Influences of antiplatelet autoantibodies on platelet function in immune thrombocytopenic purpura

We investigated the characteristics of the antiplatelet autoantibodies in 60 patients with ITP. Using flow cytometry, the binding of monoclonal antibodies to the platelet glycoprotein (GP) IIb/IIIa complex and to GPIb was examined in these patients. The extent of binding was decreased in 15 patients (anti-GPIIb/IIIa in 12 patients and both anti-GPIIb/IIIa and anti-GPIb in 3 patients). Western blotting revealed that 10 of these 15 patients had either anti-GPIIb or anti-GPIIIa and 2 had anti-GPIb autoantibodies, ADP-induced aggregation of normal platelets was inhibited by autoantibodies in 12 of 60 patients, and 11 of these had anti-GPIIb/IIIa antibodies. Ristocetin-induced aggregation was inhibited in 4 of these patients, and 2 with prominent inhibition had anti-GPIb antibodies. There was a significant relationship between platelet-associated IgG value and ATP secretion. These results suggest that some antiplatelet autoantibodies can affect platelet function and thus have an influence on the pathophysiology of ITP.     

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.     

Analysis of anti-platelet antibody and platelet volume in idiopathic thrombocytopenic purpura

We investigated platelets and plasma from patients with idiopathic thrombocytopenic purpura (ITP) to elucidate the antigenic determinants at which their autoantibodies are directed, and studied the relationship between anti-platelet antibody and platelet volume. We used flow cytometry to detect platelet-associated IgG (PAIgG), C3 (PAC3), IgM (PAIgM) and platelet volume, and also to determine the binding rate of monoclonal anti-platelet antibodies in patients with ITP. The following results were obtained. 1. Both anti-GPIIb/IIIa autoantibodies (21 of 71 patients) and anti-GPIb autoantibodies (3 of 71 patients) were found in ITP. 2. The decrease in platelet count in patients without anti-GPIIb/IIIa autoantibodies was significant. 3. The increase in platelet volume was found more frequently in patients with a platelet count less than 50,000 and in untreated patients. 4. There was a positive correlation between the platelet volume and PAIgM in patients with a platelet count less than 30,000 and high levels of PAIgM.     

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.     

Relationship between platelet volume and anti-platelet autoantibodies in idiopathic thrombocytopenic purpura

We used flow cytometry to explore the relationship between platelet volume and anti-platelet autoantibodies in 71 patients with idiopathic thrombocytopenic purpura (ITP). An increase in platelet volume was found more frequently in patients with a platelet count of less than 20,000/microliters. Platelet volume was larger in patients without anti-GPIIb/IIIa autoantibodies than in patients with these autoantibodies. Furthermore, the platelet count was significantly lower in patients without anti-GIIb/IIIa autoantibodies than in the patients with these autoantibodies. There was a positive correlation between a large platelet volume in patients with a platelet count of less than 30,000/microliters and high platelet-associated IgM levels. These results suggest that the platelet volume is related to the severity of thrombocytopenia in ITP.     

Immune thrombocytopenic purpura (ITP) plasma and purified ITP monoclonal autoantibodies inhibit megakaryocytopoiesis in vitro

To determine if megakaryocytes are targeted by immune thrombocytopenic purpura (ITP) autoantibodies, as are platelets, we have studied the effects of ITP plasma on in vitro megakaryocytopoiesis. Umbilical cord blood mononuclear cells were incubated in the presence of thrombopoietin and 10% plasma from either ITP patients (n = 53) or healthy donors. The yield of megakaryocytic cells, as determined by flow cytometry, was significantly reduced in the presence of ITP plasma containing antiplatelet glycoprotein Ib (GPIb) autoantibodies (P <.001) as compared with both the control and patient plasma with no detectable anti-GPIIb/IIIa or anti-GPIb autoantibodies. Platelet absorption of anti-GPIb autoantibodies in ITP plasmas resulted in double the megakaryocyte production of the same plasmas without absorption, whereas platelet absorption of control plasma had no effect on megakaryocyte yield. Furthermore, 2 human monoclonal autoantibodies isolated from ITP patients, 2E7, specific for human platelet glycoprotein IIb heavy chain, and 5E5, specific for a neoantigen on glycoprotein IIIa expressed on activated platelets, had significant inhibitory effects on in vitro megakaryocytopoiesis (P <.001). Taken together, these data indicate that autoantibodies against either platelet GPIb or platelet GPIIb/IIIa in ITP plasma not only are involved in platelet destruction, but may also contribute to the inhibition of platelet production.     

