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Keywords:

  • β2-glycoprotein I;
  • antiphospholipid syndrome;
  • apolipoprotein E receptor 2′ (apoER2′);
  • glycoprotein Ibα;
  • platelet adhesion

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Summary. Background: The major antigen implicated in the antiphospholipid syndrome is beta2-glycoprotein I (β2GPI). Dimerized β2GPI binds to apolipoprotein E receptor 2′ (apoER2′) on platelets and increases platelet adhesion to collagen under conditions of flow. Aim: To investigate whether the interaction between dimerized β2GPI and platelets is sufficiently strong to resist shear stresses. Methods: We studied the interaction of platelets with immobilized dimerized β2GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. Results: We found that dimerized β2GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized β2GPI was completely inhibited by the addition of soluble forms of both apoER2′ and GPIbα, and the addition of receptor-associated protein and the removal of GPIbα from the platelet surface. GPIbα co-precipitated with apoER2′, suggesting the presence of complexes between GPIbα and apoER2′ on platelet membranes. The interaction between GPIbα and dimeric β2GPI was of intermediate affinity (Kd = 180 nm) and Zn2+, but not Ca2+-dependent. Deletion of domain V from dimeric β2GPI strongly reduced its binding to both GPIbα and apoER2′. Antibodies that inhibit the binding of thrombin to GPIbα inhibited platelet adhesion to dimeric β2GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIbα had no effect. Dimeric β2GPI showed reduced binding to low-sulfated GPIbα compared to the fully sulfated form. Conclusion: We show that platelets adhere to dimeric β2GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIbα and apoER2′. These receptors are present in a complex on the platelet surface.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disease characterized by the presence of arterial and/or venous thrombotic complications, both early and late pregnancy losses and pre-eclampsia in combination with the presence of antiphospholipid antibodies in the plasma of affected patients [1]. These so-called antiphospholipid antibodies do not recognize anionic phospholipids directly but are directed against proteins bound to anionic phospholipids [2,3]. The two proteins that are responsible for binding of the majority of the antiphospholipid antibodies are beta2-glycoprotein I (β2GPI) and prothrombin [4]. The presence of antibodies directed against β2GPI that are able to induce lupus anticoagulant highly correlates with the presence of clinical symptoms, and β2GPI is therefore considered to be the most important antigen in APS [5].

β2GPI is a plasma protein present at concentrations of approximately 200 μg mL−1. It is mainly synthesized in the liver and consists of five homologous complement-binding repeats of which domain V is slightly aberrant. Domain V contains a large positively charged patch with a phospholipid insertion loop, which is responsible for binding to anionic phospholipids [6]. The interaction of plasma β2GPI with anionic phospholipids is relatively weak (Kd = 330 nm) but when β2GPI is dimerized by antibodies, its affinity for anionic phospholipids increases several hundredfold [7].

We have previously shown that dimerization of β2GPI with monoclonal antibodies results in increased platelet adhesion to collagen under conditions of flow [8]. Similar results were obtained with a recombinant protein in which β2GPI was dimerized using the apple4 domain of factor XI [9]. The increase of platelet adhesion to collagen by dimeric β2GPI could be inhibited with receptor-associated protein (RAP) [8], which is a universal inhibitor of ligand binding to members of the low-density lipoprotein (LDL) receptor family. The apolipoprotein E receptor 2′ (apoER2′) is the only member of the LDL receptor family known to be expressed by human platelets. It is a truncated splice variant of the apoER2 receptor. It lacks exon 5 and therefore LDL-binding domains 4, 5 and 6 [10].

To investigate whether the dimeric β2GPI–platelet interaction is sufficiently strong to support platelet adhesion under flow, we have studied the interaction of platelets with immobilized dimeric β2GPI in an in vitro perfusion system under conditions of shear stress.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Reagents

Monoclonal antibody 6D1 raised against GPIbα was kindly provided by Dr B. S. Coller (Mount Sinai Hospital, New York, NY, USA). Monoclonal antibody 3B7, raised against β2GPI, was developed in our laboratory using standard hybridoma cloning procedures (unpubl. data). Monoclonal antibody 21B2, raised against β2GPI, was a kind gift from Prof. J. Arnout [11]. RAP was produced as previously described [12]. Thrombin was purchased from Kordia (Leiden, the Netherlands). Monoclonal antibodies 12E4, 6B4, 2D2 and 10H9 were raised against GPIbα as published previously [13]. Monoclonal antibody AK2, raised against GPIbα was purchased from Abcam (Cambridge, UK). Snake venom NK was a kind gift from Dr Andrews (Monash University, Melbourne, Australia). A1(R543Q) is the binding domain of von Willebrand factor (VWF) to GPIbα with a type 2B substitution inducing spontaneous binding to GPIbα, and was produced in our own laboratory [14]. Monoclonal antibody directed against tissue factor (TF; MoAb 4508) was purchased from American Diagnostica Inc. (Stamford, CA, USA). Rabbit anti-apoER2 (186) was kindly provided by Dr J. Nimpf (Medical University of Vienna, Vienna, Austria).

