TF (tissue factor) is a transmembrane cofactor that initiates blood coagulation in mammals by binding Factor VIIa to activate Factors X and IX. The cofactor can reside in a cryptic configuration on primary cells and de-encryption may involve a redox change in the C-terminal domain Cys186–Cys209 disulfide bond. The redox potential of the bond, the spacing of the reduced cysteine thiols and their oxidation by TF activators was investigated to test the involvement of the dithiol/disulfide in TF activation. A standard redox potential of −278 mV was determined for the Cys186–Cys209 disulfide of recombinant soluble TF. Notably, ablating the N-terminal domain Cys49–Cys57 disulfide markedly increased the redox potential of the Cys186–Cys209 bond, suggesting that the N-terminal bond may be involved in the regulation of redox activity at the C-terminal bond. Using As(III) and dibromobimane as molecular rulers for closely spaced sulfur atoms, the reduced Cys186 and Cys209 sulfurs were found to be within 3–6 Å (1 Å=0.1 nm) of each other, which is close enough to reform the disulfide bond. HgCl2 is a very efficient activator of cellular TF and activating concentrations of HgCl2-mediated oxidation of the reduced Cys186 and Cys209 thiols of soluble TF. Moreover, PAO (phenylarsonous acid), which cross-links two cysteine thiols that are in close proximity, and MMTS (methyl methanethiolsulfonate), at concentrations where it oxidizes closely spaced cysteine residues to a cystine residue, were efficient activators of cellular TF. These findings further support a role for Cys186 and Cys209 in TF activation.
TF (tissue factor) is the primary initiator of blood coagulation in mammals . The cofactor is a transmembrane receptor that binds the blood coagulation serine protease Factor VIIa to activate Factors X and IX by discrete proteolysis . Binary TF–Factor VIIa and ternary TF–Factor VIIa–Factor Xa complexes can also cleave and activate plasma membrane protease-activated receptor 2 . The activated receptor then initiates intracellular signalling events that play a role in inflammation, tumour progression and angiogenesis.
TF is a member of the class II cytokine superfamily. The extracellular part consists of two fibronectin type III domains that are made up of two antiparallel β-sheets. The N-terminal domain contains a typical structural disulfide bond (Cys49–Cys57) that links across the two β-sheets. The disulfide bond in the C-terminal (Cys186–Cys209) or membrane-proximal domain, however, is unusual (Figure 1). It straddles the F and G strands of the same antiparallel β-sheet with what has been called a cross-strand bond [4,5]. Cross-strand disulfide bonds typically have a configuration known as the –RHStaple. This is one of 20 possible disulfide bond configurations defined by the five bond angles of the cystine residue . A defining feature of –RHStaple bonds is the close proximity of the α-carbon atoms of the two cysteine residues, which can impart a significant strain on the bond [4,6]. The –RHStaple disulfide bond has been associated with an emerging class of functional disulfide bond known as the allosteric disulfide [6,7]. For example, the functional disulfides in the immune co-receptor, CD4 , and the HIV envelope protein, gp120 , are –RHStaple bonds.
Ribbon structure of the extracellular part of TF (sTF)
The Cys186–Cys209 disulfide bond has been implicated in the functioning of TF [10,11]. In primary cells, the cofactor is usually present on the cell surface in a low activity or cryptic state that is activated by cellular disruption or exposure to calcium ionophores. TF is mostly constitutively active, however, on transformed cells or when the protein is transfected into cells . We have proposed that the Cys186 and Cys209 thiols are unpaired in cryptic TF and de-encryption involves oxidation of the thiolate ions to form the disulfide bond. A number of observations, albeit indirect, support this proposal. There are conformational changes in the vicinity of the Cys186–Cys209 disulfide bond upon activation of TF (reviewed in ) and an intact bond is required for efficient TF coagulant activity [11,13,14]. Cryptic TF contains unpaired cysteine thiols that are diminished upon activation of the cofactor, and cysteine thiols are involved in de-encryption as thiol-alkylating compounds block TF activation, while thiol-oxidizing compounds and dithiol cross-linkers activate TF .