Initial laboratory findings useful for predicting the diagnosis of idiopathic thrombocytopenic purpura

PURPOSE: To identify initial laboratory findings useful for the later diagnosis of idiopathic thrombocytopenic purpura (ITP) in adult patients with thrombocytopenia. SUBJECTS AND METHODS: We studied 62 consecutive adult patients who had thrombocytopenia and whose peripheral blood film was normal except for thrombocytopenia at presentation. Each patient underwent physical examination and routine laboratory tests and was prospectively followed for 22.5 +/- 9.8 months (range, 8 to 41 months). The frequency of antiglycoprotein (GP) IIb/IIIa antibody-producing B cells, the presence of platelet-associated and plasma anti-GPIIb/IIIa antibodies, the percentage of reticulated platelets, and the plasma thrombopoietin level were examined at the first visit. The final diagnosis was based on the clinical history, physical examination, complete blood test, bone marrow findings, and the clinical course at last observation. RESULTS: Forty-six patients were diagnosed as having ITP and 16 as having another disorder, including myelodysplastic syndrome, aplastic anemia, amegakaryocytic thrombocytopenia, and reduced platelet production, with or without other cytopenias, and without dysplasia or evidence for destruction. Six initial laboratory findings discriminated ITP from other diagnoses: the absence of anemia, absence of leukocytopenia, increased frequency of anti-GPIIb/IIIa antibody-producing B cells, increased platelet-associated anti-GPIIb/IIIa antibodies, elevated percentage of reticulated platelets, and a normal or slightly increased plasma thrombopoietin level. Three or more of these ITP-associated findings were found at presentation in 44 patients (96%) with thrombocytopenia later diagnosed as ITP, compared with only 1 patient (6%) whose disorder was non-ITP. CONCLUSION: Initial laboratory findings can well predict future diagnosis of ITP. Further studies prospectively evaluating these same diagnostic criteria on another, independent set of patients are necessary.     

The association between platelet autoantibody specificity and response to intravenous immunoglobulin G in the treatment of patients with immune thrombocytopenia

We retrospectively investigated the association between platelet autoantibody specificity and response to intravenous immunoglobulin G (IVIG) in 17 patients with immune thrombocytopenia (ITP). Platelet-associated antibodies against glycoprotein (GP) IIb/IIIa, GPIb/IX, and GPIa/IIa were detected in 13, 10, and 8 patients, respectively. A response occurred in 7 of 7 patients without anti-GPIb/IX, but in only 3 of 10 patients with anti-GPIb/IX (p<0.01). There was no difference in the response rates in patients with or without anti-GPIIb/IIIa or anti-GPIa/IIa. We conclude that ITP patients with anti-GPIb/IX may be less responsive to IVIG.     

Hypertension and diabetes mellitus increase the risk of haemorrhage in chronic idiopathic thrombocytopenic purpura

The risk factors for haemorrhage in chronic idiopathic thrombocytopenic purpura (ITP) remain poorly understood. We classified 49 patients with chronic ITP into two groups on the basis of the presence (n = 11) or absence (n = 38) of hypertension and/or diabetes mellitus, and then analyzed their clinical and immunological characteristics. The patients with hypertension and/or diabetes were older than those without these complications. There were no significant differences between the two groups with regard to platelet count or the levels of platelet-associated immunoglobulin G, platelet-associated immunoglobulin M, and platelet-associated C3. Positivity for anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib autoantibodies was also similar. However, severe purpura and a poor response to prednisolone were far more common in the patients with hypertension and/or diabetes. We conclude that ITP complicated by hypertension and/or diabetes may be resistant to prednisolone and thus require more careful treatment.     

Antiplatelet autoantibody-related microparticles in patients with idiopathic (autoimmune) thrombocytopenic purpura

Summary We used flow cytometry to detect antiplatelet antibody-related microparticles (MP) in 56 patients with idiopathic thrombocytopenic purpura (ITP). We measured MP in platelets following various types of stimulation in two experimental systems. In one system washed platelets were incubated with normal serum which included the complement system, and in the other, washed platelets were incubated with Tyrode's buffer. There were no differences between the two measurement systems in the degree of increase in MP using various agonists. An increase in MP using ITP plasma was found in 12 out of 56 patients. In particular, four patients showed a significant increase in MP in washed platelets (WP) plus serum. Furthermore, the increase in platelet-associated IgM (PAIgM) was significant in these patients. There was also a definite positive correlation between PAIgM and the percentage of MP of WP plus serum. On the other hand, no specificity for MP formation with anti-GPIIb/IIIa or anti-GPIb autoantibodies was observed. IgM antibody-related MP appear to exist in some patients with ITP.     

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.     

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.     

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.     

Two types of autoantibody-mediated thrombocytopenia in patients with systemic lupus erythematosus

