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Blood, 15 February 2005, Vol. 105, No. 4, pp. 1500-1507. Prepublished online as a Blood First Edition Paper on October 5, 2004; DOI 10.1182/blood-2004-02-0608.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY A role for the thiol isomerase protein ERP5 in platelet functionFrom the School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading, United Kingdom; and the Cardiovascular Division, King's College London, New Hunt's House, Guy's Campus, London, United Kingdom.
Formation and rearrangement of disulfide bonds during the correct folding of nascent proteins is modulated by a family of enzymes known as thiol isomerases, which include protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERP5), and ERP57. Recent evidence supports an alternative role for this family of proteins on the surface of cells, where they are involved in receptor remodeling and recognition. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding. Analysis of human platelet membrane fractions identified the presence of the thiol isomerase protein ERP5. Further study showed that ERP5 is resident mainly on platelet intracellular membranes, although it is rapidly recruited to the cell surface in response to a range of platelet agonists. Blocking cell-surface ERP5 using inhibitory antibodies leads to a decrease in platelet aggregation in response to agonists, and a decrease in fibrinogen binding and P-selectin exposure. It is possible that this is based on the disruption of integrin function, as we observed that ERP5 becomes physically associated with the integrin  3 subunit during platelet stimulation. These results provide new insights into the involvement of thiol isomerases and regulation of platelet activation.
In classical terms, reduction/oxidation systems within a cell have been represented very simply. The cytoplasmic environment is hypoxic and reducing, whereas the extracellular environment is normoxic and oxidizing. The generation of a disulfide bond from 2 cysteine residues is an oxidation reaction. To correctly generate these bonds inside the cell, there are, therefore, a group of enzymes known as the thiol isomerases. These are capable of the formation, reduction, and rearrangement of the disulfide-bonding patterns of proteins, often as part of folding of nascent proteins. The thiol isomerase enzymes are anchored to the endoplasmic reticulum via KDEL-receptor proteins.1-3 Recent studies have suggested additional functions for thiol isomerase enzymes: on the surface of cells, where they participate in receptor activation and remodeling, and substrate processing.4-6
Protein disulfide isomerase (PDI) is the best-characterized thiol isomerase to demonstrate this dual functionality. A number of cell types, including bovine aortic endothelial cells,7 rat hepatocytes,8,9 and human B cells,5,10 have been shown to secrete PDI, which associates with the cell surface. Cell-surface PDI has been implicated in the reduction of the disulfide-linked diptheria toxin heterodimer11 and events triggering the entry of HIV into lymphoid cells.6,12 On the basis of a series of investigations, initially by Detweiller and coworkers, a role for PDI in platelet physiology is now established.4,13-16 Early studies demonstrated PDI was present on the external membrane of activated and resting platelets, and proteins with thiol isomerase activity were secreted from activated platelets. Indeed, cell-surface exposure of free thiol groups, such as those from PDI, are elevated following platelet activation.17 Further studies demonstrated that inhibition of PDI with inhibitory antibodies can block a number of platelet responses, including aggregation, adhesion, fibrinogen binding, and integrin activation.16,18-20 Reagents that block cell-surface thiol groups, such as para-chloromercuriphenyl sulfonate, dithiobisnitrobenzoic acid, and bacitracin, have also been shown to inhibit these functions.19,21 This inhibition has often occurred to a greater degree than that observed for anti-PDI antibodies, indicating that additional proteins may be involved in this process.19,21 The reason for these observations has not been determined, although it has been proposed that they are based upon interaction with integrins, in particular integrins
In this study, we report the isolation of an additional thiol isomerase enzyme from human platelet membranes, which was identified as endoplasmic reticulum protein 5 (ERP5). Following platelet activation, levels of ERP5 on the platelet surface were rapidly elevated. Antibodies that block the thiol isomerase activity of ERP5 were found to inhibit platelet function. Notably, ERP5 was found to become associated with the integrin
Materials
