Biochimica et Biophysica Acta (BBA) - General Subjects
Disulfide reduction abolishes tissue factor cofactor function
Highlights
► The reduction of tissue factor (TF) disulfides abolishes TF cofactor function. ► Reduced TF binds to factor VIIa but does not increase factor VIIa activity. ► Treatment of reduced TF with PDI does not restore TF function.
Introduction
Tissue factor (TF) contains five cysteines (Cys), four of them (Cys49, Cys57, Cys186, Cys209) reside in the extracellular domain and one (Cys245) in the cytoplasmic domain. Two disulfide bridges between Cys49–Cys57 and Cys186–Cys209 have been reported [1]. Over twenty years ago, Bach et al. suggested that preservation of these disulfides is necessary for the proper folding and activity of TF [2]. Based on mutagenesis studies, a non-functional role has been assigned to the NH2-terminal disulfide between Cys49–Cys57 [3]. The formation of the Cys186-Cys209 bridge has been hypothesized to account for the “decryption” of TF during which reduced TF is oxidized and emerges from its cryptic form to the fully active decrypted form [4].
The C-terminal cysteine bridge Cys186–Cys209 of the extracellular domain of TF has been hypothesized to be an allosteric disulfide, which controls protein function by triggering conformational changes upon its reduction or oxidation [4]. Unlike a catalytic disulfide bond, which enzymatically mediates thiol-disulfide interchanges in substrate proteins, the hypothesized allosteric bond changes the intra- or inter-molecular protein structure [5]. The subsequent change in TF conformation is hypothesized to affect the intermolecular interactions between TF, an enzymatic component of the extrinsic factor (F)Xase, FVIIa, and the natural substrate FX, leading to altered dynamics of FX activation and consequential thrombus formation [6].
While there is common agreement about the leading role of TF in the initiation of blood coagulation in vivo, there are significant controversies related to the expression and regulation of TF activity on the cell surface. It has been suggested that the majority of TF molecules located on the cell surface have low activity or are “encrypted” and that “decryption” is essential for the expression of TF function [7]. Several mechanisms, often contradictory, have been suggested in an attempt to explain “encryption/decryption” of TF function [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. More recently, the role of the Cys186–Cys209 bond in the “encryption/decryption” phenomenon was suggested [4]. The presumed formation of this bond using an oxidizing agent (HgCl2) increased TF-related FVIIa activity, although the subject of the disulfide bridge formation between two unpaired cysteines by this treatment remains controversial. In studies published previously, it was concluded that HgCl2 can modify only a single thiol group [19], [20]. Moreover, an increase in TF activity on cell surfaces similar to that caused by HgCl2 can be achieved by treating TF-bearing cells with other metal compounds, such as silver nitrate and phenylmercuric acetate [21]. Additionally, several studies showed that such increase in TF function is related to the elevated exposure of phosphatidylserine (PS) [21], [22] on cell surface upon treatment with HgCl2. More recent data from Hogg's laboratory, however, suggest that HgCl2 can possibly trigger the Cys186–Cys209 bond formation in TF [23].
Protein disulfide isomerase (PDI) has been suggested as an important player in the oxidation and reduction of Cys186–Cys209 disulfide bond and consequently in the enhancement and reduction of TF activity, respectively [24], [25]. Recently, Furlan-Freguia et al. [26] and Liang et al. [23] reported their observations in support of the oxidation of Cys186 and Cys209 and its contribution to TF function. Furlan-Freguia et al. described a pathway through which TF procoagulant activity is generated via a PDI mechanism. Liang et al. studied the redox potential and spacing of the two cysteines, suggesting that TF activators enhance TF function through oxidation of Cys186 and Cys209. In contrast to these publications, lack of influence of PDI on TF function has also been reported [27], [28]. Moreover, Bach and Monroe reported that the TF Cys186–Cys209 bridge is inaccessible to PDI manipulation when the cofactor is bound to the enzyme FVIIa. As a consequence of these conflicting studies, a review on PDI and TF activity concludes that the topic itself remains “cryptic” [29].
In the current study, we analyzed the status of oxidized, reduced and reduced-carboxyamidomethylated cysteines in human placental TF (pTF) and recombinant TF (rTF1–263) proteins and evaluated their effect on membrane independent fluorogenic substrate hydrolysis and membrane dependent FXa generation. Mass spectrometry was used to assess the status of the cysteines. Our data allowed a conclusion that reduction of TF cysteines eliminates TF cofactor function in the TF/FVIIa complex.
