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Originally published In Press as doi:10.1074/jbc.M702410200 on July 5, 2007

J. Biol. Chem., Vol. 282, Issue 35, 25416-25424, August 31, 2007
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Tissue Factor Coagulant Function Is Enhanced by Protein-disulfide Isomerase Independent of Oxidoreductase Activity*

Henri H. Versteeg and Wolfram Ruf1

From the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, March 21, 2007 , and in revised form, July 5, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein-disulfide isomerase (PDI) switches tissue factor (TF) from coagulation to signaling by targeting the allosteric Cys186–Cys209 disulfide. Here, we further characterize the interaction of purified PDI with TF. We find that PDI enhances factor VIIa-dependent substrate factor X activation 5–10-fold in the presence of wild-type, oxidized soluble TF but not TF mutants that contain an unpaired Cys186 or Cys209. PDI-accelerated factor Xa generation was blocked by bacitracin but not influenced by inhibition of vicinal thiols, reduction of PDI, changes in redox gradients, or covalent thiol modification of reduced PDI by N-ethylmaleimide or methyl-methanethiosulfonate, which abolished PDI oxidoreductase but not chaperone activity. PDI had no effect on fully active TF on either negatively charged phospholipids or in activating detergent, indicating that PDI selectively acts upon cryptic TF to facilitate ternary complex formation and macromolecular substrate turnover. PDI activation was reduced upon mutation of TF residues in proximity to the macromolecular substrate binding site, consistent with a primary interaction of PDI with TF. PDI enhanced TF coagulant activity on microvesicles shed from cells, suggesting that PDI plays a role as an activating chaperone for circulating cryptic TF.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue factor (TF)2 initiates coagulation by binding and catalytically activating coagulation factor VIIa (VIIa) for efficient turnover of macromolecular substrate factor X (FX). TF plays an essential role in hemostasis (1) by serving as a hemostatic envelope around blood vessels and tissues (2). In addition, TF is present in blood, and microparticle-associated TF can be localized to thrombi and contribute to local fibrin formation (3, 4). TF also has nonhemostatic, signaling functions in angiogenesis, cancer progression, and inflammation that involve regulation of integrins and direct TF·VIIa signaling through protease-activated receptor 2 (5–7). We recently showed that cell surface protein-disulfide isomerase (PDI) is associated with inactive TF and targets the solvent-exposed, allosteric Cys186–Cys209 disulfide bond in TF to inhibit coagulation while maintaining direct TF·VIIa signaling (8). In addition, we found that a strong oxidant can activate TF procoagulant activity specifically when TF is present in a signaling complex with protease-activated receptor 2 and PDI. The thiol dependence of TF activation under these conditions (8, 9) indicated that oxidation or isomerization by PDI switches TF from a noncoagulant, reduced molecule to an active molecule with an oxidized allosteric disulfide.

However, the procoagulant activity of TF is regulated at multiple levels, including rapid feedback inhibition by TF pathway inhibitor (10) that directs TF to low density microdomains (11). A relatively small portion of total cellular TF is sufficient for procoagulant activity, and various amounts of noncoagulant or cryptic cell surface TF are present dependent on cell type, cellular differentiation, and proliferation (8, 12, 13). Location of TF to rafts or caveolae attenuates coagulant activity of TF (11, 14–16). On certain cells, TF may predominantly activate coagulation in cholesterol-rich microdomains (17). Release of TF on microvesicles appears to involve raft domains and is cholesterol-dependent, but TF requires activation dependent on fusion of microvescicles with activated platelets (16). In this and other systems (13), exposure of phosphatidylserine (PS) and increased availability of procoagulant lipids are important factors that contribute to coagulation that is initiated by TF. However, interference with redox and thiol pathways can enhance TF activity independent of PS (9, 18).

Mutational elimination of the allosteric Cys186–Cys209 disulfide abolishes TF coagulant function (8, 19). This disulfide is partially exposed to solvent and becomes buried under the Gla domain of VIIa when the TF·VIIa complex forms (20). The Cys186–Cys209 disulfide further stabilizes an extended loop that contains several residues involved in the binding of macromolecular substrate FX (21). Together, these structural features explain why affinity for VIIa is somewhat reduced and substrate FX activation is severely impaired upon breaking of the disulfide (8, 19). The activation of macromolecular substrate FX involves binding to an extended exosite that is formed by TF and a region in the VIIa protease domain distant from the catalytic center (21–23). Because these extended contacts are largely unaffected by the zymogen activation of FX to FXa (21), FXa retains a relatively high affinity for TF·VIIa (24, 25). As a consequence, product release can be a rate-limiting factor in substrate turnover by TF·VIIa. The rate-enhancing effect of PS on these reactions may be caused by facilitated substrate release (13, 26).

