Immunoglobulins targeting both GPIIb/IIIa and GPIb/IX in chronic idiopathic thrombocytopenic purpura (ITP): evidence for at least two different IgG antibodies

Antiplatelet antibodies in chronic idiopathic thrombocytopenic purpura (ITP) mainly target glycoprotein (GP) IIb/IIIa and GPIb/IX. Previous studies, employing modern antigen-specific assays, indicate that serum reactive with both GPIIb/IIIa and GPIb/IX is not an uncommon finding in chronic ITP. However, the mechanism behind this dual reactivity remains unclear. We studied sera from 72 patients with chronic ITP using modified GPIIb/IIIa- and GPIb/IX-specific MAIPA assays. Among the 34 positive sera, seven showed strong reactivity against both GPIIb/IIIa and GPIb/IX. These seven dual reactive ITP sera were further analysed by absorption studies. It was found that sera absorbed with immobilized GPIb/IX lost nearly all serum IgG specific for GPIb/IX but fully retained the IgG specific for GPIIb/IIIa. Conversely, sera absorbed with immobilized GPIIb/IIIa retained their reactivity only with GPIb/IX. These findings demonstrate that ITP sera, reactive with both GPIIb/IIIa and GPIb/IX, contain at least two different IgG antibody populations, each reactive with only one of the GP complexes.     

The IgG subclasses of platelet-associated autoantibodies directed against platelet glycoproteins IIb/IIIa in patients with idiopathic thrombocytopenic purpura

The majority of patients with idiopathic thrombocytopenic purpura (ITP) have antiplatelet autoantibodies that are most frequently directed against platelet glycoproteins IIb/IIIa or Ib/IX/V. However, there is some debate whether the immune response is oligoclonal or polyclonal in nature. We investigated the subclass distribution of anti-IIb/IIIa IgG autoantibodies in 59 prospectively studied patients with ITP. We also tested patients with a variety of thrombocytopenic disorders (n=31) and healthy controls (n=30). Platelet lysates were tested for IgG anti-IIb/IIIa autoantibodies, and the specific IgG subclass distribution was measured using antigen capture assays. All testing was done blinded to diagnosis and other assay results. After unblinding, we found that 43 of the 59 ITP patients had anti-IIb/IIIa autoantibodies (sensitivity=73%). Anti-IIb/IIIa autoantibodies were also detected in five of the 31 non-ITP patients, but in none of the 30 healthy controls (specificity=91%). The IgG subclass assay was positive in 39 of the 43 ITP patients with anti-IIb/IIIa antibodies (sensitivity=92%) and in 12 samples that had no detectable anti-IIb/IIIa antibodies including two ITP patients (specificity=83%). The most common subclass in the ITP patient samples was IgG1 (77%), either alone (n=14) or with other IgG subclass antibodies (n=19). However, there were also patients with only IgG2 (n=2), IgG3 (n=3) or IgG4 (n=3) antibodies. Our results are consistent with the hypothesis that ITP is a heterogeneous disorder and that some patients have evidence of oligoclonality, whereas other patients have polyclonal autoantibodies.     

Glycoprotein IIb/IIIa autoantigenic repertoire in chronic idiopathic thrombocytopenic purpura

Summary. The objective of the present study was to further disclose the autoantigenic repertoire carried by the platelet glycoprotein (GP) IIb/IIIa complex. IgG-F(ab')₂ fragments were prepared from two prototype ITP patients, and their ability to block the binding of GPIIb/IIIa reactive antibodies derived from other patients with ITP was evaluated using a modified MAIPA assay; a P1^A1 alloantiserum and 20 normal sera were included as controls. It was found that the two prototype IgG-F(ab')₂ fragments were each able to significantly block the binding of serum IgG to GPIIb/IIIa in six (55%) and seven (64%) out of 11 patients with chronic ITP, respectively. No significant blocking effect was observed for IgG-F(ab')2 fragments prepared from normal subjects. Also, the binding of the P1^A1 alloantiserum to its epitope on GPIIIa was not affected by any of the blocking IgG-F(ab')2 fragments exploited in the study. These data substantiate that in chronic ITP at least half of the GPIIb/IIIa reactive sera bind to homogenous autoepitopes.     

