Results of previous studies suggest that anti–β2-glycoprotein I (anti-β2GPI) antibodies in complex with β2GPI activate platelets in a dysregulated manner, potentially contributing to the prothrombotic tendency associated with the antiphospholipid syndrome (APS). We undertook this study to investigate the possible contribution of the GPIb-IX-V receptor to platelet activation mediated by the anti-β2GPI antibody–β2GPI complex.
In vitro methods were used in the present study. The interaction between β2GPI and the GPIbα subunit of the GPIb-IX-V receptor was delineated using direct binding and competitive inhibition assays. The interaction between the anti-β2GPI antibody–β2GPI complex and platelets was studied using a novel method in which the Fc portion of the antibody was immobilized using protein A coated onto a microtiter plate. Platelet activation was assessed by two methods; one involved measuring thromboxane B2 production and the other involved assessment of the activation of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3β intracellular signaling pathway. The contribution of the GPIbα receptor to platelet activation induced by the anti-β2GPI antibody–β2GPI complex was assessed by observing the influence of 2 anti-GPIbα antibodies (AK2 and SZ2) directed against distinct epitopes.
This study showed that β2GPI could bind to the GPIbα receptor. The anti-β2GPI antibody–β2GPI complex was able to activate platelets, and this effect was inhibited by anti-GPIbα antibody directed against epitope Leu-36–Gln-59, but not by anti-GPIbα antibody directed against residues Tyr-276–Glu-282.
Our findings show that inappropriate platelet activation by the anti-β2GPI antibody–β2GPI complex via the GPIbα receptor may contribute to the prothrombotic tendency associated with APS.
The antiphospholipid syndrome (APS) is characterized by the occurrence of arterial or venous thrombosis and, in female patients, recurrent fetal loss, in association with the detection of antiphospholipid antibodies (aPL) (1). The term “aPL” represents a heterogeneous group of antibodies, the most important and well-characterized in APS being anti–β2-glycoprotein I (anti-β2GPI) antibodies (2–4).
β2GPI is found in plasma at a concentration of ∼4 μM. It consists of repeating sequences comprising the form typical of complement control protein modules. It is composed of 5 domains. The first 4 domains (domains I–IV) are located toward the N-terminus (5). The fifth domain (domain V) mediates β2GPI binding to a number of molecules, including anionic phospholipids (6), heparin (7), coagulation factor XI (FXI) (8), and apolipoprotein E receptor 2′ (9). Its physiologic function has not been fully defined, although there is evidence to suggest that it may have a role in regulating FXI activation (10) and plasminogen activation (11).
In vivo experiments have demonstrated that anti-β2GPI antibodies can promote the formation of a platelet-rich thrombus (12), suggesting that they potentially may be directly pathogenic in humans. Furthermore, using an in vitro experimental system, β2GPI-dependent antibodies have been shown to be able to potentiate platelet activation by thrombin, leading to increased production of thromboxane A2 (TXA2), a major proaggregatory eicosanoid (13). These findings suggest that dysregulated platelet activation by anti-β2GPI antibodies may contribute to the prothrombotic tendency in APS. This hypothesis is supported by the finding, in a number of studies, of increased levels of thromboxane breakdown products in the urine of patients with APS (13, 14). Lutters et al (15) have suggested that crosslinking of apolipoprotein E receptor 2′ on platelets by the anti-β2GPI antibody–β2GPI complex may contribute to inappropriate platelet activation. The possibility that other receptors might be involved has not been excluded.
The GPIbα subunit of the GPIb-IX-V platelet receptor is able to bind multiple ligands, including von Willebrand factor (vWF) (16). The interaction between this receptor and vWF enables platelet adhesion under conditions of high shear stress blood flow (17). In this study we demonstrated that β2GPI is able to directly bind GPIbα and that this enables anti-β2GPI antibodies directed against domain I to activate platelets, leading to the generation of TXB2, which is the stable metabolite of TXA2. The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway mediates a role in intracellular signaling downstream of GPIbα upon ligation by vWF, contributing to platelet adhesion and aggregation (17), and we found that this pathway could be activated in an analogous manner by the anti-β2GPI antibody–β2GPI complex. These findings delineate a novel mechanism by which anti-β2GPI antibodies may predispose to platelet activation, perhaps contributing to the prothrombotic tendency associated with APS.