In addition to changes in PS exposure, TF activity in thrombosis may be regulated by PDI, because PDI is released from activated platelets and found associated with the platelet surface (27). PDI, a 64-kDa protein normally found in the endoplasmic reticulum, has both oxidoreductase and isomerase activity, thus facilitating thiol/disulfide exchange reactions during protein folding (28). In addition, PDI has chaperone function (29), and PDI has denitrosylase (30) and transnitrosylase activity that mediates cellular uptake of nitric oxide (NO) (31, 32). Cell surface PDI furthermore regulates platelet integrin activation (33–36). Because of the unique extracellular redox environment, cell surface PDI probably performs specific functions that are distinct from endoplasmic reticulum-localized PDI (37).

In this study, we show that PDI enhances TF procoagulant activity on microvesicles generated in vitro. In a defined system with purified proteins, we further demonstrate that PDI accelerates macromolecular substrate FX activation of soluble TF that is known to have low procoagulant activity (38). Our data show that PDI acts on oxidized TF independent of the oxidoreductase activity of PDI. Whereas PDI on cells inactivates the procoagulant activity of TF by NO-dependent pathways, the data presented here indicate that PDI also participates in activating cryptic TF through the chaperone activity of PDI. This study provides new insight into the interaction of PDI with the ternary TF initiation complex.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Bovine PDI, reduced (GSH) and oxidized (GSSG) glutathione, phenylarsenic oxide (PAO), methyl-methanethio-sulfonate (MMTS), N-ethylmaleimide (NEM), and bacitracin were purchased from Sigma. Bacitracin was repurified by gel filtration to eliminate enzymatic activity that degrades PDI (39). The NO-donor sodium nitroprusside was from Alexis (Carlsbad, CA), and N-(3-maleimidylpropionyl)biocytin (MPB) was from Invitrogen. The glutathione-reactive antibody ({alpha}GSH) was from Virogen (Watertown, MA).

Proteins—Monoclonal antibodies 5G9 and 10H10 to TF and polyclonal rabbit anti-TF cytoplasmic domain antibody have been previously described (8, 40, 41). Isotype-matched control antibody TIB115 to the viral large T antigen was obtained from ATCC. Soluble TF (TF-(1–218)) was expressed in Escherichia coli, as previously described (42, 43). Briefly, protein was prepared from inclusion bodies, solubilized, reduced, and refolded in 0.8 M guanidine HCl, 0.3 M NaCl, 50 mM Tris-HCl, pH 8.0, in a redox gradient of 2.5 mM GSH and 0.5 mM GSSG. Mutants with an unpaired Cys186 or Cys209 were refolded in the same buffer in the presence of sodium nitroprusside added to 2.5 mM at the beginning of the folding reaction and again 2 h later at the same concentration, based on initial observations that indicated better yield of monomeric mutants under these conditions. Refolded protein was recovered after extensive dialysis and concentrated, followed by gel filtration to separate monomeric mutant from higher molecular weight aggregates and dimers. Removal of the expression tag by thrombin was omitted to avoid degradation of the mutants. Wild-type soluble TF with an attached expression tag were used as controls. The TFK149A/D150A mutant was expressed as a fusion protein with a carboxyl-terminal leucine zipper domain (43), which was chosen to improve poor refolding efficiency of the mutant. Characterization of this mutant was in comparison with the same construct of wild-type TF. The VIIaDVQA mutant was kindly provided by Dr. Lars Petersen, Novo Nordisk. FX was purified from plasma (44), and VII contamination was eliminated by a subsequent immunopurification step, as previously described (22).

Detection of Free Thiols in Wild-type Soluble TF—Free thiol content of soluble wild type was determined by the Ellman reaction using Cys as a standard. The sensitivity of the assay would have detected the reduction of 1% of the allosteric disulfide in TF. Soluble TF was further labeled with polyethylene glycol-maleimide (PEG-mal), which reacts with free thiols to add 5 kDa per modified Cys residue on SDS-PAGE. Typically, soluble TF was labeled with 5 mM PEG-mal at 37 °C for 60 min in the presence or absence of reducing agents or PDI. Control reactions under denaturing (0.8% SDS) conditions showed no appreciable difference in the degree of PEG-mal labeling. In order to remove excess PEG-mal, TF was precipitated with acetone and resuspended in sample buffer for SDS-PAGE and Western blotting with polyclonal anti-TF.

Cell Culture and Generation of TF-positive Microparticles—Human umbilical vein endothelial cells (HUVEC) were passaged in endothelial cell basal medium, prepared with endothelial medium supplement (Cambrex, Walkersville, MD), and transduced with adenovirus encoding TF and protease-activated receptor 2, as previously described (8). After 2 days, cells were transferred to serum-free Medium 199, supplemented with 1 mM CaCl2, L-glutamine, and HEPES to collect released microparticles for 5 h. The supernatant was cleared from cellular debris at 14,000 x g for 10 min. Cleared supernatant was used without further manipulations for FXa generation assays or immunodepletion using Dynabeads coupled with anti-TF 5G9 or isotype-matched control TIB115. TF immunoprecipitated from 1 ml of supernatant was used for Western blotting with immunopurified, polyclonal goat anti-TF, or a rabbit anti-TF cytoplasmic domain antibody (40). Blots were digitized for densitometry by using Scion Image (Scion, Frederick, MD).

