Anti-β₂ glycoprotein I antibodies in complex with β₂ glycoprotein I induce platelet activation via two receptors: apolipoprotein E receptor 2' and glycoprotein I bα

Abstract

Anti-β₂ glycoprotein I (anti-β₂GP I ) antibodies are important contributors to thrombosis, especially in patients with antiphospholipid syndrome (APS). However, the mechanism by which anti-β₂GP I antibodies are involved in the pathogenesis of thrombosis is not fully understood. In this report, we investigated the role of anti-β₂GP I antibodies in complexes with β₂GP I as mediators of platelet activation, which can serve as a potential source contributing to thrombosis. We examined the involvement of the apolipoprotein E receptor 2' (apoER2') and glycoprotein I ba (GP I bα) in platelet activation induced by the anti-β₂GP I /β₂GP I complex. The interaction between the anti-β₂GP I /β₂GP I complex and platelets was examined using in vitro methods, in which the Fc portion of the antibody was immobilized using protein A coated onto a microtiter plate. Platelet activation was assessed by measuring GP II b/ III a activation and P-selectin expression and thromboxane B₂ production as well as p38 mitogen-activated protein kinase phosphorylation. Our results revealed that the anti-β₂GP I /β₂GP I complex was able to activate platelets, and this activation was inhibited by either the anti-GP I bα antibody or the apoER2' inhibitor. Results showed that the anti-β₂GP I /β₂GP I complex induced platelet activation via GP I bα and apoER2', which may then contribute to the prothrombotic tendency in APS patients.

Keywords

anti-β₂GP I /β₂GP I complex / platelet / GP I bα / apoER2' / thrombosis

Introduction

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL) in the plasma [ 1]. Of these aPLs, the most notable and well-characterized are the anti-b₂ glycoprotein I (anti-b₂GP I) antibodies. In APS patients, the presence of anti-b₂GP I antibodies in plasma strongly correlates with the presence of thrombosis [ 2, 3]; a link between anti-b₂GP I antibodies and an increased risk for thrombosis in peripheral and coronary arteries has also been demonstrated [ 4].

b₂GP I (formerly known as apolipoprotein H) consists of five domains (I–V) and is an abundant plasma protein with a concentration of about 200 mg/ml [ 5]. The positioning of domain I adjacent to domain V represents the predominant conformation of b₂GP I in normal human plasma. When b₂GP I interacts with the anti-b₂GP I antibody, b₂GP I becomes dimerized, and conformational changes are introduced into its structure, resulting in an enhanced affinity for anionic phospholipids [ 6, 7].

GP Ibα, subunit of the GP Ib-IX-V platelet, can bind multiple ligands, including von willebrand factor (vWF). Anti-b₂GP I antibodies complexed with b₂GP I increased thromboxane B₂ (TXB₂) release from platelets via GP Ib-IX-V [ 8]. ApoER2' is the only member of low-density lipoprotein (LDL) receptor family known to be expressed by human platelets. Previous studies have revealed that dimeric b₂GP I (fusing b₂GP I with the apple 4 domain of factor XI) results in an increased affinity for platelets and promoted platelet deposition to collagen, an effect that could be blocked with the apoER2' -associated protein (RAP) [ 9]. More than one receptor may be involved in the anti-b₂GP I/b₂GP I complex for induction of cell activation.

Accordingly, an important issue to address is that of a better understanding regarding the interaction between platelets and the anti-b₂GP I/b₂GP I complex. This study tested the hypothesis that the anti-b₂GP I/b₂GP I complex activation of platelets is mediated by at least two receptors, apoER2' and GP Ibα.

Materials and methods

Purification of human plasma b₂GP I

Plasma b₂GP I was isolated from fresh citrated human plasma as described previously [ 10]. Specificity of b₂GP I was determined with western blot assay. Purified plasma b₂GP I showed a single band with a molecular mass of approximately 45 kDa under non-reducing conditions. The concentration of the protein was determined with ultraviolet spectrophotometry (LKB, Sweden). Sodium dodecyl sulfated (SDS)-polyacrylamide gel electrophoresis (Bio-Rad, USA) analysis of the purified protein confirmed the protein’s purity.