MATERIALS AND METHODS
125I was purchased from Amersham Biosciences (Piscataway, NJ). Bovine serum albumin (BSA), phenylmethylsulfonyl fluoride (PMSF), protein A, and horseradish peroxidase (HRP)–conjugated anti-rabbit IgG antibody were purchased from Sigma (St. Louis, MO). ADIAgel platelet separation tubes were purchased from American Diagnostica (Greenwich, CT). Plasma-derived native β2GPI (nβ2GPI) was purified in our laboratory as previously described (2, 10) or purchased from Hematologic Technologies (Essex Junction, VT). Recombinant human β2GPI (rβ2GPI) and rβ2GPI domain deletion mutants utilized in this study were generated as previously described (18–20). The mutants contain either isolated β2GPI domains or combinations, and are labeled according to the domains they contain. The mutants used in this study contained domain I (DI mutant), domains I–II (DI-II mutant), domains I–III (DI-III mutant), domains III–V (DIII-V mutant), domains IV–V (DIV-V mutant), and domain V (DV mutant). Murine IgG1κ anti-β2GPI monoclonal antibody (mAb) 3G11C7D10 (designated “mAb number 16” in ref. 21), which binds domain I with an apparent affinity of ∼5 nM (22), was produced as previously described (21). Isotype control murine IgG1κ was purchased from BD PharMingen (San Diego, CA). Anti-GPIbα mAb AK2 (IgG1 isotype), SZ2 (IgG1 isotype), and PM6/40 (IgG1 isotype) were obtained from Biodesign International (Saco, ME). Rabbit anti-Akt antibody, anti–phosphorylated Akt (Ser-473) antibody, and anti–phosphorylated glycogen synthase kinase 3β (GSK-3β) antibody were obtained from Cell Signaling Technology (Beverly, MA). Indomethacin was purchased from Sigma. LY294002 was purchased from Cell Signaling Technology. Leupeptin was purchased from Roche (Nutley, NJ).
Glycocalicin purification and radiolabeling.
Glycocalicin (GC), the soluble form of the extracellular portion of GPIbα, which contains the NH2-terminal globular domain as well as the macroglycopeptide region (16), was purified using a minor modification of the method of Canfield et al (23), by successive chromatography on wheat germ lectin Sepharose 6 MB and jacalin agarose (23, 24). Immunoreactivity of GC was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis utilizing the anti-GPIbα antibody PM6/40 (Figure 1). GC was radiolabeled with 125I using the Iodogene method (Iodo-Beads, Pierce, Rockford, IL), as described previously (10).
The concentration of 125I-GC was determined using the Micro BCA protein assay kit according to the instructions provided by the manufacturer (Pierce). Specific radioactivity was determined as described by Baird and Walsh (25). The specific activity of 125I-GC was 1.54–3.72 × 1018 counts per minute/mole.
Saturation binding of 125I-GC to β2GPI coated onto microtiter plates.
Saturation binding of 125I-GC to immobilized rβ2GPI and nβ2GPI was performed using Lockwell microtiter plates (Nunc, Roskilde, Denmark) as described for binding of 125I-FXI to β2GPI (10). Briefly, the wells were coated with 100 μl of rβ2GPI, nβ2GPI, or BSA (100 nM) using 50 mM carbonate-bicarbonate buffer (pH 9.6) and then incubated overnight at 4°C. The plate was washed 5 times with phosphate buffered saline–0.1% Tween 20 (PBST) using an automated microplate washer (Beckman Coulter, Fullerton, CA). The wells were blocked with 2% BSA–PBST for 2 hours at 25°C. They were then washed 5 times with PBST and 5 times with PBS. One hundred microliters of various concentrations (0.078–20 nM) of 125I-GC in 0.5% BSA–PBS was then added and incubated for 5 hours at 25°C. The wells were washed 5 times with 0.5% BSA–PBS, air-dried, and radioactivity counted with a gamma counter. Bound cpm were measured and converted to fmoles of bound GC. The dissociation constant (Kd) was calculated by nonlinear regression with the GraphPad guide to analyzing radioligand binding data (GraphPad Prism 3.03; GraphPad, San Diego, CA), as previously described (26–28).