Chemical Modification of PDI with MMTS/NEM and PDI Functional Assays—PDI (16 µM) was reduced with 1 mM DTT for 1 h at ambient temperature. Reduced PDI was then reacted with 20 mM MMTS for 1 h followed by dialysis against HBS. Efficiency of MMTS modification was determined by labeling residual free thiols with 100 µM MPB for 30 min at ambient temperature, followed by Western blotting to detect biotin incorporation. For NEM modification, reduced PDI was reacted after 4-fold dilution with 1 mM NEM, followed by dialysis as above. Reductase activity of MMTS-treated PDI was measured by an insulin turbidity assay (45). Briefly, 800 nM PDI, PDI in the presence of 100 µM PAO or 3 mM bacitracin, or MMTS-modified PDI was added to a solution of 100 mM K2HPO4,2mM EDTA, 500 µM GSH, 75 µM insulin. Changes in insulin turbidity were measured for 20 min at 650 nm in a kinetic microplate reader. Chaperone activity of MMTS-modified PDI was determined by rhodanese aggregation assay with the following modifications (46). Rhodanese (1 mg/ml) was first denatured in 5.6 M urea and 10 mM DTT. Rhodanese was diluted 1:100 in 100 mM Tris, pH 8.0, to induce protein aggregation. PDI or MMTS-modified PDI (1 µM) were added, and protein aggregation was monitored at 405 nm over a 15-min time period.


Figure 1
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  		FIGURE 1.
PDI enhances TF procoagulant activity. A, Coomassie Blue-stained nonreducing SDS gels of wild-type (wt), TFC186A, and TFC209A soluble TF-(1–218). Proteins were expressed in E. coli and refolded, and monomeric soluble TF was obtained by gel filtration chromatography. B, reactivity of antibodies with purified wild type and TFC186A and TFC209A mutant soluble TF-(1–218) loaded at the indicated amounts. Blotting with a polyclonal antibody to TF-(1–218) confirms equal loading and blotting with anti-glutathione antibody ({alpha}GSH) shows that TFC186A but not TFC209A is prone to glutathionation during the refolding. Anti-TF 5G9, but not 10H10, has poor reactivity after reduction of the allosteric disulfide. C, labeling of free thiols in soluble TF with PEG-mal. Top, 200 nM soluble TF was incubated with 10 mM DTT or 150 nM PDI for 1 h prior to labeling with 5 mM PEG-mal. Bottom, 200 nM soluble TF was labeled with 5 mM PEG-mal at 37 °C in the absence or presence of 150 nM untreated or DTT-reduced PDI. VIIa was added at 200 nM where indicated. Western blots for TF are shown. D, PDI enhances coagulant activity of wild type TF but not single thiol mutants. Stoichiometric amounts of VIIa (25 nM) and of soluble wild-type TF, TFC186A,orTFC209A as well as a combination of TFC186A or TFC209A were incubated with or without 25 nM PDI and 1 µM FX. FXa generation after 30 min is shown. E, PDI-enhanced FXa generation requires TF, VIIa, and FX. FXa generation after 10 min is shown. F, PDI-enhanced TF coagulant activity is independent of TF glycosylation. PDI enhancement of FXa generation was analyzed with soluble TF (25 nM) expressed in E. coli, yeast or Chinese hamster ovary cells with VIIa (25 nM), PDI (25 nM), and FX (1 µM). G, PDI enhances FXa generation independent of the allosteric activation mechanism of VIIa. 25 nM VIIaDVQA has similar FXa generation activity as wild-type VIIa in the presence of soluble TF, but PDI has no effect on FXa generation by the free mutant. Inset, in the presence of TF-(1–218), VIIaDVQA-mediated FXa generation was enhanced by PDI. FXa generation after 3 min is shown.

 
FXa Generation and Amidolytic Assays—FXa generation assays were performed in HBS (10 mM HEPES, pH 7.4, 137 mM NaCl, 5.3 mM KCl, 1.5 mM CaCl2). Typically, 25 nM TF, VIIa, and PDI were used to activate 1 µM substrate FX, and aliquots were quenched in 20 mM Tris, pH 7.4, 150 mM NaCl, 100 mM EDTA at defined times for a chromogenic assay with Spectrozyme FXa. PDI inhibitors bacitracin (3 mM) and PAO (100 µM) were added at the start of the reaction. FXa generation assays using supernatant containing microparticles were performed with the same VIIa and FX concentrations, and PDI was added as indicated. Amidolytic activity of the TF·VIIa complex was assayed as described before (47). Briefly, complexes of the indicated concentrations of TF and VIIa were formed in HBS in the presence of 1 mM Spectrozyme FXa, and conversion of substrate by TF·VIIa was continuously monitored in a kinetic plate reader at 405 nM. All data are presented as means and S.D. from at least three repeat experiments.