OBJECTIVES: To determine whether autoantibodies to two platelet-specific antigens, glycoprotein IIb/IIIa (GPIIb/IIIa) and thrombopoietin receptor (TPOR), contribute to thrombocytopenia in patients with systemic lupus erythematosus (SLE). METHODS: Circulating B cells producing anti-GPIIb/IIIa antibodies and serum anti-TPOR antibodies were measured in 32 SLE patients with thrombocytopenia, 30 SLE patients without thrombocytopenia, 92 patients with idiopathic thrombocytopenia and 60 healthy controls. The megakaryocyte density in bone-marrow smears from all the patients with thrombocytopenia was evaluated. RESULTS: Anti-GPIIb/IIIa and anti-TPOR antibody responses were more frequent in SLE patients with thrombocytopenia than in those without thrombocytopenia (88 vs 17%, P<0.0001; and 22% vs 0%, P=0.01, respectively). The frequencies of these platelet-related antibodies were comparable between SLE patients with thrombocytopenia and patients with idiopathic thrombocytopenia. Twenty-nine (91%) SLE patients with thrombocytopenia had either anti-GPIIb/IIIa or anti-TPOR antibody, and six had both. In SLE patients with thrombocytopenia, the anti-TPOR-positive patients had significantly higher frequencies of megakaryocytic hypoplasia and poorer therapeutic responses to corticosteroids and intravenous immunoglobulin than did the anti-TPOR-negative patients, most of whom had the anti-GPIIb/IIIa antibody alone. CONCLUSIONS: Anti-GPIIb/IIIa and anti-TPOR antibodies are major factors contributing to SLE-associated thrombocytopenia, but the clinical presentations associated with these autoantibodies are different.     

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.     

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.     

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.     

Platelet-associated CD154 in immune thrombocytopenic purpura

CD40-ligand (CD154) is expressed on activated CD4+ T lymphocytes and is essential for the T cell-dependent activation of B lymphocytes. CD154 is also expressed at the activated platelet surface. In this study, we show that platelet-associated CD154 is increased in immune thrombocytopenic purpura (ITP), a disease characterized by an autoimmune response against proteins of the platelet membrane. CD154 and its messenger RNA were also present in increased amounts in the megakaryocytes of patients with ITP. We found that platelet-associated CD154 is competent to induce the CD40-dependent proliferation of B lymphocytes, and we observed an in vitro CD154-dependent production of antibodies to the GPIIb/IIIa complex (integrin alphaIIbbeta3) when platelets and peripheral blood B lymphocytes from ITP patients with circulating anti-GPIIb/IIIa antibody were cultured together. Therefore, platelet-associated CD154 expression is increased in ITP and is able to drive the activation of autoreactive B lymphocytes in this disease.     

The effect of platelet autoantibodies on the course of the disease and clinical response of patients with idiopathic thrombocytopenic purpura

In this study, we evaluated the response to treatment of 409 idiopathic thrombocytopenic purpura (ITP) patients who were tested for the presence of platelet-associated autoantibodies by direct-platelet immunofluorescence test (PIFT) and for the presence of plasma antibodies directed against the GPIIb/IIIa, GPIb and GPIa/IIa by monoclonal antibody immobilization of platelet antigens (MAIPA). In patients with platelet autoantibodies in comparison with patients without antibodies more frequently were observed the chronic form of disease (83.5%vs. 68.5%) and severe symptoms of haemorrhage diathesis (17.3%vs. 6.9%). Evaluation of the treatment response (to corticosteroids, immunosuppressive drugs and splenectomy) referred to patients with complete response, e.g. complete remission defined as platelet count of >100 x 10(9)/l for at least 2 years. The percentage of complete response in the whole population of ITP patients, both with and without autoantibodies regardless of the method of treatment, was similar (about 54%). However, the presence of platelet autoantibodies had effect on patients treated with corticosteroids: complete response approximately 71% (36/51) of patients with autoantibodies and in 60% (72/120) of patients without antibodies, as well as in patients treated with immunosuppressive drugs (cyclophosphamide, azathioprine, vincristin and vinblastin); complete response approximately 51% (11/21) of patients with autoantibodies and in 34.8% (6/17) of patients without autoantibodies. The presence of autoantibodies had no effect on the response of splenectomy patients.     

Demonstration of platelet antigens that bind platelet-associated autoantibodies in chronic ITP by direct immunoprecipitation procedure

Platelet antigens that bind platelet-associated autoantibodies in chronic idiopathic thrombocytopenic purpura (ITP) were demonstrated using a direct immunoprecipitation procedure. ITP platelets, with bound autoantibodies, were radiolabelled and solubilized, and then platelet antigen-antibody complexes adsorbed to protein A-bearing Staphylococcus aureus were analysed by 7.5% sodium dodecyl sulphate, polyacrylamide gel electrophoresis (SDS-PAGE). Direct immunoprecipitation demonstrated the presence of platelet-associated autoantibodies against glycoprotein (GP) IIb/IIIa in four of six ITP patients with an intensive band corresponding to platelet-associated IgG. These results were confirmed by indirect immunoprecipitation using ether eluates from two ITP patients. In addition, only direct immunoprecipitation demonstrated the presence of autoantibodies against an unidentified protein having a molecular mass of 56 kDa in three of the six patients. These three ITP patients having autoantibodies against GP IIb/IIIa and against the 56 kDa protein were studied after splenectomy. Two patients, showing disappearance of autoantibodies against these antigens, attained a complete remission, and one patient, with autoantibodies against the 56 kDa protein despite splenectomy, attained only partial remission. These data suggest that autoantibodies against GP IIb/IIIa and against the 56 kDa protein may play a role in platelet destruction in some ITP patients.