Lupus anticoagulants: strong association with the major histocompatibility complex class II and platelet antibodies

A relationship between the presence of platelet autoantibodies and major histocompatibilty complex class II alleles was determined in 27 patients with lupus anticoagulants. Twenty-two patients had a 'primary antiphospholipid syndrome' and five patients had lupus erythematosus (SLE). Platelet antibodies against the platelet glycoproteins (GP) IIb/IIIa were detectable in 20 patients. Anti-GPIb/IX or -GPIV antibodies were detectable only in patients with anti-GPIIb/IIIa antibodies. An increased frequency of HLA-DQB1*06 was demonstrable in the total patient population. The association between the lupus anticoagulants and HLA-DQB1*06 was even stronger if patients also had detectable platelet antibodies. This association was also seen if patients with a history of thromboembolic disease were considered separately. However, within the patient population there was no difference between frequencies of HLA alleles detectable platelet antibodies.     

Idiopathic autoimmune thrombocytopenia: evidence for redistribution of platelet antibodies into the circulation after immunoadsorption treatment

Platelet antibodies are detectable in only about 50% of patients with chronic autoimmune thrombocytopenia (AITP). We determined platelet antibodies against GPIa/IIa, GPIb/IX, GPIIb/IIIa, and GPV and reticulated platelets in three female patients with AITP, before and after immunoadsorption treatment. None of the three patients' sera contained platelet antibodies prior to treatment. Thereafter, anti-GPIIb/IIIa, anti-GPIb/IX (n = 3), and anti-GPV (n = 1) were detectable in the patients' sera. These antibody specificities were also found in the eluates from the immunoadsorption columns. Only one patient had elevated levels of reticulated platelets. Immunoadsorption treatment did not induce a sustained increase of platelet counts in any patient. Immunoadsorption treatment in AITP can induce redistribution of antibodies into the circulation.     

Recombinant human natural autoantibodies against GPIIb/IIIa inhibit binding of autoantibodies from patients with AITP

Autoimmune thrombocytopenic purpura (AITP) is caused by autoantibodies predominantly against platelet membrane glycoproteins (GP) IIb/IIIa and GPIb/IX. Naturally occurring autoantibodies have been described against a variety of autoantigens; it has been suggested that perturbation of their regulation may be associated with autoimmune diseases. Using a combinatorial Fab phagemid library from an individual immunized with human RhD⁺ red blood cells, we evaluated the presence of natural anti-GPIIb/IIIa autoantibodies as well as their relation to AITP-associated anti-GPIIb/IIIa autoantibodies. Selection on native GPIIb/IIIa and characterization of positive clones by inhibition studies against murine monoclonal anti-GPIIb/IIIa antibodies and by DNA analysis revealed the presence of two distinct recombinant anti-GPIIb/IIIa autoantibodies, which partially inhibited binding of affinity-purified platelet-associated autoantibodies from 8/12 AITP patients. Our results demonstrated that GPIIb/IIIa-specific Fab directed against conformational epitopes within the GPIIb/IIIa complex may be cloned from the genome of an individual immunized with RhD⁺ red blood cells, who was not affected by AITP. The partial inhibition of binding of platelet-associated autoantibodies from AITP patients to GPIIb/IIIa by the recombinant anti-GPIIb/IIIa phage clones suggests recognition of closely related antigenic epitopes. These phage clones may represent down-regulated, potentially pathological autoantibodies and could be used as new tools for investigation of AITP.     

Presence of cross-reactive antibody between human immunodeficiency virus (HIV) and platelet glycoproteins in HIV-related immune thrombocytopenic purpura.