Purification of monomeric β2GPI

Monomeric β2GPI was isolated from fresh citrated human plasma as described previously [15]. The purity of the protein was checked on a 4–15% sodium dodecyl sulfate (SDS) polyacrylamide gel showing a single band of 47 kD. Concentration of the protein was determined using the bicinchoninic acid protein assay.

Cloning and expression of dimeric β2GPI and deletion mutants

Recombinant dimeric β2GPI was constructed and purified as described previously [9]. Purified constructs were analyzed by SDS polyacrylamide gel electrophoresis (SDS–PAGE).

Cloning, expression and purification of soluble apoER2

Mature megakaryocytes were cultured from citrated umbilical cord blood as described previously [16]. Soluble apoER2′ (sapoER2′) was cloned as previously described [17]. Purified constructs were analyzed by SDS–PAGE. Only one band was observed at 45 kD.

Cloning, expression, and purification of soluble GPIbα

Recombinant human soluble GPIbα (sGPIbα) was cloned and expressed as previously described [14]. Purified protein was analyzed by SDS–PAGE. Only one band was observed at 35 kD.

Platelet and red blood cell preparation

Reconstituted blood and washed platelets were prepared as described by Weeterings et al. [18].

Coating of cover slips

Glass cover slips (Menzel-Galzer 40 × 50) were cleaned overnight in 80% ethanol, rinsed with distilled water and dried before coating. Coating was carried out by incubating cover slips back to back, overnight with 400 μL of either a monoclonal anti-β2GPI antibody (21B2) or monoclonal anti-TF antibody (MoAb 4508; American Diagnostica Inc., Stamford, CA, USA) at 50 μg mL−1 in a humified chamber. Cover slips were then blocked in 1% bovine serum albumin (BSA) and incubated with 25 μg mL−1 monomeric β2GPI, 25 μg mL−1 dimeric β2GPI or buffer for 1 h at room temperature (RT).

Perfusion studies

Perfusion experiments were performed in a single-pass triplo perfusion chamber consisting of a silicon sheet gasket, which maintained a flow path height of 0.125 mm and width of 2 mm [19,20]. To test shear dependency, reconstituted blood was perfused over 21B2-coated cover slips, pre-incubated with plasma β2GPI, at different shear forces (100s, 300s, 800s, 1300s and 1600s). To test the inhibitory capacity of different proteins, proteins were added to platelets and mixed carefully to avoid activation. The red blood cells were then added gently to allow them to settle on the bottom of the tube. Reconstituted, unmixed blood was incubated for 5 min at 37 °C. The reconstituted blood was then mixed gently to homogeneity and directly used for perfusions. RAP, sGPIbα and sapoER2′ were added at 35 μg mL−1 and antibodies were used at a concentration of 50 μg mL−1. NK was pre-incubated at 5 μg mL−1 with platelets for 30 min at 37 °C with 3 mm Ca2+. Reconstituted blood was perfused over coated cover slips for 5 min at a shear rate of 300 s−1 using a infusion pump (pump 22, model 2400–004; Harvard, Natrick, MA, USA). After this, the cover slips were removed from the perfusion chamber and rinsed with Hepes-buffered saline (10 mm Hepes, 150 mm NaCl, pH 7.4), fixed in 0.5% glutaraldehyde in phosphate-buffered saline (PBS), dehydrated with methanol, and stained with May-Grünwald/Giemsa as described previously [21]. Platelet deposition was evaluated with a light microscope equipped with a JAI-CCD camera (Copenhagen, Denmark) coupled to a Matrox frame grabber (Matrox Electronic Systems Ltd, QC, Canada) using optimas 6.2 software (Optimas Inc., Seattle, WA, USA) for image analysis. Perfusions were carried out with blood from three different donors, and for every donor three independent flow experiments were performed. Evaluation of platelet adhesion was performed at 20 fields and measured at five different positions spaced by 1 mm, starting at a distance of 5 mm from the blood inlet. Analysis was performed perpendicular to the flow direction. Platelet adhesion was expressed as the percentage of the surface covered with platelets relative to adhesion to dimeric β2GPI. Results are expressed as mean relative coverage (mean ± SD, n = 9). Statistical analysis was performed using the Student's t-test.