Competitive inhibition of 125I-GC binding to β2GPI, using noniodinated GC, BSA, anti-GPIbα antibody (AK2), or isotype control (IgG1κ).
Wells of Lockwell microtiter plates were coated with 100 μl of nβ2GPI (100 nM) in 50 mM carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. The plates were washed, then blocked, and then washed again as described above. Fifty microliters of 125I-GC (1 nM) and 50 μl of either noniodinated GC (50 nM), BSA (50 nM), anti-GPIbα antibody (AK2) (50 nM), or isotype control (IgG1) (50 nM) in 0.5% BSA–PBS was then added to the wells and incubated for 5 hours at 25°C. The wells were then washed, air-dried, and radioactivity counted with a gamma counter as described above. Bound cpm were then measured and converted to a percentage of 125I-GC bound; each value was expressed relative to the value obtained in control experiments using BSA as a competitive inhibitor of 125I-GC.
Competitive inhibition of 125I-GC binding to β2GPI coated onto microtiter plates, using mutants of rβ2GPI.
The effects of domain deletion mutants of rβ2GPI on the binding of 125I-GC to nβ2GPI were studied using Lockwell microtiter plates. Wells were coated with 100 μl of nβ2GPI (100 nM) in 50 mM carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. The plates were washed, then blocked, and then washed again as described above. 125I-GC (1 nM) and various preparations of the rβ2GPI mutants or BSA, in a range of concentrations (0.65–10 μM) in 0.5% BSA–PBS, were added to the wells and incubated for 5 hours at 25°C. The wells were washed, air-dried, and radioactivity counted with a gamma counter. Bound cpm were measured and converted to a percentage of total binding by dividing by the cpm bound in the absence of competitors.
Preparation of gel-filtered platelets.
After approval was obtained from the ethics committee at our institution and informed consent was obtained from the donors, venous blood from 4 healthy adult volunteers, who had not taken any medication affecting platelet function, was collected. Gel-filtered platelets were prepared using ADIAgel platelet separation tubes, based on the method developed by Walsh et al (29) and as described by Shenkman et al (30). Platelets were used at 3.5 × 108/ml for the experiments involving the assessment of TXB2, and at 1 × 108/ml for the experiments involving the assessment of platelet protein phosphorylation by Western blot analysis.
Immobilization of anti-β2GPI antibody and isotype control (IgG1κ) on microtiter plates via their Fc portion, and measurement of platelet TXB2 production in the presence of β2GPI or BSA.
The influence of anti-GPIbα antibodies (AK2 and SZ2) and LY294002 was also assessed. Individual wells were coated with 200 μl of protein A (100 μg/ml) in 50 mM carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. They were then washed 5 times with PBST as described above. The resident binding sites were blocked with 2% BSA–PBST for 2 hours at 25°C. The wells were then washed 5 times with PBS, and 25 μl of anti-β2GPI mAb (0.4 μM) or of isotype control IgG1κ (0.4 μM) was added to the relevant wells. Twenty-five microliters of rβ2GPI (0.8 μM) or of BSA (0.8 μM), suspended in calcium-free HEPES-Tyrode's buffer (126 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.38 mM NaH2PO4, 5.6 mM dextrose, 6.2 mM sodium HEPES, 8.8 mM HEPES-free acid, 0.35% BSA [pH 7.3]) (31) was then added to the relevant wells on an enzyme-linked immunosorbent assay (ELISA) plate containing the protein A–immobilized antibody (either anti-β2GPI mAb or isotype control). The plate was shaken for 15 seconds (450 shakes per minute) using an automated plate minishaker and was then left to incubate for 30 minutes at room temperature.