The endogenous thiol/disulfide-modifying molecules, PDI (protein disulfide-isomerase) [11,15,16], glutathione  and NO , have also been implicated in TF de-encryption. In particular, inhibitors of PDI, which is on the surface of platelets  and platelet microparticles , block TF-mediated thrombus formation in vivo [15,16]. PDI does not appear to control the redox state of the TF Cys186–Cys209 disulfide bond, however [19,20]. The redox model of TF de-encryption is controversial and other mechanisms have been proposed [21,22].
To better understand the role of the Cys186–Cys209 disulfide bond in TF function, we have studied its redox properties. The redox potential of the bond, the approximate spacing of the reduced cysteine thiols and their oxidation by TF activators were investigated.
MATERIALS AND METHODS
Recombinant TF protein
The cDNA encoding residues 1–219 of human TF was cloned into the BamHI and PstI sites of pQE30 (Invitrogen) to generate an N-terminally His6-tagged TF fragment. Cys49 and Cys57 or Cys186 and Cys209 were sequentially mutated to alanine using the QuikChange® XL site-directed mutagenesis kit (Stratagene). The integrity of the constructs was confirmed by automated sequencing. Expression and purification of the wild-type and mutant sTF (soluble TF) proteins was performed by the Protein Production Unit at Monash University, Clayton, Victoria, Australia. Briefly, the pQE30-TF constructs were transfected into Escherichia coli BL21(DE3) C41 cells and protein expressed in autoinduction medium at 28 °C overnight then for a further 24 h at 20 °C. The cells were lysed by sonication, and TF in the soluble fraction was purified by nickel-affinity chromatography followed by gel filtration on Sephacryl S-75. Protein purity of 95–97% was achieved for both wild-type and mutant sTF.
Determination of the redox potential of the sTF Cys186–Cys209 disulfide bond
Recombinant wild-type, C186A,C209A or C49A,C57A mutant sTF (residues 1–219) was incubated at 1 μM concentration with 0.1 M sodium phosphate buffer (pH 7) containing 0.1 mM EDTA (flushed with argon for 1 h to eliminate oxygen), 75 mM DTTox [oxidized DTT (dithiothreitol); 4,5-dihydroxy-1,2-dithiane; Sigma] and various concentrations of DTTred (reduced DTT; 1,4-dithiothreitol; Sigma) for 15 h at 30 °C. Reactions were quenched with 100 mM iodoacetamide, samples were resolved by SDS/PAGE using NuPAGE Novex 4–12% Bis-Tris gels and protein was stained with colloidal Coomassie Blue. The redox state of the Cys186–Cys209 bond was measured from the slower migration of the reduced sTF. The results were expressed as the ratio of reduced to oxidized protein and fitted to eqn (1):
where R is the fraction of reduced protein at equilibrium, A is the fraction of reduced TF in the recombinant protein preparation and Keq is the equilibrium constant. The standard redox potential (E0′) of the Cys186–Cys209 bond was calculated using the Nernst equation (eqn 2):
using a value of −307 mV for the redox potential of the DTT disulfide bond .