Immuno-precipitation: interaction of dimeric β2GPI with GPIbα

For immuno-precipitations, 500 μL aliquots of washed platelets (200 000 μL−1) resuspended in Hepes/Tyrode buffer pH 7.4 and incubated with Tris-buffered saline (TBS), monomeric β2GPI or dimeric β2GPI at 100 μg mL−1 for 5 min at 37 °C. Platelets were then lyzed on ice with 1% CHAPS solution, containing 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid, 50 mm MES [2-(N-Morpholino) ethanesulfonic acid], 3 mm CaCl2 and 150 mm NaCl, pH 7.4. GPIbα was precipitated with 1 μg mL−1 of a monoclonal anti-GPIbα (6D1) in combination with 50 μL of 10% protein G-Sepharose® slurry (Amersham Biosciences, Diegum, Belgium) and incubated for 18 h at 4 °C. Sepharose beads were then washed three times with lysis buffer, resuspended in non-reducing Laemmli sample buffer [0.001% (w/v) bromophenol blue, 2% (w/v) SDS, 10% (v/v) glycerol in 62.5 mm Tris, pH 6.8], and boiled for 5 min. The supernatant was separated on a 10% SDS–polyacrylamide gel and transferred onto an Immobilon-P polyvinylidene difluoride membrane. Blots were blocked with TBS (25 mm Tris, 150 mm NaCl) with 0.1% (v/v) Tween 20 (TBST) containing 2% (w/v) non-fat dry milk powder (MP; Nutricia, the Netherlands) for 1 h at RT. The blot was then incubated with mouse anti-β2GPI antibody (3B7, 3 μg mL−1) overnight in TBST with 1% MP and washed three times with TBST. After incubation with peroxidase labeled rabbit antimouse antibodies for 1 h at RT (1:2500; Dako, Glostrup, Denmark) blots were washed again with TBST–1%MP and developed with enhanced chemiluminescence reagent plus (PerkinElmer Life Sciences, Wellesley, MA, USA).

Interaction of apoER2′ with GPIbα

A quantity of 250 μL aliquots of washed platelets (300 000 μL−1) resuspended in Hepes/Tyrode buffer and treated with cytochalasin-D (30 μm; Sigma Aldrich, St Louis, MO, USA) for 30 min at 37 °C. Cytochalasin-D disrupts the actin cytoskeleton of the platelets and by pretreatment, co-precipitation as a result of the cytoskeleton attachment of both receptors can be excluded. Platelets were then incubated with TBS, monomeric β2-PGI or dimeric β2GPI at 100 μg mL−1 for 5 min at 37 °C. Platelets were then lyzed by adding 25 μL of 10× RIPA lysis buffer (PBS with 10% Nonidet P40, 5% octylglucoside, 1% SDS, 1.86% ethylenediaminetetraacetic acid (EDTA) 10 mm NaVO3, and SIGMA protease inhibitor cocktail according to the manufacturer's protocol). Lysate was then put on ice for 30 min and apoER2′ was precipitated with 1 μg mL−1 of a polyclonal anti-apoER2 antibody (D-18; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with 50 μL of a 10% protein G-Sepharose® slurry (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). The immunoprecipitations were incubated for 18 h at 4 °C in a head-over-head rotor, washed three times with 1× lysis buffer, resuspended in reducing Laemmli sample buffer [0.001% (w/v) bromphenol blue, 2% (w/v) SDS, 10% (v/v) glycerol, 25 mm DTT in 62.5 mm Tris, pH 6.8], and boiled for 5 min. The supernatants were analyzed on a 10% SDS–polyacrylamide gel and electro blotted on to Protran® Nitrocellulose membrane. Blots were blocked with TBS with 0.1% (v/v) Tween 20 (TBST) containing 4% (w/v) BSA (Sigma) for 1 h at RT. Incubation with monoclonal anti-GPIbα antibody AK-2 (1 μg mL−1) was performed overnight in TBST supplemented with 1% BSA. The membranes were washed three times and incubated with RAMPO (1:2500; Dako A/S, Glostrup, Denmark) in the same buffer. Bands on blots were visualized with enhanced chemiluminescence reagent plus (PerkinElmer Life Sciences, Wellesley, MA, USA).

