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Tissue Factor residue Asp44 regulates catalytic function of the bound proteinase Factor VIIa.

ABSTRACT: The coagulation pathways are initiated by the cell-surface receptor Tissue Factor (TF), which binds the serine proteinase coagulation Factor VIIa (VIIa), resulting in enhanced catalytic function, both amidolytic, towards small pseudo-substrates, and proteolytic, towards macromolecular substrates. Here we implicate Asp44 in TF as a ligand-interactive residue that, in contrast with previously characterized binding residues, is involved in the enhancement of VIIa catalytic function. Whereas charge neutralization by replacement of Asp44 with Asn did not reduce function of human TF, the exchange by Ala resulted in mutants with 8-fold reduced affinity for binding of VIIa. Enhancement of VIIa amidolytic function by TF Ala44 was reduced by 20-25% relative to wild-type and support of proteolytic function was diminished 6-fold indicating that this cofactor residue is significantly enhancing proteolysis of the macromolecular substrate by VIIa. Replacement of Asp44 by Glu, Thr, and Arg exhibited a less severe phenotype with an approx. 4-fold reduced affinity for VIIa and a 2-3 fold diminished activation of Factor X. The improved activity of these mutants as compared with the Ala replacement is consistent with functional importance of an extended side chain at this position. The specific influence of the Asp44 exchange on catalytic function of the TF x VIIa complex indicates fine specificity of the TF ligand interface in mediating receptor and cofactor function.

SUBMITTER: Kelly CR

PROVIDER: S-EPMC1217163 | biostudies-other | 1996 Apr

REPOSITORIES: biostudies-other

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Structure-Function Relationship of the Interaction between Tissue Factor and Factor VIIa.

Project description:Interactions between tissue factor and factor VIIa are the primary initiators of coagulation in hemostasis and certain thrombotic diseases. Tissue factor, an integral membrane protein expressed extensively outside of the vasculature, is the regulatory protein cofactor for coagulation factor VIIa. Factor VIIa, a trypsin-like serine protease homologous with other blood coagulation proteases, is weakly active when free in solution and must bind its membrane-bound cofactor for physiologically relevant activity. Tissue factor allosterically activates factor VIIa by several mechanisms such as active site positioning, spatial stabilization, and direct interactions with the substrate. Protein-membrane interactions between tissue factor, factor VIIa, and substrates all play critical roles in modulating the activity of this enzyme complex. Additionally, divalent cations such as Ca(2+) and Mg(2+) are critical for correct protein folding, as well as protein-membrane and protein-protein interactions. The contributions of these factors toward tissue factor-factor VIIa activity are discussed in this review.

| S-EPMC4862872 | biostudies-literature

Factor VIIa Regulates the Level of Cell-Surface Tissue Factor through Separate but Cooperative Mechanisms.

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| S-EPMC8345218 | biostudies-literature

Structural biology of factor VIIa/tissue factor initiated coagulation.

Project description:Factor VII (FVII) consists of an N-terminal gamma-carboxyglutamic acid domain followed by two epidermal growth factor-like (EGF1 and EGF2) domains and the C-terminal protease domain. Activation of FVII results in a two-chain FVIIa molecule consisting of a light chain (Gla-EGF1-EGF2 domains) and a heavy chain (protease domain) held together by a single disulfide bond. During coagulation, the complex of tissue factor (TF, a transmembrane glycoprotein) and FVIIa activates factor IX (FIX) and factor X (FX). FVIIa is structurally "zymogen-like" and when bound to TF, it is more "active enzyme-like." FIX and FX share structural homology with FVII. Three structural biology aspects of FVIIa/TF are presented in this review. One, regions in soluble TF (sTF) that interact with FVIIa as well as mapping of Ca2+, Mg2+, Na+ and Zn2+ sites in FVIIa and their functions; two, modeled interactive regions of Gla and EGF1 domains of FXa and FIXa with FVIIa/sTF; and three, incompletely formed oxyanion hole in FVIIa/sTF and its induction by substrate/inhibitor. Finally, an overview of the recognition elements in TF pathway inhibitor is provided.

| S-EPMC3383856 | biostudies-literature

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| S-EPMC5630012 | biostudies-literature

Tissue Factor Residues That Modulate Magnesium-Dependent Rate Enhancements of the Tissue Factor/Factor VIIa Complex.

