Autoantibodies to α_IIbβ₃ in patients with chronic immune thrombocytopenic purpura bind primarily to epitopes on α_IIb

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by the production of antiplatelet antibodies that bind to platelet surface membrane proteins, resulting in platelet destruction. Approximately 75% of these autoantibodies bind to platelet antigens that lie on either platelet glycoprotein (GP) IIb/IIIa (α_IIbβ₃) or GPIb/IX.1 2 In the current study, we evaluated the binding of platelet-associated anti-α_IIbβ₃ antibodies from ITP patients to Chinese hamster ovary (CHO) cells expressing either α_IIbβ₃ or the vitronectin receptor, α_vβ₃. Because these 2 integrins have the same beta chain (β₃), it allowed us to evaluate the relative importance of α_IIb and β₃ as sites of auto-epitopes in chronic ITP.

Samples were obtained from 14 patients with chronic ITP who had high-titer autoantibodies to platelet α_IIbβ₃, 2 patients with high-titer anti-GPIb/IX antibodies (one with chronic ITP and one with a drug [procainamide]-dependent antibody), and one patient with posttransfusion purpura who had anti-β₃ alloantibodies. Platelets from 3 normal subjects were obtained as controls.

Platelet-associated and plasma antibody eluates were prepared by acid elution as previously described.3 Before use in the binding assay, the eluates were ultracentrifuged for 30 minutes at 20 000 rpm and pre-adsorbed with untransfected CHO cells (2 × 10⁷ cells/mL eluate) to prevent nonspecific binding.

The α_IIbβ₃ complex-specific monoclonal antibody (mAb) AP2 was provided by Dr Thomas Kunicki (The Scripps Research Institute, La Jolla, CA), and the α_v-specific mAb LM142 was obtained from Chemicon (Temecula, CA).

Stably transfected CHO cell lines expressing wild-type α_IIbβ₃ or α_vβ₃have been described previously.4 Briefly, full-length cDNAs for α_IIb, α_v, and β₃were subcloned into the vector CDM8, and CHO cells were cotransfected with the appropriate α subunit (α_IIb or α_v) and β₃. The expressed receptors were detected, and clonal cell lines were established with the use of fluorescence-activated cell sorting (FACS) with appropriate monoclonal antibodies. Expression of both the α and β chains was confirmed using appropriate mAbs and FACS.

The CHO cells were harvested from tissue culture flasks with 0.05% trypsin–0.53 mM EDTA in Hank's balanced salt solution. After centrifugation for 5 minutes at 200g, the cells were resuspended in 10 mL Tyrode FACS buffer (0.137 M NaCl, 12 mM NaHCO₃, 2.6 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 0.1% bovine serum albumin, 0.1% dextrose, and 5 mM HEPES) containing 0.2 mg/mL soybean trypsin inhibitor and 0.2% fetal calf serum. After 2 washes in Tyrode FACS buffer, the cells were resuspended to a concentration of 10⁷/mL. Each eluate was incubated separately with A5 cells (expressing α_IIbβ₃), B10 cells (expressing α_vβ₃), or nontransfected CHO cells. Fifty-microliter aliquots of CHO cell suspension (5 × 10⁵ cells) were transferred to the required number of v-bottom microtiter wells. Autoantibody eluate (175 μL) or monoclonal antibody (100 μL of either 15 μg/mL, if purified, or 1:1000 dilution, if ascites) was added, and the mixture was incubated overnight at 4°C. After 3 washes in Tyrode FACS buffer, the pellets were resuspended in either 100 μL fluorescein isothiocyanate–conjugated goat antihuman IgG or 100 μL FITC-conjugated goat antimouse IgG (Vector Laboratories, Burlingame, CA) at a concentration of 15 μg/mL. After a 30-minute incubation on ice and 3 washes, the cells were resuspended in Tyrode FACS buffer and analyzed on a Facscalibur (Becton Dickinson, Mountain View, CA).

We studied platelet eluates from 15 patients with chronic ITP. Clinical details and antiplatelet antibody results are summarized in Table 1. There were 9 female and 6 male patients whose ages ranged from 20 to 70 years. Five patients had other autoimmune disorders, such as antiphospholipid syndrome (patients ITP-4 and -5), autoimmune hemolytic anemia (patients ITP-5, -8, -9) and Crohn disease of the colon (patient ITP-6). Thirteen of the 15 ITP patients had undergone splenectomy, and all had relapses after surgery. Fourteen patients had high-titer anti-α_IIbβ₃ antibodies, and one of these also had anti-GPIb/IX antibodies (patient ITP-9). The remaining ITP patient (patient ITP-15) had only anti-GPIb/IX antibodies. In addition, we studied platelet eluates from one patient with an anti-GPIb/IX drug antibody (procainamide), one patient with posttransfusion purpura caused by anti-Pl^A1 alloantibodies, and 3 control subjects.

Platelet eluates that had been preadsorbed with untransfected CHO cells were incubated with CHO cells expressing α_IIbβ₃, CHO cells expressing α_vβ₃, or untransfected CHO cells. Antibody binding was determined by flow cytometry. Representative studies are shown in Figure 1. Of the 14 platelet eluates from patients with chronic ITP with high-titer autoantibodies to α_IIbβ₃, 13 bound only to CHO cells expressing α_IIbβ₃. The remaining platelet eluate (patient ITP-6) bound to CHO cells expressing either α_IIbβ₃ or α_vβ₃. Eluates from 3 normal subjects and from the 2 patients with anti-GPIb/IX antibodies (ITP-15 and the patient with the drug-dependent antibody) showed no binding to either of the CHO cell lines. The eluted alloantibody from the patient with posttransfusion purpura and anti-β₃ alloantibodies bound to both α_IIbβ₃ and α_vβ₃transfected CHO cells as expected because both expressed β₃.

These results indicate that anti-α_IIbβ₃ autoantibodies from most patients with chronic ITP (13 of 14) bind to epitopes localized on α_IIb. Although it is likely that association with β₃ is required for the proper folding of α_IIb and epitope formation, it is clear from these studies that binding of these 13 autoantibodies to antigen does not occur in the absence of α_IIb or in the presence of β₃ coupled to α_v. One autoantibody, from patient ITP-6, bound to both α_IIbβ₃- and α_vβ₃-expressing cells, indicating that either (1) the autoepitope is dependent on the presence of β₃, (2) the autoepitope is present on both α_IIb and α_v because these molecules are partially homologous, or (3) the patient has multiple autoantibodies with some binding to α_IIb and others to α_v. Additional studies will be needed to clarify this.

These findings are consistent with previous results from this laboratory. In earlier studies, we produced a series of large peptides spanning the human β₃ molecule and evaluated the ability of auto-antibodies from patients with chronic ITP to bind to these peptides. Platelet-associated autoantibodies from only one of 33 patients with ITP showed convincing binding to any of these β₃ peptides.3 Earlier studies have also shown that many ITP auto-epitopes are cation dependent.5 6The formation of configurational epitopes, caused by cation-dependent folding, could depend on one or more of the 4 calcium-binding regions on α_IIb or to the cation-dependent molecular relations between α_IIb and β₃ required for complex formation. Future studies, using CHO cells expressing chimeric α_IIb-α_vβ₃ molecules, should help us to further localize these auto-epitopes.