During this period, gel-filtered platelets were prepared as described above. Platelets in calcium-free HEPES-Tyrode's buffer (pH 7.3) were then dispensed into individual polypropylene tubes (80 μl per tube). Depending on the experiment, 80 μl of either mAb AK2 (0.2 μM), mAb SZ2 (0.2 μM), or LY294002 (200 μM) in calcium-free HEPES-Tyrode's buffer (pH 7.3) was added to the relevant tube containing the platelets. To control for LY294002, 80 μl of buffer was added. The polypropylene tubes containing the platelets plus antibody, LY294002, or control were left to incubate for 10 minutes at room temperature. Forty microliters of HEPES-Tyrode's buffer (pH 7.3) containing CaCl2 (20 mM) was then added to each tube. Fifty microliters of sample from each polypropylene tube was taken and added to the relevant ELISA well which contained, depending on the experiment, immobilized protein A and either anti-β2GPI antibody or isotype control (IgG1κ), plus β2GPI or BSA. The plate was then shaken for 10 seconds (450 shakes per minute) using an automated plate minishaker. The plate was incubated for 25 minutes at 37°C for experiments involving TXB2 analysis. TXB2 production was terminated with the addition of 50 μl of indomethacin (0.3 mM) to each well. The wells were then promptly centrifuged at 10,000 revolutions per minute for 2 minutes at 4°C. The supernatant was collected from each well (150 μl/well) and stored at −20°C until assayed using a TXB2 enzyme immunoassay system according to the recommendations of the manufacturer (Amersham Biosciences).
Measurement of TXB2 production by platelets exposed to β2GPI- or BSA-coated microtiter plates.
Wells were coated with 200 μl of either rβ2GPI (0.2 μM) or BSA (0.2 μM) in 50 mM carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. The wells were subsequently washed, blocked, and washed again as described above. The isolated platelets were then added, and TXB2 production was measured using the method described above.
Western blot analysis of phosphorylated platelet proteins.
Platelets were incubated in wells containing either protein A–anti-β2GPI antibody–β2GPI or protein A–anti-β2GPI antibody–BSA for defined time periods (0, 1, 2, 5, 10, 20, or 30 minutes) at 37°C, for kinetic experiments investigating the phosphorylation of Akt and GSK-3β. The samples were incubated for 30 minutes at 37°C for experiments investigating the effects of mAb AK2, mAb SZ2, and LY294002 on Akt and GSK-3β phosphorylation.
At the defined time points the platelets were centrifuged at 8,000 rpm for 2 minutes at 4°C. The supernatant was removed, and the platelets were then lysed at 4°C in 30 μl of lysis buffer (20 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 μg/ml leupeptin, 1 mM PMSF, 10% [volume/volume] β-mercaptoethanol, 1.2M urea, 1 mM Na2VO4, and 6% SDS [pH 7.2]) per well. Platelet lysates were boiled and subjected to SDS-PAGE (NuPAGE; Invitrogen, Carlsbad, CA). Proteins were transferred to a nitrocellulose membrane, which was blocked by incubation with 2% BSA–PBST for 2 hours at room temperature. Blots were developed with rabbit anti–phosphorylated Akt (Ser-473) antibody, anti-Akt antibody, anti–phosphorylated GSK-3β antibody, and HRP-conjugated anti-rabbit antibody, using the chemiluminescence Western blotting detection system (Amersham Biosciences) and 3,3′-diaminobenzidine tetrahydrochloride (Sigma Fast; Sigma). Radiography was performed using High Performance Chemiluminescence film (Amersham Biosciences).
Data are expressed as the mean ± SD or mean ± SEM. Data on TXB2 production were analyzed by Mann-Whitney U test; all other data were analyzed by Student's unpaired t-test. P values less than 0.05 were considered significant.
Ability of β2GPI to directly bind to GPIbα.
As an initial step in this study, we investigated β2GPI binding to GC. Saturation binding experiments were performed using a fixed concentration (0.1 μM) of either rβ2GPI or nβ2GPI and variable concentrations of radioactive labeled 125I-GC (0.078–20 nM). The results (Figure 2) demonstrate that 125I-GC bound to rβ2GPI (Kd 6.83 ± 2.99 nM, Bmax 8.32 ± 3.26 fmoles) (mean ± SEM; n = 4) and to nβ2GPI (Kd 5.81 ± 2.26 nM, Bmax 4.51 ± 1.48 fmoles) (mean ± SEM; n = 4). As a comparison, a previous study has demonstrated that FXI binds to GC in the presence of ZnCl2 with a Kd of ∼10 nM (31). To confirm the specificity of the interaction between β2GPI and GC, we performed competitive inhibition experiments in the presence of either unlabeled GC or control inhibitor (BSA). The amount of binding of 125I-GC to β2GPI coated on microtiter plates in the presence of unlabeled GC was 57.40 ± 4.77% of the value obtained in the presence of BSA (mean ± SD; n = 3) (P = 0.0005). Furthermore, the amount of β2GPI binding to GPIbα was significantly reduced in the presence of anti-GPIbα antibody (AK2) compared with that obtained with isotype control antibody (IgG1) (53.63 ± 5.46% versus 111.9 ± 24.45%; P = 0.0158).