We previously reported the presence in platelet eluates of autoantibodies directed against epitopes of the platelet glycoprotein (GP)IIb/IIIa complex in acquired immunodeficiency syndrome (AIDS)-free human immunodeficiency virus (HIV)-infected patients with immunologic thrombocytopenic purpura (ITP). We investigated whether HIV antibodies recognized platelet membrane antigens to determine whether the virus might be directly or indirectly responsible for the thrombocytopenia in this context. Direct eluates of platelets from 25 patients with HIV-related ITP contained IgG reacting with HIV-GP160/120 and also, in 45% of patients, detectable antiplatelet antibodies, immunochemically characterized as anti-GPIIb and/or anti-GPIIIa in 5 patients. Furthermore, serum HIV-GP160/120 antibodies could be absorbed on and eluted from platelets from normal non-HIV-infected healthy blood donors (indirect eluates). In contrast, GP160/120 antibodies present in the serum of nonthrombocytopenic HIV-infected patients were not absorbable on normal platelets in most patients, suggesting a pathogenic role in HIV-related ITP. We performed detailed studies of a patient with the highest titer of both HIV-GP160/120 and GPIIb/IIIa antibodies in direct and indirect platelet eluates. No antibody binding to GPIIb/IIIa-deficient Glanzmann thrombasthenic platelets was detected. Furthermore, binding/elution experiments conducted with insoluble recombinant GP160 (expressed in baculovirus) and purified platelet GPIIb/IIIa demonstrated that the patient's IgG bound specifically, through the F(ab')2 portion, to a common epitope of HIV-GP160/120 and platelet GPIIb/IIIa. This common epitope was present on a recombinant GP160 expressed in baculovirus but absent from another recombinant GP160 expressed in vaccinia virus, suggesting that the cross-reactivity is dependent on the glycosylation or conformational structure of the GP. We conclude that molecular mimicry between HIV-GP160/120 and platelet GPIIb/IIIa may explain at least some cases of ITP in AIDS-free HIV-infected patients.     

Autoantibodies to platelet glycoproteins in patients with disease-related immune thrombocytopenia

Although increased platelet destruction and elevated platelet-associated IgG have been shown in patients with lymphomas and various autoimmune diseases, such as systemic lupus erythematosus (SLE), there have been few studies evaluating autoantibodies against platelet-specific antigens. We evaluated 24 patients retrospectively with disease-related thrombocytopenia (12 with lymphoproliferative diseases and 12 with various autoimmune disorders) using a recently reported antigen-specific assay. Autoantibodies against platelet GPIIb/IIIa or GPIb/IX were noted in 15 of the 24 patients (10 of 12 with autoimmune disease and five of 12 with lymphoproliferative disorders). Platelet-associated autoantibodies were present in 60% and plasma autoantibodies in 33%. Anti-GPIIb/IIIa autoantibodies were much more common than those against GPIb/IX. In one patient each with thrombocytopenia and either SLE or myasthenia gravis, absorption of plasma with platelets completely removed the anti-GPIIb/IIIa autoantibodies, but did not affect the level of anti-cochlear autoantibody involved with immune-mediated hearing loss in the SLE patient or the anti-acetylcholine receptor autoantibody in the myasthenic patient. These findings show that, in some cases of disease-related immune thrombocytopenia, autoantibodies against GPIIb/IIIa or GPIb/IX can be detected similar to those seen in chronic ITP. As shown in two patients with multiple autoimmune manifestations, the various autoantibodies have diverse specificities and do not crossreact.     

Antibodies against platelet GPIb/IX,GPIIb/IIIa,and other platelet antigens in chronic idiopathic thrombocytopenic purpura

Abstract: Antibodies involved in the pathogenesis of chronic idiopathic thrombocytopenic purpura (ITP) react most frequently with platelet glycoprotein (GP) Ib/IX and GPIIb/IIIa. However, uncertainty as to the specificity, frequency, and clinical significance of such antibodies still remains. By using a modified antigen-capture ELISA (MACE), an immunoprecipitation assay, and an immunoblot assay, sera from 60 patients with chronic ITP were analyzed. GP-specific antibodies were found in 50% (30/60) of the patients, with 14 patients having antibodies directed solely to GPIIb/IIIa, 8 holding antibodies specific only for GPIb/IX, and 8 possessing antibodies against both antigens. Serum antibodies were more frequently (p<0.01) detected in either active and/or non-splenectomized ITP patients. Moreover, in patients displaying antibodies against GPIb/IX, significantly (p<0.05) lower platelet counts were observed. Using the immunoblot assay, antibodies specific for a 30 kD platelet antigen were detected in 12 of 60 patients. This antigen could not be immunoprecipitated from surface labelled platelet membranes, indicating an intracellular location. We conclude that in chronic ITP, (1) the frequency of anti-GPIIb/IIIa antibodies is close to that of anti-GPIb/IX antibodies, (2) anti-GP antibodies are more likely to be detected in patients with an active disease status and, (3) a 30 kD internal platelet protein is another frequent antigen.     