TF Immunoprecipitation of Reaction Mixtures—25 nM TF-(1–218) and VIIa in the presence or absence of 25 nM PDI were incubated with 1 µM FX at 37 °C, in a total volume of 300 µl for 0, 5, 30, 60, or 90 min. Samples were quenched in 900 µl of 100 mM EDTA in HBS, and TF was precipitated with anti-TF 9C3 coupled to paramagnetic beads at 4 °C. TF, FX/Xa, and PDI in the immuno-precipitate were detected by Western blotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PDI Enhances TF·VIIa-dependent FXa Generation—In order to characterize the interaction of TF and PDI in a purified system, we expressed wild-type soluble TF or mutants that retained either Cys186(TFC209A) or Cys209 (TFC186A)of the allosteric disulfide. TF has an additional extracellular disulfide (Cys49–Cys57) that is buried and not required for function (19). SDS-PAGE showed homogeneity of the purified proteins (Fig. 1A). Because the refolding reaction contained glutathione, we addressed whether wild-type or mutants became glutathionated during refolding. Western blotting with anti-glutathione antibody demonstrated that no glutathione was incorporated into wild-type TF or TFC209A, but detectable in TFC186A, indicating that Cys209 is prone to glutathionation (Fig. 1B).

Disulfide formation of wild-type TF was confirmed by the Ellman reaction, which did not detect free thiols in wild-type TF. Reactivity of the conformation-sensitive antibody 5G9 to the carboxyl terminus of TF also indicated that the Cys186- Cys209 disulfide was intact. In addition, labeling of TF with PEG-mal, which increases the molecular mass of proteins by 5 kDa for each modified Cys residue, produced a molecular weight shift for only a small percentage of the wild-type soluble TF preparation (Fig. 1C), whereas 50% or more of the soluble TF preparation was modified after reduction with DTT. Note that competition of excess DTT over PEG-mal probably prevented complete modification of reduced TF. In addition, purified bovine PDI or VIIa had no appreciable effect on PEG-mal labeling of soluble TF, indicating that neither the physiological ligand nor PDI promote reduction of TF in solution.

The Cys186–Cys209 disulfide is necessary for binding of the VIIa Gla domain to TF, and consistently both soluble mutants showed decreased affinity for VIIa in an amidolytic assay that measures TF-dependent allosteric activation of VIIa catalytic activity. However, no differences were seen at saturating concentrations of mutants (data not shown). Both mutants had low activity in a macromolecular substrate FX activation assay, and the addition of purified PDI had no appreciable effect on FXa generation by the mutants alone or in combination (Fig. 1D), consistent with the accepted view that formation of the disulfide is required for efficient substrate FX binding. Unexpectedly, PDI enhanced substrate FX activation by wild-type soluble TF 5–10-fold. Elimination of VIIa or TF from the reaction showed that PDI was not contaminated with a protease or a cofactor that induced activation of FX independent of TF·VIIa (Fig. 1E).

PDI interaction with partially unfolded proteins is influenced by glycosylation, and TF is glycosylated at three N-linked sites in the extracellular domain (42). In order to exclude the possibility that the observed rate enhancing effect of PDI is an artifact of the nonglycosylated preparation of E. coli-derived soluble TF, we analyzed glycosylated soluble TF secreted from yeast or mammalian Chinese hamster ovary cell expression systems (38, 42). All three preparations had the same basal activity, and in each case PDI enhanced the activity to a similar extent (Fig. 1F). Eukaryotic soluble TF was not refolded but rather secreted from cells. These data also exclude the possibility that the refolding protocol for E. coli-derived TF introduced an artificial inactive state.

Although soluble TF has no defect in promoting the allosteric activation of VIIa catalytic activity toward small chromogenic substrates, macromolecular substrate binds to an exosite of the VIIa protease domain that is conformationally labile (23, 48, 49). One possibility was that PDI accelerated macromolecular substrate turnover by influencing allosteric activation of the VIIa exosite for macromolecular substrate. To address this issue, we first analyzed a mutant of VIIa (VIIaV21D/E154V/M156Q/K188A;VIIaDVQA) that is representative for a series of mutants that do not require allosteric activation by TF and activate FX efficiently in the absence of cofactor (50–52). PDI did not increase the activity of this mutant in the absence of TF but enhanced FXa generation when TF was added (Fig. 1G). These data show that PDI does not influence the allosteric activation of VIIa but rather has a specific effect on TF to enhance turnover of substrate FX.

PDI Specifically Targets Cryptic TF—Soluble TF has low activity that can be enhanced by phospholipids (38, 53). Increasing concentrations of phospholipids accelerated FXa generation by soluble TF and progressively diminished the rate enhancing effect of PDI (Fig. 2A). In addition, PDI had no effect on the procoagulant activity of full-length TF reconstituted into procoagulant lipid vesicles (Fig. 2B). These data show that PDI specifically acts on TF with low procoagulant activity.

On cells, the majority of TF is in a cryptic conformation with low procoagulant activity. TF can be activated by exposure to detergents, such as octyl glucopyranoside (OG). OG also increased the activation of FX by soluble TF·VIIa in a dose-dependent manner. The optimal concentration was 5–10 mM (not shown), which is similar to optimal OG concentrations required to activate cellular, cryptic TF (54). OG increased soluble TF activity to a similar extent as PDI, and the presence of both PDI and OG did not produce an additive effect on FXa generation (Fig. 2C). These data further support the concept that PDI selectively acts upon cryptic TF.