Scanning electron microscopy

Reconstituted blood was perfused over 21B2-coated cover slips, pre-incubated with plasma β2GPI, for 5 min. Adhered platelets were fixed in 3% paraformaldehyde/0.25% glutaraldehyde for 30 min and washed three times in TBS (25 mm Tris, 150 mm NaCl). Cover slips were blocked with 1% BSA + 1.1% glycine for 15 min. Cover slips were then incubated with mouse anti-GPIbα (6.30) at 1 μg mL−1 and washed three times with TBS. Cover slips were then incubated with protein A-Gold 15 nm (Dako A/S, Glostrup, Denmark) 1:100 for 30 min and washed three times with TBS. First-step immunogold labeling was fixed with 3% paraformaldehyde/0.25% glutaraldehyde for 30 min to block all free protein A binding sites. Cover slips were washed three times with TBS, and rabbit anti-apoER2 (186) 1:500 was added for 30 min. The cover slips were washed three times with TBS. After incubation with protein A-Gold 10 nm (Dako A/S, Glostrup, Denmark) cover slips were washed three times with TBS and fixed again with 3% glutaraldehyde/0.25 % glutaraldehyde. Sequential dehydration was performed with, respectively, 80%, 100% ethanol and hexamethyldisilazane. Cover slips were sputter-coated with platinum (6.5 nm) and scanning electron microscopy (SEM) was performed.

Surface plasmon resonance analysis

Surface plasmon resonance (SPR) binding assays were performed employing a Biacore 2000 system (Biacore AB, Uppsala, Sweden). For binding experiments, sGPIbα was immobilized on a CM5 sensor chip using the amine-coupling kit as instructed by the supplier (Biacore AB, Uppsala, Sweden). One channel was activated and blocked in the absence of protein and afterwards its signal was used to correct for a-specific binding. Analysis of binding was measured in TBS/0.005% Tween 20, 3 mm Ca2+ or 15 μm Zn2+ with a flow rate of 10 μL min−1 at 25 °C. All proteins were injected for 2 min, and regeneration of the surface was performed by application of 0.1 m sodium citrate containing 10 mm EDTA and 1 m NaCl, pH 5.0. Analysis of binding curves was performed using biaevaluation 3.0 and graph pad 4.1 software. Data obtained from SPR analysis were used for the calculation of the affinity constant (Kd) as follows. Responses at equilibrium (Req) derived from sensorgrams were plotted against protein concentration. The resulting binding isotherms were subsequently fitted to the following equation, for a one-site binding model:

  • image

GPIbαdimeric β2GPI interaction studied in a solid phase binding assay

Both fully sulfated and low sulfated sGPIbα were coated in TBS on 96-well Costar hydrophobic enzyme-linked immunosorbent assay (ELISA) plates (5 μg mL−1, 37 °C for 1 h). The wells were then blocked with TBS containing 4% BSA (1 h at RT). For inhibition studies, 1.25 μg mL−1 dimeric β2GPI was pre-incubated with 12.5 μg mL−1 thrombin or 12.5 μg mL−1 antibodies for 30 min at RT and the mixture was then added to the wells. The wells were then incubated with polyclonal rabbit anti-β2GPI and bound antibodies were visualized using peroxidase labeled swine antirabbit (Dako A/S, Glostrup, Denmark) followed by staining with orto-phenylenediamine. For binding curves of dimeric β2GPI to non-sulfated and fully sulfated GPIbα, serial dilutions were incubated in GPIbα-coated wells and detected as described above. Analysis of binding curves was performed using graph pad software.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

To investigate whether the interaction between blood platelets and dimeric β2GPI is sufficiently strong to support platelet adhesion under conditions of flow, perfusions were performed with reconstituted blood over different surfaces. As shown in Fig. 1A, dimeric β2GPI supported both platelet adhesion and aggregate formation at venous shear (300 s−1). Comparable results were found when monomeric β2GPI was incubated on the 21B2-coated cover slips (Fig. 1E). Maximal adhesion was obtained at a shear of 800s but only a minimal difference was observed in relative surface coverage when a shear of 300s was used (Fig. 1D). We therefore used 300s as the standard shear in this study to minimize protein usage in the inhibition experiments performed in this study. No adhesion was observed when anti-β2GPI antibody 21B2-coated cover slips were not pre-incubated with β2GPI (Fig. 1B) or when a control antibody (monoclonal anti-TF antibody) pre-incubated with β2GPI was used (Fig. 1C).

