.F.A. Maurissen ◽  
S.N. Tchaikovski ◽  
G. Tans ◽  
T.M. Hackeng
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Protein S ◽  
Tissue Factor Pathway

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 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document