In cellular systems, cryptic TF has low affinity for VIIa as compared with fully active, procoagulant TF. We tested by amidolytic assay whether PDI or OG enhance the affinity of TF for VIIa. At saturating (50 nM) concentration of TF, neither PDI nor OG had an effect on TF·VIIa amidolytic activity (Fig. 2D). However, PDI, but not OG, enhanced the amidolytic activity of 2nM VIIa at a subsaturating (2 nM) concentration of soluble TF. These data indicate that PDI has a specific direct effect on TF, which results in enhanced affinity for VIIa. However, the increase in affinity cannot account for the rate-enhancing effect of PDI on FX activation, because the FXa generation assay was performed at nearly saturating concentrations of TF for VIIa.

The complex of VIIa and a site-specific mutant of TF (TFK149A/D150A) activated FX with similar efficiency as wild type TF when PDI was absent. However, the addition of PDI produced only a <2-fold enhancement of FXa generation in the case of the mutant (Fig. 2E). Control experiments with OG confirmed that detergent activation of the mutant was unaffected, excluding the possibility that the conformation of the mutant prevents activation to full procoagulant function. Taken together, these data provide evidence that PDI has a direct effect on cryptic TF to enhance FXa generation.

PDI Facilitates Ternary Complex Formation and Substrate Turnover—In a soluble system, the rate of FXa generation is dependent on the encounter of three reactants: cofactor, enzyme, and substrate. Consequently, the reaction rate will increase at a fixed enzyme concentration if cofactor concentration is raised to stoichiometry with substrate, as shown in Fig. 3A. This model predicts that the addition of cofactor that is devoid of enzyme binding cannot enhance the reaction rate. Indeed, a site-specific mutant of TF, TFD44A, that activates and binds VIIa poorly (55) did not enhance FXa generation when added at substrate concentration to 25 nM wild-type TF·VIIa complex (Fig. 3A). PDI may enhance ternary complex formation without accelerating dissociation of product or, alternatively, accelerate both aspects of substrate turnover. In the former case, one would expect that increasing TF concentrations progressively diminish the PDI effect, because ternary complex formation is close to optimal at saturating cofactor. However, the rate-enhancing effect of PDI was observed throughout the TF concentration range (Fig. 3B), indicating that PDI facilitates both ternary complex formation and product release.


Figure 2
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  		FIGURE 2.
PDI targets cryptic TF. A, the addition of phosphatidylcholine/PS vesicles overcomes PDI-mediated enhancement of TF procoagulant activity. Activation of 1 µM FX by 25 nM VIIa and TF-(1–218) with or without 25 nM PDI was characterized in the presence of increasing concentrations of phosphatidylcholine/PS (80/20, w/w) vesicles. FXa generation after 2 min is shown. B, PDI does not enhance activity of relipidated, full-length TF. FX (100 nM) activation by 0.1 nM TF reconstituted in 80% phosphatidylcholine, 20% PS vesicles and 10 nM VIIa was analyzed in the absence or presence of 25 nM PDI. C, PDI and activating detergent OG similarly enhance TF coagulant function. Activation of 1 µM FX by 25 nM TF-(1–218) and VIIa was analyzed without or with 25 nM PDI and/or 7.5 mM OG. D, PDI, but not OG, increases affinity of TF for VIIa. Amidolytic activities of VIIa (2 nM) with subsaturating concentrations (2 nM) or saturating concentrations (50 nM) of soluble TF were determined in the presence or absence of OG and/or PDI (50 nM). E, OG, but not PDI, induces activation of TFK149A/D150A. FXa generation by 25 nM VIIa and TFK149A/D150A or wild-type TF was analyzed in the presence of 25 nM PDI or 7.5 mM OG. Wild type (wt) and the TFK149A/D150A mutant, which shows inefficient folding, were expressed as carboxyl-terminal Leu zipper fusion proteins for improved refolding. Note that the carboxyl-terminal fusion increases FX activation by ~5-fold relative to soluble TF-(1–218), as previously characterized (43).

 
In order to directly demonstrate the association of TF and PDI, we subjected reactions to immunoprecipitations with anti-TF. TF, PDI, and FX/Xa in the immunoprecipitates were then detected by Western blotting. PDI co-precipitated with TF at the start of the reaction, but PDI did not influence the amount of FX that was initially associated with TF (Fig. 3C). In the absence of PDI, the faster migrating FXa accumulated in the TF immunoprecipitates. In contrast, FXa appeared to be more readily released from TF in reactions with PDI. At later times, FXa association with TF increased in the PDI reactions, which probably resulted from a combined effect of increased product concentration and reduced PDI binding to TF. The partial degradation of PDI in some samples suggested the possibility of PDI inactivation by prolonged exposure to proteases in the reaction. The substantial amount of FXa detected in the reactions without PDI indicates that the poor activity of soluble TF may in part be caused by slow product release. Taken together, these data support a mechanism by which PDI enhances ternary complex formation and facilitates the release of product FXa.