image

Figure 1.  Dimeric beta2-glycoprotein I (β2GPI) is able to support platelet adhesion and aggregate formation via apolipoprotein E receptor 2′ (apoER2′) and glycoprotein Ibα (GPIbα). Glass cover slips were coated with either anti-β2GPI or antitissue-factor (anti-TF) antibodies (control antibody). Cover slips were then blocked and incubated with monomeric β2GPI (E), dimeric β2GPI (A, C, E, F) or buffer (B). Reconstituted blood was then perfused over 21B2 incubated with dimerized β2GPI, 21B2 incubated with buffer or a control IgG incubated with dimeric β2GPI, and pictures were taken from the present platelet adhesion (Figs. 1A, 1B and 1C, respectively). Maximal surface coverage was measured at a shear of 800s (D). Experiments were performed at a shear of 300s. Surface coverage on dimeric β2GPI was set at 100%. (E) shows the relative coverage of platelets to anti-β2GPI incubated with dimeric β2GPI, anti-β2GPI or control antibody (antitissue factor) incubated with dimeric β2GPI relative to the percent of the total surface of dimeric β2GPI-coated cover slips covered with platelets after 5 min. (F) shows the relative coverage after pre-incubation with a panel of potential inhibitors compared to the coverage on dimeric β2GPI (F). Data are presented as mean ± SD, n = 9.

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To test whether apoER2′ was involved in platelet adhesion and aggregate formation on a dimeric β2GPI-coated surface under conditions of flow, perfusions were repeated in the presence of RAP or sapoER2′. Addition of RAP and sapoER2′ inhibited platelet adhesion and aggregate formation completely (Fig. 1F).

We furthermore tested whether other receptors play a role in the adhesion to dimeric β2GPI. Addition of sGPIbα almost completely inhibited platelet deposition to dimeric β2GPI (Fig. 1F). Also, cleavage of GPIbα from the surface of platelets using snake venom NK completely inhibited the adhesion of platelets to dimeric β2GPI (Fig. 1F). We subsequently tested a set of well-characterized antibodies against GPIbα for their capacity to inhibit adhesion of platelets to dimeric β2GPI. Anti-GPIbα antibody 10H9, inhibiting thrombin binding to GPIbα, almost completely blocked adhesion of platelets to dimeric β2GPI, whereas an antibody directed against the VWF binding site on GPIbα (12E4) did not affect platelet adhesion and aggregate formation. The addition of VWF domain A1 expressing a type 2B VWD mutation (R543Q) also did not inhibit platelet adhesion to dimeric β2GPI (Fig. 1F).

The capability of sGPIbα to completely abolish platelet adhesion to dimeric β2GPI suggests a direct interaction between dimeric β2GPI and GPIbα. In order to probe such a direct interaction, pull-down experiments were performed using an antibody directed against GPIbα (6D1) in order to detect co-precipitation of dimeric β2GPI with GPIbα from washed platelets. Dimeric β2GPI, but not monomeric β2GPI, could be co-precipitated with GPIbα, indicating that a direct interaction between GPIbα and dimeric β2GPI occurs when platelets are incubated with dimeric β2GPI (Fig. 2A).

image

Figure 2.  Dimeric beta2-glycoprotein I (β2GPI) binds to glycoprotein Ibα (GPIbα) present on the platelet surface and a complex is formed with GPIbα and apolipoprotein E receptor 2′ (apoER2′). Washed platelets were incubated with cytochalasin-D to disrupt the actin cytoskeleton. Platelets were then incubated with Tris-buffered saline (TBS), monomeric β2GPI or dimeric β2GPI for 5 min. Suspensions were then lyzed and incubated with anti-GPIbα or anti-apoER2′ in combination with protein G-Sepharose® beads. Beads were then washed and immuno-precipitations were separated on SDS–PAGE under reducing and non-reducing conditions. Proteins were blotted and incubated with mouse anti-β2GPI (A) or with mouse anti-GPIbα (B). Blots were incubated with rabbit antimouse–horseradish peroxidase and proteins were visualized using chemiluminescence ECN. Immuno-scanning electron microscopy was performed showing GPIbα and apoER2′ to be present in close proximity on the platelet surface (C).