Ability of β2GPI to bind GPIbα via domain V, but not domain I.
We carried out fluid-phase inhibition experiments with various mutants of rβ2GPI. With each mutant, the experiments were performed in triplicate. The mutant of β2GPI containing DV was able to inhibit 125I-GC binding to nβ2GPI, whereas the mutant containing DI did not inhibit binding (Figure 3). These results demonstrate that β2GPI binding to GC involves domain V, but not domain I.
In this set of experiments we also demonstrated that the DI-II, DI-III, DIII-V, and DIV-V mutants were able to inhibit 125I-GC binding to nβ2GPI as well (Figure 3). These results suggest that β2GPI domains other than DV may be involved in binding to GC. The finding that DI did not inhibit 125I-GC binding to nβ2GPI suggests that the binding involving the DI-II and DI-III mutants is not due to domain I, but is more likely due to domain II and/or domain III.
Immobilized β2GPI in the absence of anti-β2GPI antibodies does not induce increased TXB2 production.
The mean ± SD amount of TXB2 produced by platelets exposed to microtiter plates coated with rβ2GPI was 0.123 ± 0.029 pg/μl (n = 3), and that produced by platelets exposed to BSA-coated wells was 0.117 ± 0.025 pg/μl (n = 3). The difference in TXB2 production between the 2 groups was not significant (P = 0.778).
Protein A–immobilized anti-β2GPI antibody induces the production of TXB2 only in the presence of the β2GPI molecule.
Platelets exposed to the immobilized anti-β2GPI antibody–β2GPI complex exhibited significantly more TXB2 production (17.44 ± 6.569 pg/μl, mean ± SD; n = 12) compared with platelets exposed to immobilized anti-β2GPI antibody plus BSA (0.6717 ± 0.4102 pg/μl; n = 12), or immobilized isotype control antibody (IgG1) in the presence of β2GPI (0.4503 ± 0.2990 pg/μl; n = 12) (P < 0.0001) (Figure 4). These results demonstrate that platelet activation was not due to the immobilized anti-β2GPI antibody alone, and that the presence of β2GPI is an absolute requirement.
Platelet activation by immobilized anti-β2GPI antibody–β2GPI is inhibited significantly more by the anti-GPIbα antibody AK2 compared with anti-GPIbα antibody SZ2.
We performed the platelet activation experiments in the presence of anti-GPIbα mAb that bind to different epitopes on the receptor. Antibody AK2 is directed against residues 36–59, which correspond to the leucine-rich repeat region (32). Antibody SZ2 is directed against residues 276–282, which correspond to the sulfated tyrosine region on the receptor (16). The amount of TXB2 produced in the presence of AK2 was significantly lower (4.941 ± 1.349 pg/μl, mean ± SD; n = 12) compared with that produced in the presence of SZ2 (12.48 ± 4.102 pg/μl; n = 12) (P < 0.0001) (Figure 4). These results, along with the data pertaining to AK2 in the competitive inhibition experiments discussed above, suggest that this antibody may block the β2GPI binding site on GPIbα.
The protein A–immobilized anti-β2GPI antibody–β2GPI complex induces activation of the PI 3-kinase pathway downstream of the GPIb-IX-V receptor.
The PI 3-kinase pathway mediates an important role in platelet activation downstream of the GPIb-IX-V receptor (17). Our kinetic experiments demonstrated increased phosphorylation of Akt and GSK-3β in the anti-β2GPI antibody–β2GPI–treated group compared with control (anti-β2GPI antibody plus BSA) (Figure 5A). In the anti-β2GPI antibody–β2GPI–treated group, Akt was phosphorylated at 5 minutes and phosphorylation peaked at 20 minutes. GSK-3β also was phosphorylated at 5 minutes with a peak at 20 minutes. These proteins did not become phosphorylated to a similar degree in the control group of platelets exposed to protein A–immobilized anti-β2GPI antibody and BSA (Figure 5A).