Labelling of the unpaired Cys186 and Cys209 thiols of sTF
Recombinant sTF (5 mg/ml) in 0.1 M sodium phosphate buffer (pH 7) containing 0.1 mM EDTA was incubated with 0.1 mM iodoacetamide for 24 h at room temperature (22 °C) to block any unpaired thiols in the sTF preparation and the unreacted iodoacetamide was separated from the protein using a PD-10 Sephadex G25 desalting column. The Cys186–Cys209 disulfide bond was reduced with 10 mM DTT at room temperature for 30 min and the unreacted DTT was removed using a PD-10 Sephadex G25 desalting column. The reduced sTF (1 μM) was incubated alone or with 10 μM HgCl2 for 20 min at room temperature in the phosphate buffer containing 2 mM CaCl2. GSAO [4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide] and GSAOB [4-(N-(S-(N-(6-((6-((biotinoyl)amino)hexanoyl)amino)hexanoyl)-glutathionyl)acetyl)amino)phenylarsenoxide] was prepared as described in . GSAOB (100 μM) or dibromobimane (100 μM; Sigma) was added and incubated at room temperature for 30 min. The sTF samples (30 μl) were resolved by 4–12% Bis-Tris SDS/PAGE under non-reducing conditions and either stained with colloidal Coomassie Blue and the UV fluorescence of the dibromobimane measured or the proteins were transferred on to PVDF membrane and blotted for biotin. Biotin-labelled sTF was blotted with streptavidin peroxidase (Dako) and detected using chemiluminescence (Pierce). In some cases, sTF protein was immunoblotted with 1 μg/ml polyclonal goat anti-human anti-TF antibody and a 1:4000 dilution of horseradish peroxidase-conjugated rabbit anti-goat antibody (Dako). Chemiluminescence was detected using Western Lightning Plus ECL solution (PerkinElmer) and blots were developed on Amersham Hyperfilm (GE Healthcare).
TF activity assays
The TF procoagulant activity of human Jurkat T-cells was measured by a one-stage clotting assay using a ST Art mechanical clot detection coagulometer (Diagnostica Stago). Cells were resuspended in fresh medium at 106 cells/ml the day before assaying. On the day of assay, cells were stimulated for 6 h with 1 μM PMA (Sigma) to induce TF expression, then washed three times with 10 mM Hepes, pH 7.4, buffer containing 137 mM NaCl, 5.38 mM KCl, 5.55 mM glucose and 12.5 mM CaCl2 (Hepes-buffered saline). Cells were resuspended in Hepes-buffered saline to 106 cells/ml, aliquots (200 μl) equilibrated at 37 °C for 3 min and then incubated with PAO (phenylarsonous acid) (0–1 mM), GSAO (0–5 mM) or MMTS (methyl methanethiolsulfonate) (0–10 mM) for 30 or 60 s. Clot formation was initiated by the addition of 100 μl of a 1:1 mixture of pooled normal human plasma and PBS. Control reactions with vehicle only (DMSO) were run in parallel. Clotting times were converted into arbitrary units of TF procoagulant activity from a standard curve constructed using Innovin (Dade Behring). Results are expressed as the change in TF activity.
Oxidation of the Cys186 and Cys209 thiols of sTF
The Cys186–Cys209 disulfide bond of sTF was reduced with 10 mM DTT at room temperature for 30 min and the unreacted DTT was removed using a PD-10 Sephadex G25 desalting column. The reduced sTF (1 μM) was incubated alone or with 100–200 μM HgCl2 for 20 min at room temperature in the phosphate buffer containing 2 mM CaCl2. Samples of the reactions were resolved on NuPAGE Novex 4–12% Bis-Tris gels under non-reducing conditions and stained with colloidal Coomassie Blue.
Results are presented as means±S.D. or S.E.M. All tests of statistical significance were two-sided, and P values < 0.05 were considered statistically significant.
RESULTS AND DISCUSSION
The standard redox potential of the Cys186–Cys209 disulfide bond was determined using DTTox and DTTred. Recombinant wild-type sTF (residues 1–219) or C186A,C209A mutant sTF was incubated with increasing ratios of DTTred to DTTox and the protein was resolved by SDS/PAGE and stained with colloidal Coomassie Blue (Figure 2A). The slightly slower migration of the reduced protein was used to calculate the ratio of reduced to oxidized sTF as a function of the ratio of DTTred to DTTox (Figure 2B). There was no change in the migration of the C186A,C209A mutant sTF in the presence of DTT (Figure 2A), indicating that reduction of the Cys186–Cys209 bond was being measured in this reaction. A standard redox potential of −278 mV was calculated using the Nernst equation.