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To test whether GPIbα and apoER2′ are present in a complex or that a complex is formed on the surface of platelets upon incubation with dimeric β2GPI, immuno-precipitations were performed using an antibody directed against apoER2 (D-18) with a-specific rabbit immunoglobulin G (IgG) as negative control (goat antimouse IgG-PE; BD Technologies, Alphen ad Rÿn, the Netherlands). GPIbα was co-precipitated with apoER2′, indicating a complex of GPIbα and apoER2′ present on the membrane of platelets (Fig. 2B). However, the complex between GPIbα and apoER2′ already exists on the platelet membrane surface in the absence of dimeric β2GPI (Fig. 2B). The interaction between GPIbα and apoER2′ was independent of an intact cytoskeleton, because pre-incubation of the platelets with cytochalasin-D did not disrupt the complex (Fig. 2B). Immuno-gold labeling for SEM also showed apoER2′ and GPIbα to present in close proximity of each other on the surface of the adhered platelets (Fig. 2C). No data were obtained from the SEM experiments in relation to relative distances between both receptors in complex.

To further characterize the interaction between dimeric β2GPI and GPIbα, sGPIbα was coupled to a CM5 SPR chip. Dimeric β2GPI bound to immobilized sGPIbα (Fig. 3) and this interaction was dependent on the presence of Zn2+ (Fig. 3), whereas the presence or absence of Ca2+ did not affect the binding of dimeric β2GPI to GPIbα (Fig. 3, inset). The binding of dimeric β2GPI to GPIbα increased with increasing concentrations of Zn2+ and no saturation was reached when Zn2+ concentrations exceeded the plasma concentrations (15 μm). No binding to GPIbα was observed with monomeric β2GPI.

image

Figure 3.  Binding of dimeric beta2-glycoprotein I (β2GPI) to glycoprotein Ibα (GPIbα) is dependent on the presence of Zn2+. Soluble GPIbα was immobilized on a CM5 chip using the amine-coupling kit. Dimeric β2GPI was injected over the surface at 10 μL min−1 in the presence of increasing concentrations of either Zn2+ (1 = 60 μm; 2 = 30 μm; 3 = 15 μm; 4 = 7.5 μm; 5 = 3.75 μm) or Ca2+ (inset; concentrations range from 50 to 3.125 mm) (RU denotes arbitrary response units.)

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To determine which domain of dimeric β2GPI binds to GPIbα, surface plasmon experiments were performed with domain deletion mutants. The different proteins were injected at different concentrations and saturation curves were plotted (Fig. 4). The affinity of dimeric β2GPI for GPIbα was calculated to be 180 ± 20 nm. No differences in affinity and Bmax were observed upon deletion of domain I. The curves for dimeric β2GPI and dimeric β2GPI δD1 were best described employing a one-site binding model. When domains II and III were deleted from dimeric β2GPI, a slight non-significant decrease in Kd was observed for both deletion mutants (280 ± 80 nm and 280 ± 15 nm, respectively). When domain V was deleted from dimeric β2GPI, the affinity reduced from 180 ± 20 nm to 590 ± 160 nm, indicating that the major binding site of dimeric β2GPI for GPIbα resides in domain V.

image

Figure 4.  Dimeric beta2-glycoprotein I (β2GPI) binds to glycoprotein Ibα (GPIbα) via domain V. Increasing concentrations of dimeric β2GPI and domain deletion mutants of dimeric β2GPI were injected over a CM5 chip coupled with GPIbα at 10 μL min−1 in the presence of Zn2+ (15 μm):□, dimeric β2GPI; ♦, dimeric β2GPI δD1; bsl00066, dimeric β2GPI δD2; bsl00072, dimeric β2GPI δD3; bsl00001, dimeric β2GPI δD5. Saturation curves were plotted and affinities were calculated from these curves. Curves are presented as mean ± SD (n = 3).

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To determine the binding site for domain V of dimeric β2GPI located in GPIbα, several antibodies against GPIbα were tested for the capacity to block dimeric β2GPI binding to GPIbα. GPIbα was coated on an ELISA plate and dimeric β2GPI was pre-incubated with a tenfold molar excess of antibodies of which the epitopes have been previously determined. Monoclonal antibodies that inhibit VWF binding to GPIbα (12E4, 6B4, 6D1) did not inhibit dimeric β2GPI binding to sGPIbα, and even showed a slight increase in binding of dimeric β2GPI to GPIbα. Monoclonal antibodies that inhibit thrombin binding to GPIbα (2D2, 10H9) inhibited dimeric β2GPI binding to sGPIbα by 50%. When purified thrombin was added in a 10 times molar excess, binding was also inhibited by 50% (Fig. 5A).