PDI Chaperone Function Is Sufficient to Enhance TF Coagulant Activity—We next determined whether PDI-enhanced FXa generation was dependent on its oxidoreductase, isomerase, or chaperone activity. Bacitracin inhibits all functions of PDI, which is thought to be an effect resulting from occupancy of the PDI hydrophobic pocket. Bacitracin completely blocked the effect of PDI on TF·VIIa-mediated FX activation without influencing TF function in the absence of PDI (Fig. 4A). Vicinal thiols are typically required for reductase and isomerase activity of PDI. However, blockade of vicinal thiols with PAO did not reduce coagulant activity in the presence of PDI. In control experiments, we confirmed that PAO also was without effect when PDI was reduced immediately prior to the assay.

In order to address the oxidoreductase function of PDI in this system further, we incubated PDI for 30 min in reducing (4:1 GSH/GSSG) or oxidizing (1:4 GSH/GSSG) gradients of glutathione. There was no appreciable effect of redox gradients on PDI-mediated enhancement of TF-dependent FXa generation (Fig. 4B). In addition, we preincubated soluble or relipidated TF with 1 mM DTT in the presence or absence of PDI for 1 h and determined FXa generation after dilution to 0.5 mM DTT. Reducing conditions diminished basal FXa generation for both soluble and relipidated TF, but DTT did not inhibit the rate-enhancing effect of PDI that is specific for soluble TF (Fig. 4C). PDI also maintained rate-enhancing activity after reduction with 8 mM DTT and dilution to 1 mM (data not shown). Taken together, these data indicate that reductase and oxidase function are not required for the rate-enhancing effect of PDI on TF.


Figure 3
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  		FIGURE 3.
PDI associates with the ternary complex. A, effect of saturating TF concentrations on FXa generation in solution. Activation of 1 µM FX by 25 nM VIIa was characterized at stoichiometric concentrations of TF with either enzyme or substrate using 25 or 1 µM wild-type TF-(1–218) or 1 µM VIIa binding-deficient soluble TFD44A (55) in the presence and absence of 25 nM wild-type (wt) TF-(1–218). B, PDI increases FX activation independent of the TF concentration. Activation of 1 µM FX by 25 nM VIIa with or without 25 nM PDI was determined in the presence of various TF-(1–218) concentrations. FXa generation at 4 min is shown. C, PDI co-precipitates with a TF·FX complex. Xa generation by 25 nM VIIa and TF in the presence or absence of 25 nM PDI was stopped after 0, 5, 30, 60, and 90 min. Co-precipitation of FX/Xa and PDI with TF was assessed by Western blotting.

 
To further address the role of free thiols in this reaction, we incubated each of the reactants separately with 20 mM MMTS, which in our previous experiments prevented TF activation by strong oxidants on cells (8, 9). After pretreatment with 20 mM MMTS, samples were diluted 100-fold into the FXa generation assay (Fig. 5A). Blocking free thiols of reactants individually did not inhibit the rate-enhancing effect of PDI. We further reduced PDI with 1 mM DTT and reacted free thiols with 20 mM MMTS, followed by extensive dialysis to remove MMTS. Quantification of residual free thiols by labeling with MPB showed that reduction with DTT led to a 2.5-fold increase in MPB labeling of PDI free thiols, whereas subsequent MMTS treatment abolished free thiols with >95% efficiency (Fig. 5B). MMTS-treated PDI was indistinguishable from unmodified PDI in enhancing TF-dependent FX activation (Fig. 5C). MMTS-modified PDI displayed significantly diminished reductase activity as determined by an insulin denaturation assay, and reductase activity of PDI was similarly inhibited by PAO or bacitracin (Fig. 5D). Importantly, MMTS-modified PDI retained chaperone activity similar to that of unmodified PDI toward denatured rhodanese (Fig. 5E). Because dithiolmethane catalytic Cys residues in PDI rapidly convert to disulfides (56), we further covalently modified DTT-treated PDI with NEM. MPB labeling confirmed that free thiols were efficiently blocked (Fig. 5F), and the rate enhancing effect of PDI was not diminished. These data provide additional evidence that oxidoreductase activity is not required for the effect of PDI on TF and suggest that PDI acts through its chaperone activity to enhance TF procoagulant activity.


Figure 4
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  		FIGURE 4.
PDI enhanced TF activity is not dependent on oxidoreductase activity. A, bacitracin, but not PAO, inhibits PDI-enhanced TF activity. Stoichiometric amounts of VIIa (25 nM) and soluble TF were incubated with or without 25 nM of PDI and 1 µM FX, in the presence or absence of 100 µM of the vicinal thiol blocker PAO or a 3mM concentration of the PDI inhibitor bacitracin. B, redox gradients do not affect PDI-mediated enhancement of TF-dependent FXa generation. PDI was preincubated for 30 min in control buffer or redox gradients (6.4 mM GSH plus 1.6 mM GSSG or 1.6 mM GSH plus 6.4 mM GSSG) and then diluted to the indicated final concentration for a reaction with 25 nM VIIa and soluble TF with or without 25 nM PDI and 500 nM FX. FXa generation after 10 min is shown. C, reduction does not abolish the enhancing effect of PDI on TF activity. Soluble (100 nM) or relipidated (0.5 nM)TF was incubated with 1 mM DTT in the presence or absence of PDI for 1 h. VIIa (100 and 10 nM, respectively) and substrate FX (2 µM) were added, and FXa generation was determined after 1 min.

