Disulfide reduction abolishes tissue factor cofactor function

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Abstract

Background

Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties.

Methods

The status of disulfides in recombinant TF1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function.

Results

In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function.

Conclusion

The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis.

General significance

Results of this study advance our knowledge on TF structure/function relationships.

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.

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