image

Figure 5.  Dimeric beta2-glycoprotein I (β2GPI) binds in close proximity of the thrombin-binding site in glycoprotein Ibα (GPIbα). Soluble GPIbα was coated on an enzyme-linked immunosorbent assay (ELISA) plate and the wells were blocked with bovine serum albumin (BSA; A). Dimeric β2GPI was pre-incubated with a panel of GPIbα binding inhibitors in a ratio of 1:10 (μg:μg). This mixture was then incubated in the GPIbα coated wells and binding of dimeric β2GPI was detected using a monoclonal anti-β2GPI and rabbit antimouse–horseradish peroxidize (RAM–HRP). Orto-phenylenediamine was used for staining (A). Statistical analysis was performed using the Student's t-test. High (2 or 3) or low (0 or 1) sulfated soluble GPIbα was coated on an ELISA plate and wells were then blocked with BSA. Wells were then incubated with an increasing concentration of dimeric β2GPI and binding was detected using the same system as described above. bsl00001 denotes dimeric β2GPI binding to high sulfated GPIbα, and bsl00066 denotes low-sulfated GPIbα.

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As the main thrombin-binding site on GPIbα resides in sulfated tyrosines (residues 276–282), we tested the involvement of the sulfated region in GPIbα in the interaction with dimeric β2GPI. Low-sulfated and fully sulfated GPIbα were coated on an ELISA plate and incubated with dimeric β2GPI. The binding of dimeric β2GPI to low-sulfated GPIbα was 50% lower than the binding to fully sulfated GPIbα. The resulting affinity for dimeric β2GPI was reduced 2-fold when tyrosine residues 276, 278 and 279 in sGPIbα were low sulfated (Fig. 5B).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

We now accept that not antibodies against anionic phospholipids but rather antibodies against β2GPI are the important pathological antibodies present in the APS. However, we lack a generally accepted model for how these antibodies cause the clinical manifestations observed in this syndrome. It is now evident that these so-called ‘antiphospholipid’ antibodies dimerized β2GPI, resulting in a strongly increased affinity of β2GPI for cells such as platelets and endothelial cells.

Recently, we have shown that apoER2′ present on platelets is involved in the activation of platelets by dimerized β2GPI. In this paper, we have shown that there is a second receptor for dimeric β2GPI present on platelets, notably GPIbα. The interaction between dimeric β2GPI and platelets under conditions of flow was inhibited with both sapoER2′ and sGPIbα. Both the cleavage of GPIbα from the platelet surface and blocking apoER2′ using RAP completely abrogate platelet adhesion and aggregate formation under conditions of flow. Furthermore, antibodies against the thrombin-binding site but not antibodies against the VWF binding site within GPIbα are able to block platelet adhesion and aggregate formation on dimeric β2GPI. Similar results were found in a solid phase binding assay where binding of dimeric β2GPI to GPIbα could be substantially inhibited by thrombin itself and by antibodies blocking thrombin binding to GPIbα (e.g. 10H9, 2D2). Antibodies blocking VWF binding to GPIbα (12E4, 6B4, 6D1) did not inhibit dimeric β2GPI binding to GPIbα. The tyrosine-sulfated region of GPIbα, representing the main thrombin binding site on GPIbα, seems to be important for optimal interaction of dimeric β2GPI, as highly sulfated GPIbα (two or three sulfated tyrosine residues) binds dimeric β2GPI much stronger than GPIbα with no or only one sulfated tyrosine residue.

Dimeric β2GPI binds in close proximity of the thrombin-binding site present on GPIbα. Just as for apoER2′, the main binding site for GPIbα within dimeric β2GPI is located in domain V, as shown by SPR experiments using deletion mutants of dimeric β2GPI. The interaction between dimeric β2GPI and GPIbα appeared to be Zn2+-dependent and not Ca2+-dependent. Whether this represents an effect of Zn2+ on β2GPI or on GPIbα remains unclear and requires further investigation. Domain V of β2GPI contains a large positively charged patch and one might speculate that the interaction of this patch with the negatively charged sulfate groups on the tyrosines is important for the binding of dimeric β2GPI to GPIbα. Taken together, these results show that there are two receptors for dimeric β2GPI on the platelet membrane: apoER2′ and GPIbα. The interaction of dimeric β2GPI with apoER2′and GPIbα not only results in adhesion of platelets but also in activation of platelets as aggregates were observed on the dimeric β2GPI-coated surfaces.