Platelet agonists were collagen (Horm, type I from equine tendon; Nycomed, Munich, Germany); thrombin (Sigma, Poole, United Kingdom) and convulxin (convulxin was purified from snake venom and was a generous gift from Drs M. Leduc and C. Bon [Institut Pasteur, Paris, France]). Two venom preparations, with different potencies, were used. Horseradish peroxidase–conjugated secondary antibodies and the enhanced chemiluminescence detection system were from Amersham Biosciences (Buckinghamshire, United Kingdom); RNAse, from Roche (Lewes, United Kingdom); and bovine serum albumin, from First Link (Birmingham, United Kingdom). Anti- ERP5 purification Platelet membranes prepared from approximately 5 U blood (provided by Dr P. Smethurst, University of Cambridge, United Kingdom) were solubilized in buffer containing 1% (vol/vol) Triton X-100. Affinity chromatography was performed with 50 µg convulxin coupled to 200 µL sepharose 4B. Eluted fractions were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), Western blotted onto polyvinylidene difluoride (PVDF) membrane, and stained. Bands of approximately 50 kDa were subjected to protein sequencing by Edman degradation. Antibody generation Full-length ERP5 gene (cDNA clone provided by Prof M. Kikuchi, Ritsumeikan University, Japan)26,27 was cloned into the pGEX4T2 expression vector to generate an ERP5–glutathione S-transferase (ERP5-GST) fusion protein. Recombinant protein was located in inclusion bodies from Escherichia coli and isolated via urea solubilization and refolding to greater than 98% purity, as estimated by SDS-PAGE. Polyclonal antibodies were raised against the fusion protein and purified by means of protein G–sepharose. Specificity was determined by immunoblotting platelet lysates and comparing with anti-PDI, anti–calcium-binding protein 1 (anti-CaBP1) (anti-CaBP1 antibody to the rat homolog of ERP5 was a gift from Dr D. Ferrari, Max Planck Institute, Göttingen, Germany), and with antibody neutralized with recombinant ERP5. Polyclonal anti-PDI antibodies were raised in rabbits with the use of purified recombinant PDI (human PDI expression vector [pLWRP62] was provided by Dr L. W. Ruddock, Biocenter Oulu, Oulu, Finland). Preparation of highly purified platelet plasma membranes (PMs) and intracellular membranes (IMs) Platelet PMs and IMs were prepared as described in detail previously.28 Briefly, platelets were separated from human blood and treated with neuraminidase (type X, 0.05 U/mL) for 20 minutes at 37°C. After washing, platelets were disrupted by sonication, and the platelet homogenate was centrifuged at 42 000g for 90 minutes on a linear (1 to 3.5 M) sorbitol density gradient to obtain a mixed membrane (MM) fraction (free of granular contamination). MMs were separated into PMs and IMs by free-flow electrophoresis with an Octopus apparatus (FEE Weber, Planegg, Germany) running at 750 V, 100 mA. Two discrete peaks comprising PMs and IMs (more electronegative) were obtained. Tops of peaks were pooled, centrifuged (100 000g for 60 minutes), and resuspended in 0.4 M sorbitol, 5% glycerol, and 10 mM triethanolamine (pH 7.2). Preparation and stimulation of washed platelets Human platelets from drug-free volunteers were freshly prepared by differential centrifugation as described previously29 and resuspended in modified Tyrode/HEPES (Tyrode/N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, and 1 mM MgCl2, pH 7.3). Stimulation of platelets with collagen, convulxin, and thrombin was performed in an optical aggregometer (Chrono-log) at 37°C with continuous stirring. For platelet aggregation and flow cytometry studies, platelets were stimulated at 4 x 108/mL; for immunoprecipitation studies, platelets were stimulated at 1 x 109/mL. Where necessary, nonaggregating conditions were maintained by addition of EGTA (ethylene glycol tetraacetic acid) (1 mM), and secondary stimulation with released thromboxane A2 or secreted adenosine diphosphate (ADP) was prevented by inclusion of indomethacin (10 µM) and apyrase (2 U/mL), respectively. Coimmunoprecipitation Standard procedures for immunoprecipitation were followed.30 Platelets were lysed with ice-cold Nonidet P40 (NP-40) buffer (300 mM NaCl, 20 mM Tris [tris(hydroxymethyl)aminomethane], 10 mM EDTA [ethylenediaminetetraacetic acid], 2% NP-40, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 10 µg/mL leupeptin, 10 µg/mL aprotonin, and 1 µg/mL pepstatin A, pH 7.3). Precleared lysates were incubated with specific antibodies (1 µg) and protein A (or G)–sepharose at 4°C with rotation for 90 minutes. Following SDS-PAGE and Western blotting onto PVDF, immunoblotting was performed by means of standard procedures.29 Primary antibodies were used at a concentration of 1 µg/mL. Horseradish peroxidase–conjugated secondary antibodies were diluted 1:10 000. Blots were stripped and reprobed to verify equivalent levels of protein loading. Enhanced chemiluminescence (ECL) images were collected on x-ray film. Flow cytometry Platelets were stimulated with agonist in the presence of EGTA (1 mM), indomethacin (10 µM), and apyrase (2 U/mL). Stimulation was terminated by addition of modified Tyrodes/HEPES buffer containing 1% (wt/vol) bovine serum albumin, 1 mM EGTA, and 200 µM sodium azide. Primary antibody was added at appropriate dilutions, and the buffer was incubated for 1 hour on ice. Secondary antibody (fluorescein isothiocyanate–conjugated immunoglobulin G [IgG]) was used at 1:2000 dilution and incubated for 1 hour on ice in the dark. Data were collected and analyzed by means of a Becton Dickinson (Oxford, United Kingdom) FACScan flow cytometer and CELLQuest software. Fibrinogen binding
Fluorescein isothiocyanate (FITC)–labeled human fibrinogen binding was measured as for flow cytometry with the omission of EGTA from all buffers. Where required, samples were incubated with RNAse activity assay Thiol isomerase activity was assessed by the ability to renature reduced and denatured RNAse (rdRNAse). The assay was performed as outlined by O'Neill et al25 and Pigiet and Schuster.32 Reactivated RNAse was assayed by the degradation of cyclic 2'-3'cytidine monophosphate (cCytP), measured by increase in absorbance at 284 nm in refolding buffer (50 mM NaPO4, pH 7.2; 50 mM NaCl; 1.5 mM glutathione; 500 µM glutathione disulfide; 400 µM CaCl2; and 400 µM MgCl2). Controls of rdRNAse only, cCytP substrate only (blank), protein, and cCytP substrate but no rdRNAse were run with each set of experiments. The activity was expressed relative to native RNAse, or as a percentage of inhibition of activity for antibody-blocking experiments. Calcium mobilization assay Mobilization of calcium from intracellular stores was measured by means of a fast-filter ratiometric technique based on fluorescence of the calcium-binding compound Fura-2.31,33 Platelets (approximately 5 x 109/mL) were loaded with Fura-2-AM/HEPES (5 µM) and resuspended at 4 x 108/mL in Tyrodes/HEPES buffer.31,33 Prior to stimulation, EGTA (1 mM) was added to each sample. Data were collected on a Perkin Elmer (Beaconsfield, United Kingdom) LS50B fluorimeter and analyzed by means of FLWinLab software as described prevously.31,33 Data analysis Data were analyzed with SPSS (Chicago, IL) software to calculate 2-tailed paired t test at a 95% confidence value.
ERP5 is present in human platelets and is associated with intracellular and plasma membranes An unknown protein was purified from human platelet membrane fractions by means of a convulxin affinity column. Convulxin, isolated from the venom of the rattlesnake, possesses a high affinity for the platelet glycoproteins (GPs) GPVI and GPIb.34,35 N-terminal sequence data were obtained for the first 15 residues of the protein (LYSSSDDVIELTPSN). A BLAST search36 revealed this to be identical to the sequence of ERP5 following cleavage of a predicted signal sequence. ERP5 was cloned by Hayano and Kikuchi26 from a placental cDNA library while screening for proteins related to protein disulfide isomerase, PDI. The gene sequence encodes a 48-kDa protein containing 2 active thioredoxin domains (containing CGHC motifs) that share 47% amino acid sequence identity with human PDI, a C-terminal peptide-binding domain, and a KDEL sequence for retention in the endoplasmic reticulum.26 Sequence alignment studies by Ferrari and Soling (Ferrari and Soling2; Kramer et al37) suggest that ERP5 and PDI share a similar domain structure. Given its expected restriction to the ER, the presence of ERP5 in membrane fractions was confirmed by immunoblot analysis (not shown). The membrane association was examined more closely with the use of IMs and PMs from resting platelets isolated by free-flow electrophoresis (Figure 1A). ERP5 was present on both intracellular and plasma membranes. The IM fraction contains membranes of intracellular origin such as endoplasmic reticulum, but excludes granule membranes and surface-connected membranes. The higher loading of PMs indicates that the predominant location of ERP5 in resting platelets is the intracellular membranes. The purity of platelet membrane fractions was assessed by complete separation of the 2 peaks following free-flow electrophoresis, the absence of sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase 2b (SERCA 2b) in PM, and the absence of GP1b in IM (not shown). Full characterization of these fractions has been extensively reported previously.28,38 ERP5 was detected with the use of polyclonal antibodies raised to a GST-fusion protein containing full-length human ERP5. On Western blots (Figure 1B), a protein was detected of the same mobility as that recognized by antibodies to CaBP1, the rat homolog of ERP5 (not shown).37 These antibodies showed no cross-reactivity with human recombinant and platelet PDI (Figure 1B).