 


Figure 5
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  		FIGURE 5.
PDI enhances TF coagulant activity through chaperone function. A, PDI-enhanced FXa generation is not dependent on preexisting free thiols in TF, VIIa, PDI, or FX. Soluble TF, VIIa, PDI, or FX were pretreated with 20 mM MMTS and diluted 100-fold into the final reaction. 25 nM TF and VIIa in the absence of PDI (no PDI) or presence of PDI (control) were used to activate 1 µM FX. Reactions in the presence of PDI were also performed after pretreatment of each reactant. B, free thiol detection in reduced and MMTS-modified PDI. PDI was reduced with 1 mM DTT and subsequently reacted with 20 mM MMTS. Free thiols were detected in nontreated and DTT- and MMTS-treated PDI by labeling with 100 µM MPB and Western blotting with horseradish peroxidase-conjugated streptavidin (SA-HRP). Densitometric quantitation is shown, mean and S.D., n = 3. C, effect of MMTS-modified PDI on TF-dependent FXa generation. Activation of 1 µM FX by 25 nM TF-(1–218) and 25 nM VIIa was analyzed in the absence or presence of 25 nM unmodified PDI, PDI reduced with 1 mM DTT, or MMTS-modified PDI. D, oxidoreductase activity was determined by an insulin turbidity assay in the presence of 800 nM MMTS-modified PDI or unmodified PDI in the absence or presence of 100 µM PAO or 3 mM bacitracin. E, MMTS-modified PDI retains chaperone activity. Chaperone activities of 1 µM PDI and MMTS-modified PDI were compared by a rhodanese aggregation assay. F, effect of blocking free thiols of PDI with NEM on TF-dependent FXa generation. Free thiols after NEM modification were quantified by densitometry of MPB labeling. Activation of 1 µM FX by 25 nM TF-(1–218) and VIIa was analyzed in the absence or presence of 25 nM unmodified PDI or NEM-modified PDI.

 
PDI Enhances Microparticle TF Procoagulant Activity—PDI specifically enhanced the activity of cryptic TF. TF in microvesicles has low activity, and TF in such a membrane environment may represent a likely target for PDI. To test this hypothesis, we generated microparticles under serum-free conditions from HUVECs in which TF was overexpressed by adenoviral transduction. Supernatants were cleared from cellular debris, and TF shedding into the supernatant was confirmed by immunoprecipitation of TF by anti-TF antibody coupled to paramagnetic beads (Fig. 6A). Western blotting with an antibody against the TF cytoplasmic domain showed that full-length TF is released into the supernatant. Detection of flottilin specifically in the TF immuno-precipitate is consistent with a previous study (16) that suggested a shedding mechanism of TF from raft domains.

Supernatants were used for an FXa generation assay in the presence or absence of PDI. PDI dose-dependently increased FXa generation in microvesicle-containing culture supernatant (Fig. 6B). Depletion of TF from the supernatant by monoclonal antibody beads prevented FXa generation both in the absence and presence of PDI (Fig. 6C), demonstrating that shed TF is the primary target for PDI. Consistent with the data using purified soluble TF, blockade of free thiols by MMTS or of vicinal thiols by PAO did not reduce the enhancing effect of PDI on TF coagulant activity (Fig. 6D). These data further support our overall conclusion that PDI specifically regulates the activity of cryptic TF.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We had identified PDI as a regulator of TF functions on cells, but how PDI contributes to activation of cryptic, noncoagulant forms of TF remains incompletely understood. Here, we show in a purified protein system that PDI can enhance coagulant activity of oxidized TF. Blockade of PDI free thiols, which are essential for the oxidoreductase and isomerase function of PDI, did not reduce PDI chaperone function. Nevertheless, MMTS- and NEM-modified PDI fully enhanced TF-dependent FXa generation, indicating that chaperone function of PDI is sufficient for enhancement of TF coagulant activity. The rate-enhancing effect of PDI is dependent on specific TF residues that either directly or indirectly support the interaction with PDI. Unlike detergent activation, PDI not only facilitates substrate turnover but also enhances affinity for VIIa. High affinity for VIIa and efficient FX turnover are hallmarks of procoagulant TF. Thus, these data strongly implicate PDI as a physiologically relevant regulator for the conversion of cryptic to coagulant TF.