The affinity of dimeric β2GPI for apoER2′ is higher (12 ± 1 nm, unpublished data) than the affinity for GPIbα (180 ± 20 nm). However, the copy number of GPIbα on the platelet surface is much higher. We therefore propose, in analogy to activation of platelets by thrombin [22], a mechanism in which GPIbα serves as a docking site for dimeric β2GPI, after which, through positioning and concentrating dimeric β2GPI on the platelet surface, binding of dimeric β2GPI to apoER2′ and subsequent signaling occurs. In this respect, it is interesting to note that apoER2′ is present in complex with GPIbα on the platelet membrane, as demonstrated by pull-down experiments in the presence or absence of dimeric β2GPI. Recent publications have shown that GPIbα can form receptor complexes with several other platelet receptors such as GPV, GPVI, FcγRIIa and PAR1, resulting in different properties of GPIbα [23–25]. In this paper, apoER2′ is added to the list of co-receptors for GPIbα. We speculate that this complex is involved in the pathogenesis of the APS (Fig. 6).

image

Figure 6.  A schematic model of events occurring on the platelet surface. The exposure of negatively charged phospholipids facilitates beta2-glycoprotein I (β2GPI) binding to platelets and subsequent formation and stabilization of dimeric β2GPI. Dimeric β2GPI then can bind to both glycoprotein Ibα (GPIbα) and apolipoprotein E receptor 2′ (apoER2′) on the platelet surface, leading to subsequent increased activation as a result of a second stimulus. Sensitization by dimeric β2GPI leads to an enhanced activation. ApoER2′ becomes phosphorylated and adhesion to a collagen surface increases in a thromboxane A2 (TxA2) dependent way (9). Activation of GPIbα can lead to GPIbα and 14-3-3-ζ translocation to the cytoskeleton, P38MAPK phosphorylation and TxA2 synthesis. TxA2 and P38MAPK phosphorylation have been described to be involved in signaling events downstream of both apoER2′ and GPIbα (28–30).

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One of the clinical criteria of the APS is the occurrence of both arterial and/or venous thrombosis. Shear forces tested in this study represent both arterial and venous flow conditions. The role of platelets in the formation of arterial thrombosis is well established, but a role for platelets in the development of venous thrombosis is normally not anticipated. We have no information of the patho-physiology of deep venous thrombosis induced by antiphospholipid antibodies.

The data presented in this paper support the so-called second hit model in which dimeric β2GPI functions as a sensitizer or pre-activator of blood platelets. This sensitization or pre-activation results in an increased response to a second stimulus such as the exposure of collagen upon vascular damage.

We propose a mechanism by which dimeric β2GPI acts as a ‘cross-linker’ between receptors on platelets resulting in increased adhesion and aggregate formation. Both GPIbα and apoER2′ are required for this process, because the blockade of either one of the receptors completely abolishes platelet adhesion and aggregate formation.

A possible mechanism in which endothelial cells are involved is conceivable. As several members of the LDL-receptor family are present on the surface of endothelial cells, activation of endothelial cell by dimeric β2GPI can be anticipated. Padilla et al. [26] have shown that activation of endothelial cells results in the secretion of ultra large VWF, which sticks to the endothelial surface via P-selectin. As such, platelets adhered to endothelial cells would deliver an ideal pro-coagulant surface for coagulation. This hypothesis is presently under investigation.

We have not observed any difference in platelet adhesion and aggregate formation on cover slips incubated with either monomeric β2GPI or dimeric β2GPI while only dimeric β2GPI and not monomeric β2GPI binds to GPIbα. We have coated monomeric β2GPI to the glass cover slip via an antibody, and we propose that the antibody dimerized β2GPI, making it indistinguishable from dimeric β2GPI. Direct coating of monomeric β2GPI or dimeric β2GPI to the cover slip showed varying adhesion results, probably because β2GPI binds preferentially with its positively charged domain V to the negatively charged glass cover slips. The data presented in this paper have been presented in part at the XXth Congress of the International Society of Thrombosis and Haemostasis (abstract number OR256) and after submitting this paper, the group of Krilis [27] also showed an interaction between β2GPI and GPIbα.

In conclusion, we have shown here that there are two receptors present on the surface of platelets for dimeric β2GPI, apoER2′ and GPIbα, which are present in a complex. The individual roles of both receptors in adhesion and activation of the platelets need further studies. The possible involvement of GPIbα in the activation of platelets by β2GPI could open new avenues in the treatment of the APS.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

This research was supported by research funds from the Netherlands Organization for Health Research and Development (ZonMW, grant 902-26-290). K. VanHoorelbeke is a postdoctoral fellow of the ‘fonds voor wetenschappelijk onderzoek Vlaanderen’, Belgium. R. T. Urbanus is a PhD student funded by the Dutch Heart Foundation, Grant 2003B74.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The authors state that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
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