Flow cytometry was employed to confirm cell-surface expression of ERP5 and investigate whether this was a static or dynamic process. Washed platelets were stimulated with agonists convulxin, collagen, or thrombin, and the concentration- and time-dependent patterns of cell-surface exposure for ERP5 were studied (Figure 2). Low levels of cell-surface ERP5 were found to be substantially and rapidly increased following stimulation with each agonist in a concentration-dependent manner. To investigate the trends observed in time-dependence profiles, data were normalized to the greatest response within an individual experiment and averaged to overcome donor variability (Figure 2C). All 3 agonists demonstrate biphasic profiles, where there is an initial rapid increase in cell-surface exposure, which peaks at approximately 45 seconds, with substantial increases seen as rapidly as 15 seconds. For convulxin and thrombin, this is followed by a lag phase where exposure levels are maintained or dip slightly between 60 and 120 seconds before beginning to rise again over 150 to 300 seconds. These data suggest there are secondary effectors or mechanisms present for promoting a second wave of cell-surface exposure of ERP5 in response to these agonists. These experiments were performed in the presence of EGTA, apyrase, and indomethacin, which block the second wave of platelet aggregation responses based on fibrinogen, ADP, and thromboxane A2. Microaggregates based upon
ERP5 protein has thiol isomerase activity
Based upon refolding of reduced denatured RNAse, previous studies have demonstrated thiol isomerase activity for bovine liver ERP5, CaBP1, and human PDI.37 To verify that human ERP5 is a functionally active thiol isomerase, we analyzed the recombinant fusion protein in this assay system (Figure 3A). The protein was found to possess thiol isomerase activity, with activity approximately 70% of that measured for molar equivalents of the PDI recombinant fusion protein. Human ERP5 immunoprecipitated from platelet samples with the use of a nonfunction-blocking antibody also demonstrated thiol isomerase activity (not shown). It was found that, under the assay conditions employed, thiol isomerase activity of both ERP5-GST and PDI-histidine was dependent on divalent cations and inhibited in the presence of EDTA (not shown). This is opposite to the observed thiol isomerase activity profile for the integrin subunit
The effect on enzymatic activity of ERP5 by antibodies to ERP5 was investigated. Antibodies raised in sheep against recombinant ERP5 were found to inhibit enzyme activity, whereas preimmune IgG and monoclonal antibodies against human PDI displayed no effect on activity (Figure 3B). In addition, anti-ERP5 antibodies showed no inhibitory effect on the thiol isomerase activity of recombinant human PDI (not shown). It was not possible to completely block the thiol isomerase activity of ERP5-GST, even at very high antibody concentrations, consistent with studies performed on PDI with function-blocking antibodies. Platelet aggregation is inhibited by inhibition of ERP5 activity
Activity-blocking anti-ERP5 antibodies were used to investigate the potential involvement of ERP5 in the regulation of platelet function. Platelets were stimulated with collagen or convulxin following incubation with anti-ERP5 antibodies or control IgG purified from preimmune serum from the animal used to raise the antibodies. Prior to addition of inhibitory antibodies, platelets were incubated with saturating concentrations of F(ab') fragment of the monoclonal antibody IV.3 to prevent signaling through the Fc
Agonist-stimulated mobilization of calcium from intracellular stores was measured to determine whether anti-ERP5 antibodies affected platelet activation signaling itself and thereby reduced platelet activation. Mobilization of calcium from intracellular stores was not affected by incubation of platelets with anti-ERP5 antibodies (Figure 4C). Platelet activation signaling per se is therefore unaffected by anti-ERP5 treatment, but partial inhibition of ERP5 affects downstream functional responses that lead to aggregation. ERP5 is implicated in the regulation of fibrinogen binding
In view of the inhibition of aggregation observed following blockade of ERP5 and previous studies that have reported anti-PDI–mediated inhibition of fibrinogen binding,4,19 we investigated the ability of platelets to bind fibrinogen in the presence of inhibitory anti-ERP5 antibodies. Flow cytometry was used to measure the binding of FITC-labeled fibrinogen to collagen- and convulxin-stimulated platelets (Figure 5). A greater shift in fluorescence is observed by flow cytometry at higher agonist concentrations. Therefore, to increase the dynamic range for these experiments, higher agonist concentrations relative to the aggregation studies were used. Stimulation of platelets resulted in an increase in the level of binding of FITC-fibrinogen, consistent with an increase in affinity of integrin
P-selectin exposure, a marker of
The data obtained in the thiol isomerase assay (Figure 3; also O'Neill et al25) demonstrate that inhibitory antibodies are incapable of completely blocking enzyme activity. Therefore, even in the presence of high concentrations of inhibitory antibodies, there will still be low levels of thiol isomerase activity on resting and activated platelets. Thus, it is hard to determine the relative contributions of ERP5 and PDI proteins to fibrinogen binding and P-selectin exposure assays used here. However, these data strongly implicate cell-surface ERP5 and PDI in the regulation of platelet thrombus formation.