International study to compare antigen-specific methods used for the measurement of antiplatelet autoantibodies

Platelet-associated and plasma autoantibodies against platelet glycoproteins (GP) have been demonstrated in patients with autoimmune thrombocytopenia (AITP) using various methods. Eight laboratories in seven countries participated in this international study to evaluate the interlaboratory agreement using glycoprotein-specific immunoassays for these autoantibodies. The participating laboratories received blind samples of frozen washed platelets and plasma from 22 normal donors and 22 AITP patients. Platelet-associated and plasma autoantibodies against GPIIb–IIIa and GPIb–IX were measured by MAIPA, immunobead assay or modified antigen capture assay. Of the control samples, 96.0% and 97.2% of all results for platelet-associated and plasma autoantibodies to GPIIb–IIIa/GPIb–IX, respectively, were negative. The mean variation coefficient of the control samples of platelet-associated and plasma autoantibodies was 89.5% (range 11.1–272.9%) and 46.5% (range 21.0–78.0%), respectively. In 20/22 patient samples, platelet-associated autoantibodies to either glycoprotein were noted by at least two laboratories. The mean degree of agreement in these samples was 74.0%. There was a significant correlation in the individual antibody measurements between all laboratories (Kendall coefficient of concordance 0.60 and 0.38, P < 0.001; Spearman rank order test, range of correlation coefficient 52.3–94.0% and 42.2–85.0%, P < 0.05, for anti-GPIIb–IIIa and anti-GPIb–IX, respectively). In contrast, plasma autoantibodies to either glycoprotein were noted by at least two laboratories in only 13/22 patient samples. Moreover, the degree of agreement was poor (50.1%) and a significant correlation was noted between only six pairs of laboratories. We conclude that methods used in this study yield good interlaboratory agreement in measuring platelet-associated autoantibodies against GPIIb–IIIa and GPIb–IX. In contrast, poor agreement was found in detecting plasma autoantibodies to the same glycoproteins.     

Plasma autoantibodies against platelet glycoprotein IIb/IIIa from patients with autoimmune thrombocytopenic purpura may recognize different antigenic determinants

Abstract: Autoantibodies against platelet glycoprotein (GP) GPIIb/IIIa have been demonstrated in patients with autoimmune thrombocytopenic purpura. Recently, it has been shown that plasma autoantibodies from some patients bind to the cytoplasmic domain of GPIIIa. Our aim was to evaluate further the binding specificity of these plasma autoantibodies. From 7 patients with detectable plasma antibodies against intact GPIIb/IIIa, 1 showed strong antibody binding to a synthetic C-terminal peptide of GPIIIa. Ig class analysis of affinity purified anti-GPIIb/IIIa autoantibodies from this patient revealed an IgM antibody that reacted with intact GPIIb/IIIa as well as with recombinant GPIIb/IIIa lacking the C-terminal domains, and an IgG antibody that bound to intact GPIIb/IIIa but not to GPIIb/IIIa lacking the C-terminal region. These data indicate that this patient has at least 2 autoantibodies, an IgG directed against the cytoplasmic domain of GPIIIa and an IgM reacting with the extracellular part of GPIIIa. This may support the hypothesis that plasma IgG antibodies directed against the C-terminal domain of GPIIIa may be due to the exposition of cytoplasmic epitopes of GPIIIa as a result of increased cell lysis by IgM autoantibodies.     

Anti-platelet antibodies in patients with systemic lupus erythematosus and the primary antiphospholipid antibody syndrome: their relationship with the observed thrombocytopenia