Redox potential of the Cys186–Cys209 disulfide bond of sTF
Analysis of the DTT titration results indicated that ~10% of the recombinant wild-type sTF contained unpaired Cys186/Cys209 thiols (variable A in eqn (1), Figure 2B). This result was confirmed using 5,5′-dithiobis(2-nitrobenzoic acid) to quantify accessible free thiols in the sTF. A mean of 0.12 mol of thiols per mol of sTF was measured. These observations indicate that the Cys186–Cys209 bond does not form during maturation of a small fraction (≤10%) of the recombinant protein.
A standard redox potential of −278 mV for the Cys186–Cys209 bond is in the range of the standard potentials of three other –RHStaple disulfides; −229 mV for the Cys103–Cys109 DsbD (disulfide bond oxidoreductase D) bond , −241 mV for the Cys130–Cys159 CD4 bond  and −261 mV for the Cys149–Cys202 bond engineered into green fluorescent protein .
The effect of ablation of the N-terminal Cys49–Cys57 disulfide bond by mutagenesis on the redox potential of the Cys186–Cys209 disulfide bond was also determined. Removal of the N-terminal disulfide increased the redox potential of the C-terminal Cys186–Cys209 bond by 36 mV to −242 mV (Figures 2C and 2D). Presumably, ablation of the N-terminal bond transmits a conformational change into the C-terminal domain that impacts on the Cys186–Cys209 bond. The two disulfide bonds are separated by a distance of approx. 40 Å in the crystal structure, however, and the β-strands that the bonds link are not contiguous with the strand that links the two fibronectin type III domains (see Figure 1). It is therefore not apparent how cleavage of the N-terminal bond would influence the structure of the C-terminal domain. The bonds are on the same face of the molecule, however, which may underlie the influence of the N-terminal bond on the potential of the C-terminal bond. It is interesting to note that the Cys49–Cys57 disulfide bond was found to exist in bound and cleaved forms in one of the crystal structures [5,27]. Redox change of the N-terminal disulfide bond may be involved in the control of the redox state of the C-terminal bond, although there is currently no evidence for this in cellular TF. Ablation of the Cys49–Cys57 disulfide bond in cellular TF does not affect TF pro-coagulant activity .
The spatial proximity of the reduced Cys186 and Cys209 thiols of sTF was determined using As(III) or dibromobimane as molecular rulers for two closely spaced sulfur atoms. As(III) reacts with two closely spaced protein thiols, forming high-affinity cyclic dithioarsinite complexes in which both sulfur atoms are bonded to arsenic . The optimal spacing of cysteine sulfur atoms for reaction with As(III) is 3–4 Å [28,29]. Dibromobimane contains two equivalent bromomethyl groups that cross-link cysteine thiolates that are within 3–6 Å . The cross-linker is non-fluorescent in solution but becomes fluorescent (λex 385 nm, λem 477 nm) when both of its alkylating groups have reacted. The Cys186–Cys209 disulfide bond of sTF was reduced with DTT, the unreacted compound was removed by gel filtration and the reduced protein was incubated with As(III) (in the form of the biotinylated tripeptide tervalent arsenical compound GSAOB) (Figure 3A) or dibromobimane (Figure 3B). Both compounds reacted with reduced sTF, but not with oxidized protein or reduced sTF treated with HgCl2. These observations indicate that the unpaired Cys186/Cys209 thiols in sTF are within 3–6 Å of each other and are therefore close enough to reform the disulfide bond.