Purification of anti-b₂GP I antibodies from APS patients’ sera

IgG from APS patients’ sera was purified by applying sera, diluted 1:4 in phosphate-buffered saline (PBS), to a Hi trap protein G column (Webster, China). IgGs were affinity purified on b₂GP I and N-hydroxysuccinimide activated sepharose (Webster, China). The coupling of b₂GP I was performed according to the manufacturers’ instructions (Sino Biological Inc., China). Anti-b₂GP I antibodies were recovered by acid elution with 0.1 mol/L glycine, pH 2.5, and 500 mmol/L NaCl and were stored at -20 °C for analysis. Specificity of anti-b₂GP I antibodies was determined with western blot assay. Protein concentrations were determined with ultraviolet spectrophotometry (LKB, Sweden). Sodium dodecyl sulfated (SDS)-polyacrylamide gel electrophoresis (Bio-Rad, USA) analysis of the purified protein confirmed its purity. The sera from patients used in this report were positive for both lupus anticoagulant and anti-b₂GP I antibodies. The presence of lupus anticoagulant and anti-b₂GP I antibodies was detected as described previously [ 11]. Samples from patients were collected with approval of the Harbin University Institutional Ethics Committee, and informed consent was obtained in accordance with the Helsinki Declaration.

Binding ratio of anti-b₂ GP I antibodies and b₂ GP I

A total of 50 ml b₂GP I (100 mg/ml) diluted in Tris-buffered saline (TBS), pH 7.4, was added to 96-well polyvinyl microtiter plates (Beyotime, Shanghai, China) and incubated overnight at 4 °C. The plate was blocked with the addition of 150 ml per well of 4% bovine serum albumin (Beyotime, Shanghai, China) in TBS for 2 h at 37 °C. After five washings of the wells with TBS, 50 ml dilutions of anti-b₂GP I antibodies (2–100 mg/ml) were added to the wells and incubated for 2 h at 37 °C. After removal of the unbound antibodies, 50 ml of goat anti-human IgG alkaline phosphatase-conjugated antibodies (Sino Biological Inc., China), diluted 1:2500 in TBS, was added to the wells and incubated for 1 h at 37 °C. After three washings with TBS, 50 ml per well of phosphatase substrate was added, and color development was stopped after 30 min by addition of 50 ml per well of 1.0 mol/L sulfuric acid. The optical density was measured at 450 nm.

Platelet preparation and stimulation

Fresh blood was drawn via venipuncture from healthy volunteers who had no autoimmune disease and had not received any medication for at least 10 days. Blood was collected in sodium citrate anticoagulant. Plasma-rich platelets (PRPs) were obtained via centrifugation for 10 min × 1000 r/min at 25 °C. Then, the PRPs were centrifuged at 3800 r/min for 10 min at 25 °C and resuspended in Tyrode’s buffer (10 mmol/L HEPES, 137 mmol/L NaCl, 2.7 mmol/L KCl, and 12 mmol/L NaHCO₃, pH 7.4, 5 mmol/L glucose). Platelet samples (0.2–0.5 ml, 2 × 10⁸ platelets/ml) were incubated at 37 °C and then stimulated with anti-b₂GP I/b₂GP I complex (10/100 mg/ml), anti-b₂GP I/BSA complex (10/100 mg/ml), IgG/b₂GP I complex (10/100 mg/ml) or thrombin (20 mmol/L, Beyotime, Shanghai, China) for 30 min. In some experiments, platelets were pre-incubated with 0.45 mmol/L RAP (Sino Biological Inc., China) for 5 min or 0.2 mmol/L AK2 (Sino Biological Inc., China) for 10 min. Pre-incubation with 20 mmol/LSB203580 was performed for 10 min.

Flow cytometry

Human platelets were treated with phycoerythrin (PE)-labeled anti-human P-selectin (CD62P) and the FITC-labeled anti-human GP II b/III a complex (PAC-I) (BD Biosciences, USA) for 20 min. Platelets were analyzed via flow cytometry using a FACSCalibur instrument (BD Biosciences, USA). Antibody binding was determined by calculating the percent of positive platelets over a gated fluorescence threshold versus a platelet population stained with the isotype-matched control IgG. The experiments were conducted and analyzed in triplicate.

ELISA

The amount of TXB₂ secreted into the platelet culture media was measured using a TXB₂ ELISA kit (Boster, China) according to the manufacturer’s instructions. The TXB₂ level was expressed as pg/ml in cell culture media.

Western blot

All human platelets were subsequently lysed with Laemmli buffer (Bio-Rad, Richmond, CA) and 2-mercaptoethanol followed by centrifugation for 10 min at 12 000 r/min. The lysates were heated at 95 °C for 5 min. These samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a 12% gel. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA) at 300 V for 50 min using a semidry transfer cell. The membrane was blocked in fresh 5% dry skim milk in 0.05% Tween-20 (TBST) for 1 h at room temperature, washed 3 times with TBST, and then incubated in the primary antibodies against anti-human b-actin or P-p38MAPK (Cell Signaling, Beverly, MA, USA) overnight at 4 °C. Following three washes with TBST, the membranes were incubated in horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Cell Signaling, Beverly, MA, USA) for 1 h at 37 °C. Finally, the immunoblots were developed using electrochemiluminescence western blot detection reagents (Beyotime, Shanghai, China) and then imaged and quantitated using a Bio-Rad Fluor-S MultiImager (CHAMGTE15500).