The role of antiphospholipid antibodies in the pathogenesis of the thrombocytopenia observed during primary antiphospholipid antibody syndrome (APAS) and systemic lupus erythematosus (SLE) remains controversial. We have used the MAIPA test to examine the frequency and specificity of anti-platelet antibodies directed against the major platelet membrane glycoproteins (GP IIb–IIIa, GP Ib–IX, GP Ia–IIa and GP IV) in patients where SLE and APAS were associated or not with thrombocytopenia. Results were compared with a series of 26 ITP patients, 46% of whom were shown to possess anti-platelet antibodies directed against one or more of the platelet surface glycoproteins. When APAS was associated with thrombocytopenia, 7/10 patients possessed antibodies against GP IIb–IIIa and/or GP Ib–IX. For SLE patients with thrombocytopenia, 6/10 patients were shown to have antiplatelet antibodies against GP IIb–IIIa, GP Ib–IX or GP IV. In contrast, for APAS ( n =11) and SLE patients ( n =11) without thrombocytopenia, only one patient had an antibody directed against GP IIb–IIIa and one patient had an antibody to GP IV. Our results suggest that antibodies directed against major platelet membrane glycoproteins may play a role in the thrombocytopenia that is seen during SLE and APAS.     

Autoantibodies against platelet GPIIb/IIIa in chronic ITP react with different epitopes

Some patients with chronic immune thrombocytopenic purpura have autoantibodies to the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) complex. To determine whether these autoantibodies are directed towards the same or different epitopes, we evaluated the ability of four murine monoclonal anti-GPIIb/IIIa antibodies specific for different epitopes to block autoantibody binding. We noted a variation in blocking patterns among autoantibodies from patients with chronic ITP. In addition, we were able to map the relative epitope locations of both the autoantibodies and the monoclonal antibodies. These data show that the anti-GPIIb/IIIa monoclonal autoantibodies in chronic ITP are directed towards different epitopes.     

Determination of platelet-bound glycoprotein-specific autoantibodies by flow cytometric immunobead assay in primary immune thrombocytopenia

Objectives: Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by premature platelet destruction induced by autoantibodies directed against platelet glycoproteins (GPs). Despite being a clinically important disorder, ITP lacks a feasible diagnostic assay for routine clinical use. This study was meant to evaluate a newly developed flow cytometric immunobead assay for determination of platelet‐bound GP‐specific autoantibodies in comparison with indirect monoclonal antibody‐specific immobilization of platelet antigen (MAIPA) in the diagnosis of ITP. Methods: Platelet‐bound and plasma GPIIb/IIIa and GPIb/IX autoantibodies were determined by flow cytometric immunobead assay and indirect modified MAIPA, respectively. The average fluorescence level for platelet‐bound, GP‐specific autoantibodies was given as a ratio to three normal controls tested simultaneously. Results: The median value of platelet‐bound GPIIb/IIIa and GPIb/IX autoantibodies in ITP group were 3.09 (range 0.78, 30.2) and 3.09 (range 0.72, 19.2), respectively, which were significantly higher than non‐ITP group [1.01 (0.67, 5.59) and 1.01 (0.79, 5.56), respectively, P < 0.001] and normal controls [1.02 (0.72, 1.76) and 1.03 (0.79, 1.73), respectively, P < 0.001]. The receiver‐operating characteristics curve analysis showed an area under the curve of 0.895 for GPIIb/IIIa autoantibody and 0.859 for GPIb/IX autoantibody, respectively. Combined detection of GPIIb/IIIa or GPIb/IX autoantibodies by flow cytometric immunobead assay showed a sensitivity of 82.11% for ITP diagnosis. Conclusion: This study demonstrated that determination of platelet‐bound, GP‐specific autoantibodies by flow cytometric immunobead assay was a convenient, sensitive, and specific test for the differential diagnosis of thrombocytopenic patients.     

Inhibition of autoantibody binding to platelet glycoprotein IIb/IIIa by anti-idiotypic antibodies in intravenous gammaglobulin

