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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by McMillan, R. Articles by Loftus, J. C. Search for Related Content PubMed PubMed Citation Articles by McMillan, R. Articles by Loftus, J. C. Related Collections Hemostasis, Thrombosis, and Vascular Biology Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 April 2001, Vol. 97, No. 7, pp. 2171-2172 BRIEF REPORT Autoantibodies to IIb3 in patients with chronic immune thrombocytopenic purpura bind primarily to epitopes on IIb Robert McMillan, Jennifer Lopez-Dee, and Joseph C. Loftus From the Department of Molecular and Experimental Medicine, Room 215, The Scripps Research Institute, La Jolla, CA, and the Department of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, AZ.     Abstract Top Abstract Introduction Study design Results and discussion References Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disease caused by platelet destruction resulting from autoantibodies against platelet surface proteins, particularly platelet glycoprotein IIb/IIIa (IIb3). To localize the auto-epitopes on platelet IIb3, the binding of autoantibodies to Chinese hamster ovary (CHO) cells expressing either IIb3 or v3 was studied. Thirteen of 14 ITP autoantibodies bound only to CHO cells expressing IIb3. Because these 2 integrins have the same beta chain (3), these results show that most epitopes in chronic ITP are dependent on the presence of glycoprotein IIb. (Blood. 2001;97:2171-2172) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References 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 (IIb3) or GPIb/IX.1,2 In the current study, we evaluated the binding of platelet-associated anti-IIb3 antibodies from ITP patients to Chinese hamster ovary (CHO) cells expressing either IIb3 or the vitronectin receptor, v3. Because these 2 integrins have the same beta chain (3), it allowed us to evaluate the relative importance of IIb and 3 as sites of auto-epitopes in chronic ITP.     Study design Top Abstract Introduction Study design Results and discussion References Samples were obtained from 14 patients with chronic ITP who had high-titer autoantibodies to platelet IIb3, 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-3 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 IIb3 complex-specific monoclonal antibody (mAb) AP2 was provided by Dr Thomas Kunicki (The Scripps Research Institute, La Jolla, CA), and the v-specific mAb LM142 was obtained from Chemicon (Temecula, CA). Stably transfected CHO cell lines expressing wild-type IIb3 or v3 have been described previously.4 Briefly, full-length cDNAs for IIb, v, and 3 were subcloned into the vector CDM8, and CHO cells were cotransfected with the appropriate  subunit (IIb or v) and 3. 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  and  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 IIb3), B10 cells (expressing v3), 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).     Results and discussion Top Abstract Introduction Study design Results and discussion References 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-IIb3 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-PlA1 alloantibodies, and 3 control subjects.                                View this table: [in this window] [in a new window]   Table 1. Patient clinical characteristics Platelet eluates that had been preadsorbed with untransfected CHO cells were incubated with CHO cells expressing IIb3, CHO cells expressing v3, 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 IIb3, 13 bound only to CHO cells expressing IIb3. The remaining platelet eluate (patient ITP-6) bound to CHO cells expressing either IIb3 or v3. 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-3 alloantibodies bound to both IIb3 and v3 transfected CHO cells as expected because both expressed 3. View larger version (28K): [in this window] [in a new window]   Figure 1. Binding of antibodies to CHO cells expressing IIb3 or v3. CHO cells, expressing either IIb3 (left column) or v3 (right column), were incubated with platelet-associated autoantibody from ITP patients (ITP-4, -5, -6) or allo-antibody from a patient with posttransfusion purpura (PTP-1), and bound antibody was detected by FACS analysis using FITC-antihuman IgG. In each panel, binding to CHO cells, expressing the glycoprotein complex (shaded profile), is compared to binding to native CHO cells (open profile). These results indicate that anti-IIb3 autoantibodies from most patients with chronic ITP (13 of 14) bind to epitopes localized on IIb. Although it is likely that association with 3 is required for the proper folding of 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 IIb or in the presence of 3 coupled to v. One autoantibody, from patient ITP-6, bound to both IIb3- and v3-expressing cells, indicating that either (1) the autoepitope is dependent on the presence of 3, (2) the autoepitope is present on both IIb and v because these molecules are partially homologous, or (3) the patient has multiple autoantibodies with some binding to IIb and others to 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 3 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 3 peptides.3 Earlier studies have also shown that many ITP auto-epitopes are cation dependent.5,6 The formation of configurational epitopes, caused by cation-dependent folding, could depend on one or more of the 4 calcium-binding regions on IIb or to the cation-dependent molecular relations between IIb and 3 required for complex formation. Future studies, using CHO cells expressing chimeric IIb-v3 molecules, should help us to further localize these auto-epitopes.     Footnotes Submitted June 30, 2000; accepted November 22, 2000. Supported by National Institutes of Health grants HL61809 (R.M.) and HL42977 (J.C.L.). The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Robert McMillan, the Department of Molecular and Experimental Medicine, Rm 215, The Scripps Research Institute, 10550 North Torrey Pines Rd, La Jolla, CA 92037; e-mail: mcmillan{at}scripps.edu.     References Top Abstract Introduction Study design Results and discussion References 1. McMillan R, Tani P, Millard F, Berchtold P, Renshaw L, Woods VL. Platelet-associated and plasma anti-glycoprotein autoantibodies in chronic ITP. Blood. 1987;70:1040-1045[Abstract/Free Full Text]. 2. Kiefel V, Santoso S, Kaufmann E, Mueller-Eckhardt C. Autoantibodies against platelet glycoprotein Ib/Ix: a frequent finding in autoimmune thrombocytopenic purpura. Br J Haematol. 1991;79:256-262[Medline] [Order article via Infotrieve]. 3. Bowditch RD, Tani P, McMillan R. Reactivity of autoantibodies from chronic ITP patients with recombinant glycoprotein IIIa peptides. Br J Haematol. 1995;91:178-184[Medline] [Order article via Infotrieve]. 4. Loftus JC, O'Toole TE, Plow EF, Glass A, Frelinger AL III, Ginsberg MH. A 3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation. Science. 1990;249:915-918[Abstract/Free Full Text]. 5. Fujisawa K, McMillan R. Platelet-associated antibody to glycoprotein IIb/IIIa from chronic immune thrombocytopenic purpura patients often binds to divalent cation-dependent antigens. Blood. 1993;81:1284-1289[Abstract/Free Full Text]. 6. Kosugi S, Tomiyama Y, Shiraga M, et al. Platelet-associated anti-glycoprotein (GP) IIb-IIIa autoantibodies in chronic immune thrombocytopenic purpura mainly recognize cation-dependent conformations: comparison with epitopes of serum autoantibodies. Thromb Haemost. 1996;75:339-345[Medline] [Order article via Infotrieve]. 7. Fujisawa K, Tani P, Piro L, McMillan R. The effect of therapy on platelet-associated autoantibody in chronic immune thrombocytopenic purpura. Blood. 1993;81:2872-2877[Abstract/Free Full Text]. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by McMillan, R. Articles by Loftus, J. C. Search for Related Content PubMed PubMed Citation Articles by McMillan, R. Articles by Loftus, J. C. Related Collections Hemostasis, Thrombosis, and Vascular Biology Brief Reports Social Bookmarking                   What's this?

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