Figure 6
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  		FIGURE 6.
PDI enhanced TF coagulant activity on microparticles. A, release of full-length TF into the supernatant of HUVECs. Serum-free culture supernatant from TF transduced HUVEC was immunoprecipitated with immobilized 5G9 or isotype-matched control antibody (TIB115), and TF immunoprecipitates were analyzed by Western blotting with polyclonal anti-TF, anti-TF cytoplasmic domain antibody, or anti-flottilin (FLT) antibody as a marker for raft domains. The arrow indicates the correct molecular mass of FLT (48 kDa). B, PDI dose-dependently enhances TF activity on microparticles. Supernatant containing shed TF was incubated with 10 nM VIIa and 100 nM FX in the presence of increasing concentrations of PDI. FXa generation after 15 min is shown. C, PDI specifically targets TF on microvesicles. Microparticle-containing supernatants were depleted using immobilized 5G9 or control beads and analyzed for FXa generation in the presence of 100 nM PDI. D, PDI chaperone function is sufficient for microparticle TF activation. Supernatant containing TF was analyzed for FX (100 nM) activation by VIIa (10 nM) in the absence or presence of 25 nM MMTS-treated PDI or unmodified PDI with or without 100 µM PAO. FXa generation was determined after 15 min.

 
The chaperone function of PDI is generally thought to be independent of vicinal, catalytic thiols. Chemical thiol blockade by methylation or mutation of vicinal thiols in PDI does not impair chaperone function (57, 58). This is consistent with our finding that chemical blockade of PDI vicinal thiols with MMTS did not block PDI chaperone activity (Fig. 5E). Although modification of PDI free thiols with MMTS may result in the spontaneous oxidation of vicinal thiols (56), we show that chemical blockade of PDI with NEM, which irreversibly modifies free thiols, similarly did not block the rate-enhancing effect of PDI on FXa generation.

The presented data demonstrate that PDI acts on oxidized TF with an intact allosteric Cys186–Cys209 disulfide by thiol exchange-independent mechanisms. However, PDI also regulates TF activity through thiol-dependent pathways. On cells, blockade of free thiols can increase TF activity, and these changes are independent of procoagulant PS exposure (9, 18), indicating that PDI dynamically inactivates a proportion of TF through reductive modification. Consistent with this notion, blockade of free thiols with MMTS can prevent the activation of noncoagulant, PDI-associated pools of TF when the redox environment of the cell surface is changed by strong oxidants (8, 9).

Using the immortalized keratinocyte HaCaT, we found that incubation with GSH did not reduce TF coagulant activity (8), indicating that PDI does not act simply as a reductase for TF on the cell surface. In the presented purified system, this concept is further supported by the finding that neither DTT nor changes in redox gradients resulted in inactivation of TF by PDI. However, TF activity on cells was diminished by incubation with GSH in the presence of the nitric oxide donor sodium nitroprusside, suggesting a role for S-nitrosylation to regulate TF activity. The experimental system presented here should be applicable to further studies that aim to elucidate the NO-dependent reaction by which PDI inactivates TF.

Macromolecular substrates make extended contacts with the TF·VIIa complex, and conversion to product has little impact on these contact sites (21). Our data show that PDI reduces FXa that coprecipitates with TF, suggesting a mechanism of PDI action that involves facilitated dissociation of the product complex. This model is consistent with the finding that PDI has little effect on where product release is facilitated by lateral diffusion of lipids (13, 26). PDI regulation of TF activity is therefore of significance only in cellular microdomains with low PS content, such as rafts, or on relative quiescent and nonapoptotic cells (11, 13–15, 17). In addition, TF can be shed from raft domains in a poorly coagulant form that becomes active in the presence of stimulated platelets (16). Upon activation of platelets, up to 20% of total platelet PDI is locally secreted (59), and up-regulation of TF activity in association with activated platelets may depend on secreted PDI.

Our data suggest a mechanism that can activate cryptic pools of TF on shed microparticles. Platelets also express PDI on the cell surface (27), which emphasizes that the activation of TF may begin prior to the exposure of procoagulant phospholipids during platelet activation. NO regulates redox pathways during platelet activation (60). The finding that PDI may inactivate TF in the presence of NO donors under reducing conditions (8) indicates additional layers of regulatory control. These may become important for the extent of TF-driven thrombosis, depending on whether platelets are activated on NO-producing endothelial cells versus exposed vessel wall matrix components. Taken together, our study provides new evidence that PDI not only switches the specificity of TF functions on cells but can serve as a modulator in trans to activate cryptic pools of circulating TF of relevance for thrombosis and hemostasis.


   FOOTNOTES
 
* This work was supported by NHLBI, National Institutes of Health, Grant HL-31950 (to W. R.) and American Heart Association Grant 0625192Y (to H. H. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037.Tel.: 858-784-2748; Fax: 858-784-8480; E-mail: ruf{at}scripps.edu.

2 The abbreviations used are: TF, tissue factor; DTT, dithiothreitol; HUVEC, human umbilical vein endothelial cell; MPB, N-(3-maleimidylpropionyl)-biocytin; MMTS, methyl-methanethiosulfonate; NEM, N-ethylmaleimide; PAO, phenylarsine oxide; PDI, protein-disulfide isomerase; OG, octyl glucopyranoside; PS, phosphatidylserine; VIIa, factor VIIa; FX, factor X; PEG-mal, polyethylene glycol-maleimide; HBS, Hepes-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Pablito Tejada, Jennifer Royce, Cindi Biazak, and David Revak for excellent assistance. We thank Dr. Lars Petersen for providing the factor VIIa mutant.



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