Intravenous immunoglobulin (IVIgG) causes an acute rise in the platelet count in the majority of patients with chronic immune thrombocytopenic purpura (ITP) but the mechanism(s) of action is still unknown. We evaluated the ability of three different IVIgG preparations to inhibit the in vitro binding of autoantibody to platelet glycoprotein (GP) IIb/IIIa. ITP plasma, known to contain anti-GPIIb/IIIa antibodies, was incubated overnight with either IVIgG or bovine serum albumin (BSA) followed by measurement of the autoantibody titer. Binding of autoantibody from eight ITP patients was inhibited by IVIgG in proportion to the IVIgG concentration. Using 3.2% IVIgG, compatible with therapeutic concentrations expected in vivo, mean inhibition of autoantibody binding ranged from 20.2% to 41.3%. No inhibition by IVIgG of alloantibody binding to the same or different molecules was detected (five patients with anti-GPIIb/IIIa and two with anti-HLA alloantibodies). F(ab')2 fragments of IVIgG also inhibited the binding of both plasma autoantibodies and purified anti-GPIIb/IIIA autoantibodies prepared by elution from antigen affinity columns. A portion of the anti-idiotypic antibodies could be adsorbed from IVIgG using insolubilized, purified anti-GPIIb/IIIa autoantibody. These results show that IVIgG preparations from normal donors contain anti-idiotypic antibodies directed against idiotypes located on GPIIb/IIIa autoantibodies but do not have anti-idiotypes to platelet alloantibodies against the same or different molecules. The importance of these anti-idiotypic antibodies in the therapeutic response to IVIgG remains to be established.     

Platelet-associated antibody to glycoprotein IIb/IIIa from chronic immune thrombocytopenic purpura patients often binds to divalent cation- dependent antigens

Chronic immune thrombocytopenic purpura (ITP) is a syndrome of destructive thrombocytopenia due to autoantibodies against platelet- associated antigens. These antigens are most commonly located on the platelet glycoprotein (GP) IIb/IIIa complex. In the present studies, we show that many platelet-associated anti-GPIIb/IIIa autoantibodies from chronic ITP patients depend on conformationally intact GPIIb/IIIa for maximal binding. We studied anti-GPIIb/IIIa autoantibodies from 19 ITP patients (15 platelet-associated, 8 plasma) and alloantibodies from three patients with posttransfusion purpura (anti-PIA1). Antibodies were preincubated with purified intact GPIIb/IIIa, EDTA-dissociated GPIIb/IIIa, GPIIIa, or GPIIb for 2 hours and then residual antibody was measured in an antigen capture assay. The binding results were compared with those obtained using antibody preincubated in buffer. Of the 15 platelet-associated autoantibodies studied, the intact GPIIb/IIIa complex resulted in greater inhibition of antibody binding than the EDTA-dissociated complex, with a mean inhibition ratio (intact/dissociated) of 7.9 (range, 1.4 to 30.3). Little inhibition was noted using either GPIIb or GPIIIa. Conversely, plasma anti-PIA1 alloantibodies or plasma autoantibodies from ITP patients against the c- terminal region of GPIIIa were more efficiently inhibited by the dissociated complex or purified GPIIIa. We conclude that platelet- associated anti-GPIIb/IIIa autoantibodies in chronic ITP are frequently directed to cation-dependent conformational antigens.     

Autoantibodies against platelet glycoprotein Ib/IX: a frequent finding in autoimmune thrombocytopenic purpura

Many autoantibodies involved in the pathogenesis of autoimmune thrombocytopenic purpura (AITP) are directed against epitopes on platelet glycoproteins (GP). These autoantibodies are a specific diagnostic characteristic of patients with AITP. In this study, the relative frequency of antibodies against GPs IIb/IIIa and Ib/IX was assessed in sera from 81 AITP patients with a glycoprotein-specific enzyme immunoassay (MAIPA assay) using monoclonal antibodies against these platelet GPs. All sera contained platelet-specific antibodies which had been detected by platelet immunofluorescence. Of the 81 antibodies tested, 58 (72%) reacted with at least one of the platelet GPs studied. Autoantibodies against GPIb/IX were as common as antibodies against the GPIIb/IIIa complex. The same ratio of specificities was observed on autologous platelets of an independent cohort of 29 patients. The epitope of three autoantibodies against GPIb/IX and of mab Gi10, a monoclonal antibody, which inhibits binding of these autoantibodies, was further characterized. Severity of thrombocytopenia was not related to the GP specificity of the autoantibody. The observation that in 23 (28%) of these sera the antigenic determinants could not be assigned to the glycoproteins under investigation suggests that platelet autoantibodies may react with other GPs or other membrane constituents, e.g. glycolipids.     