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| S-EPMC4862878 | biostudies-literature

A serine loop in tissue factor mediates substrate selectivity by the tissue factor-factor VIIa complex.

Project description:Essentials How the tissue factor-factor VIIa complex selects between different substrates is not well understood. We investigated a serine loop in tissue factor and its role in substrate selectivity. The tissue factor serine loop is selective for factor X over factor IX. Substrate selectivity is facilitated by differential regulation of the nearby tissue factor exosite. ABSTRACT: Background The tissue factor-factor VIIa (TF-FVIIa) complex is the physiologic activator of blood clotting and plays a major role in many thrombotic diseases. TF-FVIIa drives clotting through proteolytic cleavage of its major protein substrates, factor IX (FIX) and factor X (FX). However, it remains unclear how TF-FVIIa exhibits selectivity between these substrates. We previously showed that TF residues adjacent to the putative substrate binding site of TF ("exosite") facilitate FX activation, but the role of these residues in substrate selectivity had not been tested. Objectives We hypothesized that a TF serine loop (residues S160-S163) mediates substrate selectivity by the TF-FVIIa complex. Methods We generated TF serine loop and exosite mutants. The mutants were tested in FIX and FX enzyme activation assays as well as thrombin generation assays. Results Changes in the length of the serine loop affected rates of FIX and FX activation very differently. FX activation was decreased by up to 200-fold when the loop length was changed by just one residue. In contrast, FIX activation was largely unaffected. Substrate selectivity was also detected in thrombin generation assays. Activation assays with TF serine loop and exosite double mutants revealed that the serine loop has no effect on the exosite during FIX activation. In contrast, the serine loop regulates the exosite during FX activation. Conclusions Our results provide new insights into how the TF-FVIIa complex actively selects between its major protein substrates, which is mediated by a TF serine loop.

| S-EPMC7790960 | biostudies-literature

Small peptides blocking inhibition of factor Xa and tissue factor-factor VIIa by tissue factor pathway inhibitor (TFPI).

Project description: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.

| S-EPMC3894350 | biostudies-literature

Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity.

Project description:The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.

| S-EPMC4813490 | biostudies-literature

Modulation of tissue factor-factor VIIa signaling by lipid rafts and caveolae.

Project description:ObjectiveCoagulation factor VIIa (VIIa) binding to its cellular receptor, tissue factor (TF), not only initiates the coagulation cascade but also induces cell signaling by activating G-protein coupled protease-activated receptors. The objective of the present study is to investigate the role of lipid rafts and caveolae in modulating TF-VIIa signaling and coagulant functions.Methods and resultsTF-VIIa coagulant function was measured in factor X activation assay and the signaling function was evaluated in phosphoinositide hydrolysis and IL-8 gene induction. Buoyant density gradient centrifugation and immunofluorescence confocal microscopy were used to determine cellular localization of TF and protease-activated receptor 2. The data show that a substantial fraction of TF and protease-activated receptor 2 resides in lipid rafts/caveolae, and disruption of lipid rafts by cholesterol depletion or modification reduced TF-VIIa-induced cell signaling. Disruption of caveolae with caveolin-1 silencing had no effect on the TF-VIIa coagulant activity but inhibited the TF-VIIa-induced cell signaling.ConclusionOverall our data show that lipid raft/caveolae play a selective role in modulating the TF-VIIa signaling function without affecting the TF-VIIa coagulant activity.

| S-EPMC2647778 | biostudies-literature

Effective function annotation through catalytic residue conservation.

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| S-EPMC1178014 | biostudies-literature

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