Akt and GSK-3β are predominantly known to become activated downstream of the PI 3-kinase pathway (33, 34). We conducted activation experiments in the presence of LY249002, a specific inhibitor of PI 3-kinase (35). Phosphorylation of Akt and GSK-3β was abrogated in the platelets treated with the inhibitor (Figure 5B), thus confirming that PI 3-kinase activation is required and that downstream effectors in this signaling pathway are also activated.
We further investigated the activity of this pathway in the presence of the antibodies SZ2 and AK2. AK2 was able to inhibit the phosphorylation of Akt and GSK-3β induced by the anti-β2GPI antibody–β2GPI complex, whereas there was no discernible inhibition with SZ2 (Figure 5C).
The results of this study show that the GPIbα subunit of the platelet adhesion receptor GPIb-IX-V can mediate a role in the dysregulated activation of platelets induced by antibodies directed against β2GPI. These findings may potentially have important implications regarding the pathophysiology underlying the prothrombotic tendency in APS.
We initially demonstrated that β2GPI is able to directly bind to GPIbα with a Kd of 5–7 nM (Figure 2), comparable with the Kd for binding of FXI and GPIbα in the presence of ZnCl2 (∼10 nM) (31). This interaction is specific, since 125I-GC binding to β2GPI is inhibited by unlabeled GC. The anti-GPIbα antibody AK2, which recognizes Leu-36–Gln-59, an epitope within the leucine-rich repeat sequence of GPIbα (32), is also able to inhibit 125I-GC binding to β2GPI, suggesting that this epitope may be an important binding site for β2GPI.
Domain V of β2GPI appears to mediate an important role in binding to GPIbα, whereas domain I of the β2GPI molecule is not involved (Figure 3), leaving it free to potentially be bound by anti-β2GPI antibodies directed against epitopes in this region. This is consistent with studies showing that the presence of anti-β2GPI antibodies directed against the epitope encompassing Gly-40–Arg-43 in domain I correlates with clinical manifestations of APS (18, 20, 36). The novel possibility raised by the present findings is that domains II, III, and IV may also be involved in binding to GPIbα, although site-directed mutagenesis studies involving β2GPI are needed to confirm this hypothesis.
A simple method to activate platelets using the anti-β2GPI antibody–β2GPI complex was developed. This was achieved by immobilizing the anti-β2GPI antibody via its Fc portion using plates coated with protein A, to which β2GPI and subsequently gel-filtered platelets were added. The utility of this method to achieve platelet activation in a specific manner by the anti-β2GPI antibody–β2GPI complex (i.e., compared with isotype control antibody plus β2GPI, or anti-β2GPI antibody plus BSA) was confirmed by measuring TXB2 production (Figure 4) and the activation of the PI 3-kinase/Akt/GSK-3β pathway (Figure 5A). Furthermore, we demonstrated that immobilized β2GPI per se does not induce TXB2 production, emphasizing the necessity of β2GPI and anti-β2GPI antibodies to form a complex on the cell surface in order to achieve platelet activation.
The importance of the GPIbα receptor in platelet activation induced by the anti-β2GPI antibody–β2GPI complex was established with the observation that antibody AK2, which inhibits β2GPI binding to GPIbα, is able to significantly inhibit TXB2 production (Figure 4) and activation of the PI 3-kinase/Akt/GSK-3β pathway (Figure 5C) compared with antibody SZ2, which is directed against a different epitope, Tyr-276–Glu-282 on the GPIbα receptor (16).
Ligation of the GPIb-IX-V receptor by vWF can lead to generation of TXA2 (17). The intracellular pathway involves p38 MAPK, which leads to the activation of phospholipase A2, which then converts arachidonic acid to TXA2 (17). Of interest, in a previous study p38 MAPK–induced activation of phospholipase A2 was shown to be responsible for TXA2 production induced by protein G–affinity-purified antibodies from patients with APS, compared with identically purified antibodies from healthy subjects (37). The antibodies from the APS patients were shown, by ELISA, to have reactivity against β2GPI (37). Hence, p38 MAPK signaling may mediate an important role in TXA2 production, downstream of the interaction between GPIbα and the anti-β2GPI antibody–β2GPI complex (Figure 6), although confirmation by further experimentation is needed.