Autoantibodies and autoantigens in chronic immune thrombocytopenic purpura

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder in which antiplatelet autoantibodies bind to antigens on the surface of platelets, resulting in their destruction. The newer antigen-specific (phase III) assays can detect platelet-associated and plasma autoantibodies in approximately 75% and 50% of patients, respectively. Antiplatelet autoantibodies bind to both platelets and megakaryocytes and preliminary evidence suggests that they not only cause platelet destruction but can also decrease platelet production either by interfering with megakaryocyte proliferation/maturation or by causing intramedullary platelet destruction. Autoantibodies are capable of activating complement and causing platelet phagocytosis both in vitro and in vivo. Many platelet-associated and plasma autoantibodies from ITP patients are light chain-restricted, which suggests a clonal origin. Approximately 75% of platelet autoantigens are localized to either the platelet glycoprotein (GP) IIb/IIIa or Ib/IX complex. Inhibition of the binding of autoantibodies from several ITP patients by either another ITP autoantibody or by a monoclonal anti-GPIIb/IIIa antibody suggests that the antigenic repertoire in chronic ITP may be limited. Most autoantigens on GPIIb/IIIa appear to be conformational since they are dependent on the presence of divalent cations. A variety of new investigative techniques have localized a few autoantigens to specific regions of the cytoplasmic or extracellular regions of both GPIIb/IIIa and GPIb/IX.     

Platelet-associated and plasma anti-glycoprotein autoantibodies in chronic ITP

Chronic immune thrombocytopenic purpura (ITP) is due to platelet destruction by circulating antiplatelet antibody. Although autoantibodies against the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) complex and GPIb have been demonstrated using various methods, practical assays for detection of platelet-associated or plasma autoantibodies have not been available. We studied 59 patients with chronic immune thrombocytopenic purpura in whom platelet-associated and plasma autoantibodies against the GPIIb/IIIa complex and GPIb were measured using a newly developed immunobead assay and a previously reported microtiter-well assay. Platelet-associated autoantibody was detected using the immunobead assay in 21 of 28 patients (75.0%; 13 with anti-GPIIb/IIIa, 8 with anti-GPIb). Plasma autoantibodies were noted in 34 of 59 patients (57.6%; 21 with anti-GPIIb/IIIa, 11 with anti-GPIb, and 2 with both). Positive results were noted in 30 of 59 patients using the immunobead assay and in only 14 of 59 using the microtiter-well assay, suggesting that solubilization of the platelets prior to antibody addition, as in the microtiter-well assay, alters epitope stability. Of the 31 thrombocytopenic control patients studied, all gave negative results using both assays. We conclude that these clinically adaptable assays allow detection of autoantibodies in most patients with chronic ITP, confirming the presence of an autoimmune process.     

Platelet autoantibodies are common in hepatitis C infection, irrespective of the presence of thrombocytopenia

OBJECTIVE: To investigate the generation of platelet antibodies in hepatitis C virus (HCV)-infected individuals and their relation to the development to thrombocytopenia with the aim of using their detection as a diagnostic aid of immune thrombocytopenia in these patients. MATERIALS AND METHODS: We tested by the monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA) for the presence of platelet antibodies against specific glycoprotein (GP) targets (GPIIb/IIIa, GPIb/IX, GPIa/IIa, GPIIIb, GPV, and FcRgammaIIa) in 48 HCV-infected individuals of various stages of disease and compared the results with those from 35 patients with alcoholic liver cirrhosis. RESULTS: Thirty-two HCV-infected individuals (66%) had detectable platelet antibodies. The most common target was GPIIb/IIIa, but all other GP were also targets. Results were not different from patients with alcoholic liver cirrhosis. There was no correlation between antibodies and platelet counts, or the stage of disease, or the viral genotype, or a discernible influence of treatment with alpha-interferon. CONCLUSION: While platelet autoantibodies are common in individuals with HCV infection, their detection does not assist in the diagnosis of immune thrombocytopenia.     