Spatial proximity of the reduced Cys186 and Cys209 thiols of sTF
HgCl2 is a very efficient activator of cellular TF [10,12]. We have proposed that it functions by oxidizing Cys186 and Cys209 to a cystine residue . This reaction chemistry is described in Figure 4(A). The hypothesis was tested by examining oxidation of the Cys186–Cys209 dithiol in sTF by HgCl2. Wild-type sTF was reduced with DTT, incubated with increasing concentrations of HgCl2, and oxidation of the Cys186–Cys209 disulfide bond measured by the disappearance of the slower-migrating reduced protein on SDS/PAGE (Figure 3B). There was dose-dependent oxidation of the Cys186/Cys209 dithiol with HgCl2, which has been proposed as the mechanism of activation of cellular TF .
HgCl2 mediates oxidation of the Cys186 and Cys209 thiols of sTF
The finding that As(III) can cross-link the Cys186 and Cys209 thiols (Figure 3A) suggested that this metalloid might activate cellular TF. The reaction of As(III) with two closely spaced cysteine thiols is described in Figure 5(A). The tervalent organoarsenical compound PAO (Figure 5B), but not the larger GSAO (Figure 5B), activated TF on Jurkat T-cells (Figure 5C). The extent of activation (~25-fold at 0.1 mM concentration) was comparable with that achieved with ionomycin (~15-fold) or HgCl2 (~20-fold). TF activation with PAO is in accordance with the activation by bismaleimides , which cross-link closely spaced cysteine thiols. It is possible that forced approximation of the Cys186/Cys209 thiols by PAO and the bismaleimides enables productive binding of Factor X and its efficient activation by TF–Factor VIIa. This theory is supported by the lack of activation with the larger GSAO. GSAO contains a tripeptide that may sterically restrict productive binding of Factor X to TF–Factor VIIa when it is bound to the Cys186–Cys209 dithiol. This finding further supports the hypothesis that TF activation involves oxidation of cysteine residues to a cystine residue.
Activation of cellular TF with a trivalent arsenical
The reaction of MMTS with two closely spaced cysteine thiols has two possible outcomes depending on its concentration relative to the protein thiols present. At high relative concentrations of MMTS, both thiol groups of the protein dithiol are converted into their methyldisulfide derivatives, whereas at lower MMTS concentrations, the protein is oxidized to its disulfide instead . This reaction chemistry is described in Figure 6(A). We have shown previously that a high concentration of MMTS (10 mM) blocks activation of cellular TF by HgCl2 or ionomycin [10,12]. The question we asked in the present study was whether lower concentrations of MMTS would activate TF. Indeed, 0.1 and 1 mM MMTS-activated cellular TF (Figure 6B) and the extent of activation (~25-fold at 0.1 mM concentration) was comparable with that achieved with ionomycin (~15-fold), HgCl2 (~20-fold) or PAO (25-fold).
Activation of cellular TF with MMTS
The results presented herein and previously [10,11] make a compelling case that TF activation involves oxidation of two cysteine residues to a cystine residue. The simplest explanation of the results is oxidation of the TF Cys186 and Cys209, and this is the hypothesis we have tested. Proof of their role, however, is reliant on a direct method of measuring the redox state of Cys186 and Cys209in vivo, which is currently not available. This is complicated by the fact that TF is expressed at very low levels in untransformed cells as only a few molecules of the cofactor are required to initiate coagulation because of its catalytic nature. We cannot rule out that the redox change occurs in or between other proteins that participate in TF activity. There is currently no evidence that the known TF-interacting proteins (Factors VII, X and IX) undergo redox changes, though there may be an, as yet, unidentified protein that is involved in TF activation.
soluble tissue factor
Helena Liang and Teresa Brophy performed all of the experiments and contributed to the experimental design. Philip Hogg conceived the study and wrote the manuscript.
We thank Fi-Tjen Mu and Michael Berndt (Monash University, Melbourne, Australia) for construction of the wild-type and C186A,C209A mutant sTF expression vectors, and Neil Donoghue for advice on thiol chemistry.
This study was supported by grants from the National Health and Medical Research Council of Australia [grant number 455395] the Cancer Council NSW [grant number PG 06-07] and the Cancer Institute NSW.
These authors contributed equally to this work.