Statistical analysis

The data are presented as means±SD. Data on TXB₂ production were analyzed by Mann-Whitney U tests. All other data were analyzed by one-way ANOVA. All statistical analyses were performed using SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). P values less than 0.05 were required for results to be considered statistically significant.

Results

Effective ratio for binding of anti-b₂GP I antibodies to b₂GP I

The concentration and purity of b₂GP I (0.42±0.15 mg/ml,>89%) and anti-b₂GP I antibodies (0.72±0.20 mg/ml,>82%) were detected primarily after being purified. Subsequently, we examined whether purified b₂GP I or anti-b₂GP I antibodies specifically combined with the anti-b₂GP I antibodies or b₂GP I as provided by the manufacturers (Fig. 1A). Although anti-b₂GP I antibodies complexed with b₂GP I play more important role in pathogenesis of APS than anti-b₂GP I antibodies, we were interested in the effective ratio for binding of anti-b₂GP I antibodies to b₂GP I. Anti-b₂GP I antibodies were diluted in different concentrations prior to incubation with b₂GP I. We found that the best ratio at which anti-b₂GP I antibodies bound to b₂GP I was 1/10 (10/100 mg/ml) (Fig. 1B).

Protein A-immobilized anti-b₂GP I antibody induced GP II b/III a activation and P-selectin expression along with TXB₂ production of platelets only in the presence of b₂GP I

Given the previous evidence of the ability of the anti-b₂GP I/b₂GP I complex to promote other cell type activation such as monocytes and endothelial cells, we focused on platelets for the key experiment of this study. Therefore, we were concerned about whether the complex may play a specific role in the process. Platelets exposed to the immobilized anti-b₂GP I/b₂GP I complex exhibited a greater degree of GP II b/III a activation and P-selectin expression as well as TXB₂ production as compared with platelets exposed to immobilized IgG in the presence of b₂GP I or immobilized anti-b₂GP I antibody and BSA (Fig. 2A and 2B). These results demonstrate that platelet activation was not due to the formation of IgG/b₂GP I or anti-b₂GP I/BSA complexes and that the presence of the anti-b₂GP I antibody and b₂GP I complex is an absolute requirement for this activation.

Platelet activation by the immobilized anti-b₂GP I/b₂GP I complex is dependent on both GP Ibα and apoER2'

In platelets, b₂GP I can combine with many receptors on the cell surface, such as apoER, GP Ib/IX/V, and TLR4. Platelet activation experiments were performed in the presence of anti-GP Ibα antibody or the apoER2' inhibitor. Antibody AK2 is directed toward recognizing Leu-36-Glin-59, an epitope within the leucine-rich repeat sequence of GP Ibα. RAP is a universal inhibitor of ligand binding to members of the low-density lipoprotein (LDL) receptor family. The percent of GP II b/III a activation and P-selectin expression and the amount of TXB₂ produced in the presence of AK2 or RAP were significantly decreased as compared with platelets treated with the anti-b₂GP I/b₂GP I complex, which were similar to that of the control group (Fig. 3A and 3B). These results suggest that either AK2 or RAP may inhibit the activation of platelets induced by the anti-b₂GP I/b₂GP I complex.

Protein A-immobilized anti-b₂GP I/b₂GP I complex induction of p38MAPK phosphorylation of platelets via apoER2' and GP Ibα

The p38MAPK pathway plays an important role in platelet activation, and our present results reveal that the anti-b₂GP I/b₂GP I complex phosphorylates p38MAPK in a temporally dependent manner. Specifically, we observed that p38MAPK phosphorylation was initiated within 5 min and peaked at 30 min after stimulation (Fig. 4A). The specificity of this anti-b₂GP I/b₂GP I complex in p38MAPK phosphorylation was examined in additional studies. Compared with controls, the anti-b₂GP I/b₂GP I complex increased p38MAPK phosphorylation, but neither the anti-b₂GP I/BSA nor IgG/b₂GP I complex (Fig. 4B).

Activation experiments performed in the presence of SB203580, a specific inhibitor of p38MAPK, revealed that phosphorylation of p38MAPK and TXB₂ production were abrogated in platelets treated with this inhibitor (Fig. 4C and 4D). The activity of this pathway was investigated further in the presence of AK2 and RAP. The presence of either alone or their combination was able to completely inhibit the phosphorylation of p38MAPK induced by the anti-b₂GP I/b₂GP I complex (Fig. 5). These findings confirmed that p38MAPK activation is required and that downstream effectors in this signaling pathway are also activated.