ERP5 associates with integrin
To investigate whether there was a direct association between ERP5 and the fibrinogen receptor, integrin
Similar experiments were performed to examine the potential interaction of PDI with
Recent studies have developed the concept of redox-controlled receptor remodeling as part of the activation process in platelets. It has been proposed that these reactions are based on thiol isomerase activity: the ability to generate, reduce, or rearrange disulfide bonds in proteins.23-25 Resting platelets display low levels of thiol isomerase activity on the cell surface, and these levels are enhanced dramatically when platelets are stimulated by agonists.17 The functional importance of this activity is demonstrated by the fact that blocking thiol isomerases inhibits a number of key events in the platelet activation process, including adhesion, aggregation, fibrinogen binding, and P-selectin exposure.4,19,20 The only thiol isomerase enzyme previously characterized in platelets is PDI. We report the presence of an additional thiol isomerase enzyme, ERP5, on the surface of platelets. The contribution to the cell-surface thiol isomerase activity by other enzymes, such as ERP5, could be the basis for the observation that chemical modification reagents consistently inhibit platelet activation markers to a greater extent than specific antibodies that inhibit PDI. In theory, a small number of thiol isomerases could activate a large number of receptors, as they do not have to form long-term stable complexes. The balance in this scenario will be time, because fewer proteins will take longer to activate all receptors. Indeed, limiting surface expression could be seen as another form of setting the gain, or threshold, for platelet activation by modulating the response time for complete activation. These characteristics of extended periods of shape change and slower onset of aggregation are observed when platelets are incubated with low levels of inhibitory antibodies.
Shuttling of receptors between internal organelles and the cell surface is a common phenomenon, and recent studies have shown that cell-surface expression of GluR5 kainate receptors is regulated by an endoplasmic reticulum retention signal.44 Both ERP5 and PDI are recruited to the cell surface, as shown in Figure 2 and Burgess et al,17 respectively, although the intracellular source, whether
Until recently, it would have been easy to attribute the differences observed in the time-dependent exposure profiles to the fact that these agonists stimulate platelets through different signaling pathways: thrombin through G-protein–coupled receptor pathways via protease-activated receptor 1 (PAR1) and PAR4,45,46 collagen through GPVI and the integrin
The observation that small, thiol-reactive reagents modulate platelet function suggests the enzymatic activity of the proteins underlie their function on the cell surface.18 Little is known of the mechanism by which this occurs. Essex et al18 have proposed PDI acts downstream of the primary activation process, but prior to activation of the integrin receptor
The ability of ERP5 to regulate the binding of fibrinogen, cell-surface exposure of P-selectin, and coassociation of
Remodeling of the integrin
We wish to thank Dr Gwenda Graham (University of Reading) and Dr Sheila Hassock (King's College) for technical assistance.
Submitted February 18, 2004; accepted September 27, 2004.
Prepublished online as Blood First Edition Paper, October 5, 2004; DOI 10.1182/blood-2004-02-0608.
Supported by grants from the British Heart Foundation (K.S.A., J.M.G.), the Biotechnology and Biological Sciences Research Council (BBSRC) (J.M.G.), and the Medical Research Council (MRC) (J.M.G.).
An Inside Blood analysis of this article appears in the front of this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jonathan M. Gibbins, School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading, RG6 6AJ, United Kingdom; e-mail: j.m.gibbins{at}reading.ac.uk.
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Abstract
Introduction
2
RIIA receptor. Saturating concentrations were determined as the amount of IV.3 F(ab') fragment, preincubated with platelets, required to completely inhibit platelet aggregation caused by subsequent incubation with whole IV.3 and cross-linking using anti–mouse IgG2a F(ab')2 fragment.
) or collagen (4 µg/mL, 
). Prior to stimulation, platelets were incubated with anti-ERP5 antibodies or control antibodies (24 µg/mL) from preimmune sera. Data are presented as mean ± SE for 3 separate experiments.