Reactivity of autoantibodies from chronic ITP patients with recombinant glycoprotein Ilia peptides

Summary Chronic immune thrombocytopenia is an autoimmune disorder characterized by destructive thrombocytopenia due to the formation of autoantibodies against platelet-associated antigens. Most antiplatelet autoantibodies react with either the platelet glycoprotein IIb/IIIa or Ib/IX complex, whereas some plasma autoantibodies react with glycoprotein IIIa. Previous studies from our laboratory suggested that most platelet-associated autoantibodies to platelet GPIIb/IIIa, which bind to the intact complex, bind much less avidly to the EDTA-dissociated complex, suggesting that the epitopes were complex-dependent. To evaluate this further we have studied the binding of platelet-associated autoantibody and plasma auto- and alloantibody eluates to large recombinant GPIIIa peptides: peptide 1 (GPIIIa Gly¹-Val²⁰⁰); peptide 2 (GPIIIa Arg¹⁵⁰-Glu⁴⁰⁰); peptide 3 (GPIIIa Lys³⁵⁰-Asp⁵⁵⁰); peptide 4 (GPIIIa Asn⁴⁵⁰-Val⁷⁰⁰) and peptide 5 (GPIIIa Trp⁷¹⁵-Thr⁷⁶², cytoplasmic fragment). Of the 33 platelet-associated antibody eluates tested, all bound avidly to the GPIIb/IIIa complex, but only one showed significant binding (>3 SD above control values) to one of the immobilized peptides (peptide 3). Conversely, antibodies known to bind to specific regions of GPIIIa (murine monoclonal antibody, anti-LIBS2; plasma autoantibody against the GPIIIa cytoplasmic fragment and anti-P1^A1 antibody) all bound avidly to the GPIIIa peptide containing the appropriate epitope. Based on these and our previous results, we conclude that platelet-associated antibodies from chronic ITP patients rarely bind to epitopes localized to GPIIIa alone.     

Platelet autoantibodies (IgG,IgM, IgA) against glycoproteins IIb/IIIa and Ib/IX in patients with thrombocytopenia

 Autoimmune thrombocytopenic purpura (AITP) is most frequently induced by platelet-specific autoantibodies against epitopes on platelet GP Ib/IX or GP IIb/IIIa. These antibodies are reliably detected on the patients' autologous platelets. So far, studies on the characterization of platelet autoantibodies have been restricted to IgG antibodies. We used the monoclonal antibody immobilization of platelet antigens assay (MAIPA) in a modified version to detect GP-specific IgG, IgM, and IgA antibodies. Platelets of 46.2% of patients carried elevated amounts of IgG antibodies. IgM and IgA antibodies were observed less frequently and showed only weak OD signals in the MAIPA assay. Circulating IgG antibodies in serum were found in 11.5% of patients. Circulating IgM autoantibodies were observed in 8.9% and IgA antibodies in no patient with AITP. Results of direct MAIPA assay were compared with the reactivity of eluates in the platelet adhesion immunofluorescence assay and were found to be highly concordant. Patients with AITP in remission carried high percentages of anti-GP IIb/IIIa. Findings made in this study suggest that autoantibodies of the IgM and IgA classes play only a minor role in the pathogenesis of AITP. Received: 14 December 1995 / Accepted: 24 January 1996     

Quantitation of platelet-specific autoantibodies in platelet eluates of ITP patients measured by a novel ELISA using the purified glycoprotein complexes GPIIb/IIIa and GPIb/IX as antigens

Immune thrombocytopenic purpura (ITP) is a disorder caused by anti-platelet autoantibodies (Ab), most of which are directed against epitopes on platelet membrane glycoprotein complexes GPIIb/IIIa and GPIb/IX. To detect platelet Ab, reliable techniques, such as MAIPA or immunobead assay, have been developed. They all achieve their selective specificity by the use of monoclonal antibodies (MoAb) against defined glycoproteins of the platelet membrane. In order to determine the most frequent Ab-specificities, a novel enzyme-linked immunosorbent assay, named platelet-glycoprotein-ELISA (P-GP-ELISA), has been developed. It uses purified GPIIb/IIIa and GPIb/IX complexes, respectively, as antigens and enables determination of platelet-associated as well as circulating Ab (IgG, IgM). MoAbs are not required and therefore there is no risk of competition between MoAb and Ab. Levels of Ab in patients with the clinical diagnosis of an idiopathic thrombocytopenic purpura were analysed. 92.7% (76/82) platelet eluates with significantly increased levels of Ab against at least one of the glycoproteins were found, whereas no sample from healthy volunteers (0/37) gave a positive result, pointing to a high sensitivity and specificity of the test system. Since its application is also easy and quick, P-GP-ELISA should facilitate detection of Ab against platelet membrane proteins in routine determinations.