Discussion

In this study, we have found that b₂GP I interacts specifically with anti-b₂GP I antibodies and that combining anti-b₂GP I antibodies enables b₂GP I to activate platelets in an apoER2'- and GP Ibα-dependent manner. ApoER2' and GP Ibα were also required as anti-b₂GP I/b₂GP I complex failed to induce p38MAPK activation in receptor-blocked platelet (as summarized in Fig. 6). Such findings have important implications regarding the pathophysiology underlying the prothrombotic tendency in APS.

Anti-b₂GP I antibodies are well-known prothrombotic factors that contribute significantly to the development of arterial and/or venous thrombosis in APS patients. Based on data from previous studies, the concentration of b₂GP I in human plasma is approximately 200 mg/ml, and one antibody must bind two b₂GP I molecules to obtain sufficient avidity, which is known as the “dimerization theory” [ 12, 13]. However, anti-b₂GP I/b₂GP I complexes are insufficient to induce thrombosis in most cases, as patients with circulating anti-b₂GP I antibodies do not develop thrombosis in the short term. Given the relatively stable concentration of b₂GP I in human plasma, we hypothesized that anti-b₂GP I antibodies will not bind to b₂GP I unless an appropriate concentration is achieved. We found that a 1:10 ratio of 10 mg/ml anti-b₂GP I antibody to 100 mg/ml b₂GP I provides for an effective proportion to form complexes in vitro. These relationships will require confirmation as established through extensive clinical research.

Antibody binding to a cellular surface has been shown to lead to cellular activation via Fc receptors, and based on this observation, a simple method to culture platelets was developed. This approach consisted of immobilizing the anti-b₂GP I antibody via its Fc portion using plates coated with protein A, to which b₂GP I and previously prepared platelets were added. The utility of this method to achieve platelet activation in a specific manner by the anti-b₂GP I/b₂GP I complex was confirmed by measuring GP II b/III a activation and P-selectin expression as well as TXB₂ production. Such a conclusion has been supported by the report of Zhou [ 14, 15], who demonstrated that mononuclear activation and tissue factor expression, which are involved in thrombosis, are induced by the anti-b₂GP I/b₂GP I complex but not the IgG/b₂GP I or anti-b₂GP I/BSA complexes.

Results from previous studies have demonstrated that b₂GP I can combine with many receptors on the cell surface, including, apoER, GP Ib/IX/V, and TLR4 [ 8, 9, 15, 16]. We show that the increase in platelet responses was directly inhibited with either the apoER2' inhibitor RAP or the GP Ibα antibody AK2. Although the copy number of GP Ibα on the platelet is much greater than apoER2', recent studies have shown that GP Ibα can form complexes with a number of other platelet receptors, such as FcgRIIa, GPV, GPⅥ, and RAR1, which can then produce the variety of GP Ibα effects observed [ 17– 19]. In this paper, we observed that apoER2' has the potential of being present in the GP Ibα complex and proposed a mechanism through which b₂GP I can function as a “cross-link” between anti-b₂GP I antibodies and receptors on platelets to produce this increase in activation. This speculation has received support from the work of Pennings et al. [ 20] who demonstrated that GP Ibα could co-precipitate with apoER2', indicating that a complex of GP Ibα and apoER2' was present on these platelet membranes. This complex is likely involved in the pathogenesis of thrombosis in APS (Fig. 6).

Activation of MAPK induced by anti-b₂GP I antibodies is also involved in the pathogenesis of thrombosis in APS patients [ 21]. The predominant MAPK member found in platelets is p38MAPK. Our current findings reveal that the anti-b₂GP I/b₂GP I complex specifically enhances p38MAPK phosphorylation in a temporarily dependent manner. The importance of GP Ibα and apoER2' in p38MAPK pathway was established with the observation that AK2 and RAP are able to significantly inhibit activation of the p38MAPK and reduce production of TXB₂, the downstream, stable metabolite of TXA₂. These studies suggest that p38MAPK serves as a conjunct downstream signaling pathway of apoER2' and GP Ibα.

The possible involvement of apoER2' and GP Ibα complexes in the activation of platelets induced by the anti-b₂GP I/b₂GP I complex results in cell signaling pathway activation and the induction of a prothrombotic cellular phenotype. The newly identified role of apoER2' and GP Ibα in this thrombotic mechanism may be a potentially new and important therapeutic target for treatment of the thrombotic manifestations in APS patients.

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