Section snippets
Proteins
rTF1–263 was a gift from Dr. Jenny and sheep anti-human TF polyclonal antibody (Ab) was purchased (Haematologic Technologies Inc, Essex Junction, VT). Anti-TF-5 monoclonal antibody (mAb) and anti-FVII-1 mAb were produced and purified in house. rFVIIa was a gift from Dr. Hedner (Novo Nordisk, Denmark). Human FX was isolated from fresh frozen plasma using an anti-FX mAb-coupled Sepharose [30]. Streptavidin-horse radish peroxidase (HRP), HRP-goat anti-mouse Ig and bovine serum albumbin (BSA) were
Sample preparation
The extent of reduction, as calculated by MS fractional abundance (represented as % of reduced species in a given sample), of TF proteins at 25 °C and 57 °C with 8 mM DTT and 20 mM IAA was 32% and 36% of reduced species, respectively (Table 1). The percent of reduced species increased to 66% in a sample reduced at 37 °C followed by dialysis at pH 6.0. Increasing DTT to 20 mM (37 °C followed by dialysis in pH 6.0) and IAA to 50 mM did not increase the fractional abundance of the reduced species (50%).
Discussion
The data of this study indicate that: 1) cysteines of the extracellular domain of TF play a role in TF cofactor function; 2) in freshly purified human pTF, cysteines of the extracellular domain exist in the oxidized (disulfide) form, as no alkylation is detectable without prior chemical reduction of the protein. However, it does not rule out the possibility that in a native environment these cysteines could exist in a completely or partially reduced form with oxidation occurring during pTF
Acknowledgements
This work was supported by P01 HL46703 and by 8P20GM103449 grants from the National Institutes of Health. We thank Drs. Ula Hedner and Rick Jenny for providing rFVIIa and rTF1–263. We also thank Matthew Gissel for his help in data analyses.
References (42)
- et al.
Purification and characterization of bovine tissue factor
J. Biol. Chem.
(1981) - et al.
The integrity of the cysteine186-cysteine209 bond of the 2nd disulfide loop of tissue factor is required for binding of factor VII
J. Biol. Chem.
(1991) - et al.
Allosteric disulfide bonds in thrombosis and thrombolysis
J. Thromb. Haemost.
(2006) - et al.
The effect of calcium ionophore A23187 on tissue factor activity and mRNA in endothelial cells
Thromb. Res.
(1994) - et al.
Calcium ionophore-induced de- encryption of tissue factor in monocytes is associated with extensive cell death
Thromb. Res.
(2007) - et al.
Fibroblast tissue factor: calcium and ionophore induce shape changes, release of membrane vesicles, and redistribution of tissue factor antigen in addition to increased procoagulant activity
Blood
(1994) - et al.
Lipid rafts are necessary for tonic inhibition of cellular tissue factor procoagulant activity
Blood
(2004) - et al.
Acute cholesterol depletion impairs functional expression of tissue factor in fibroblasts: modulation of tissue factor activity by membrane cholesterol
Blood
(2005) - et al.
Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site
J. Biol. Chem.
(1989) - et al.
Response: Tissue factor de-encryption: the cell model system
Blood
(2008)
Tissue factor activation: is disulfide bond switching a regulatory mechanism?
Blood
Bovine protein disulfide isomerase-enhanced tissue factor coagulant function: is phospholipid contaminant in it the real culprit?
Blood
Role of PDI in regulating tissue factor: FVIIa activity
Thromb. Res.
Role of the membrane surface in the activation of human coagulation factor X
J. Biol. Chem.
Carbohydrates and activity of natural and recombinant tissue factor
J. Biol. Chem.
Complete abolishment of coagulant activity in monomeric disulfide-deficient tissue factor
Blood
Cystine 186-cystine 209 disulfide bond is not essential for the procoagulant activity of tissue factor or for its de-encryption
Blood
Human tissue factor contains thioester-linked palmitate and stearate on the cytoplasmic half-cysteine
Biochemistry
Evidence for activation of tissue factor by an allosteric disulfide bond
Biochemistry
Allosteric disulfide bonds
Biochemistry
Tissue factor encryption
Arterioscler. Thromb. Vasc. Biol.
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