The PI 3-kinase/Akt/GSK-3β intracellular pathway was found to be activated downstream of the GPIbα receptor upon binding of the anti-β2GPI antibody–β2GPI complex. This is consistent with previous reports that this pathway becomes operational upon ligation of the GPIb-IX-V receptor by vWF (17). PI 3-kinase and its downstream effectors play an important role in the activation of the aIIbβ3 receptor, which is crucial for platelet aggregation (17). In the case of vWF interacting with GPIb-IX-V, the 14-3-3ζ adaptor molecule appears to play a role in linking the intracellular portion of the GPIbα subunit with PI 3-kinase (17). Espinola et al (38) have previously demonstrated that antibodies with anti-β2GPI activity from patients with APS are able to induce expression of the aIIbβ3 receptor on platelets treated with a subactivating dose of a thrombin agonist. The mechanism(s) of this effect was not determined. We speculate that aIIbβ3 activation may have resulted from the anti-β2GPI antibody–β2GPI complex interacting with the GPIbα subunit, leading to activation of the PI 3-kinase/Akt pathway (Figure 6).
β2GPI attached to GPIbα appears to act as a conduit enabling antibodies directed against domain I to crosslink the receptor (Figure 6). The question of whether β2GPI interacts with GPIbα via a single binding site (Leu-36–Gln-59) or multiple binding sites is an area for future study.
The GPIbα subunit is a multiligand receptor, which is able to interact not only with vWF, but also with P-selectin, Mac-1, FXI, and thrombin (17). Binding of vWF to GPIbα occurs only when blood is flowing at high shear rates, as in arterial vessels of a small diameter (16). Binding between the GPIbα subunit on platelets and P-selectin on endothelial cells, and between the GPIbα subunit on platelets and Mac-1 on white blood cells, may contribute to platelet–endothelium and platelet–white blood cell interactions, respectively (17). FXI and thrombin binding to GPIbα allows for efficient generation of activated FXI on the platelet surface during thrombus propagation (31). Furthermore, thrombin binding to GPIbα allows for activation of protease-activated receptor 1 on platelets (17).
Whether the anti-β2GPI antibody–β2GPI complex, by binding and crosslinking GPIbα, is able to interfere with or potentiate any of the above-mentioned interactions still remains to be determined. Clinically, it is known that the anti-β2GPI antibody–β2GPI complex does not induce a bleeding diathesis, and we therefore hypothesize that it is very unlikely that it interferes with the interaction between vWF and GPIbα under high shear blood flow conditions associated with exposure of the subendothelium. However, under conditions associated with low shear rates, as occur in the venous circulation, or under high shear conditions not associated with exposure of the subendothelium, as occur in an intact, undamaged artery or arteriole, vWF does not interact with GPIbα (16). We can speculate that, in these situations, the anti-β2GPI antibody–β2GPI complex may interact with GPIbα, potentially causing low-grade platelet activation and thus increased susceptibility to thrombosis.
Arvieux et al (39) have demonstrated that anti-β2GPI antibodies in patients with APS display heterogeneous affinity for β2GPI. Furthermore, as alluded to above, anti-β2GPI antibodies in patients with APS tend to be directed against an epitope in domain I (18, 20, 36), in contrast to the anti-β2GPI antibodies associated with childhood atopic dermatitis (40) and leprosy (41), which tend to be directed against domain V. In this study we utilized a well-characterized murine monoclonal antibody directed against domain I of the β2GPI molecule (21) to establish the validity of the concept that β2GPI bound to GPIbα enables antibodies directed against β2GPI to activate platelets. Murine anti-β2GPI antibodies tend to have higher affinity than those derived from patients with APS (22). To establish the importance of the mechanism delineated in this study in relation to APS pathophysiology, it will be necessary to test anti-β2GPI antibodies from a large cohort of patients, with and without thrombosis, to determine whether they are able to activate platelets via the GPIbα receptor.
In summary, we have described a potentially significant interaction between the anti-β2GPI antibody–β2GPI complex and the GPIbα receptor on platelets. This opens a new avenue for exploration of the pathophysiology of the antiphospholipid syndrome.
We are grateful to Marcus Cremonese (Medical Illustrations Unit, The Prince Of Wales Hospital, Sydney, New South Wales, Australia) for his invaluable help with the figures. We would also like to thank Alex Denangle for secretarial assistance.