Evidence for a light chain restriction of glycoprotein Ib/IX and Ilb/IIIa reactive antibodies in chronic idiopathic thrombocytopenic purpura (ITP)

To address the assumption of clonally restricted antibodies in immune thrombocytopenias we studied sera from 19 patients with chronic ITP known to possess antibodies reactive with glycoprotein (GP) Ib/IX and/or GPIIb/IIIa. These sera were re-analysed using the standard monoclonal antibody immobilization of platelet antigens (MAIPA) assay and 16 patients exhibited IgG antibodies reactive with GPIIb/IIIa; seven patients showed also a reactivity with GPIb/IX. Employing a light-chain-specific MAIPA assay, 75% (12/16) of these sera displayed GPHb/ Ilia-specific antibodies that were light chain restricted; only 13% (2/16) of the GPIIb/IHa reactive sera showed a mixed kappa and lambda phenotype. A light-chain-restricted phenotype was also seen for the GPIb/IX reactive antibodies. To further substantiate these findings, the MAIPA assay was modified in order to avoid interference from human anti-mouse antibodies. A high frequency of light-chain restricted platelet antibodies was also found using the modified MAIPA technique. These results support the hypothesis of a clonal B-cell expansion in immune thrombocytopenias, producing antibodies with a restricted idiotype repertoire and reacting with a limited number of epitopes.     

Effect of Helicobacter pylori eradication on platelet recovery in Japanese patients with chronic idiopathic thrombocytopenic purpura and secondary autoimmune thrombocytopenic purpura

The prevalence of Helicobacter pylori infection and the effect of its eradication on platelet count in 48 Japanese patients with autoimmune thrombocytopenic purpura (AITP), including 40 chronic idiopathic thrombocytopenic purpura (ITP) and eight secondary AITP, were investigated. H. pylori infection was found in 25 ITP patients (62.5%) and in two secondary AITP (25%). H.pylori eradication was obtained in 19 of 19 infected ITP patients (100%), who were not in remission (platelets < 100 x 109/l) at the time of infection assessment. During follow-up (median 14.8 months), 12 of 19 H. pylori-eradicated patients (63.2%) showed a significant increase in platelet count accompanied by a significant decrease of platelet-associated immunoglobulin G (IgG). This response was maintained in all responding patients throughout the follow-up period. However, two infected patients with secondary AITP did not show platelet increase after eradication. The assessment of H. pylori infection and its eradication should be attempted in ITP as this approach could be an effective strategy, at least for some of these patients.     

Dapsone for refractory chronic idiopathic thrombocytopenic purpura

Summary. Fifteen patients with refractory chronic idiopathic thrombocytopenic purpura (ITP) were treated with dapsone (lOOmg/d) for 1-31 months. The overall response rate to dapsone was 40%. Five patients responded in 1 month and one patient in 2 months. No pretreatment characteristics -sex, age, platelet count or duration of ITP - were correlated with response to dapsone. Treatment was well tolerated. The most frequent adverse effect was dose-related haemolytic anaemia. In our experience, dapsone provides an inexpensive and well-tolerated alternative for patients with ITP who had inadequate reponses to conventional therapy.     

Pulsed intravenous high-dose dexamethasone in adults with chronic idiopathic thrombocytopenic purpura

The role of pulsed high-dose dexamethasone (DXM) in the treatment of patients with chronic idiopathic thrombocytopenic purpura (ITP) is still uncertain. Following an early report in which it was described as an effective and well-tolerated treatment with a sustained platelet response in 100% of cases, a number of subsequent studies have failed to confirm such favorable results. As all these studies were conducted on small numbers of patients, we investigated further the effectiveness and side effects of this therapeutic modality in a larger cohort. Thirty-two patients with chronic ITP were scheduled to receive six monthly courses of intravenous DXM at the dose of 40 mg/day for 4 consecutive days. All patients had ITP that had been resistant to between two and five different therapeutic regimens, including 9 patients who had already failed splenectomy. All patients had to be seen 2 weeks after each cycle to asses their response as well as secondary effects. Three patients failed to respond and clinically required other therapy. Thirteen patients (41%) had a partial (platelet count between 50 and 100 x 10(9)/liter) or complete (platelet count >100 x 10(9)/liter) response to treatment, responses being mostly transient. Responses were observed early during the course of treatment, usually right after the first cycle of DXM. There were no late responses. Side effects were mild and did not require discontinuation of treatment. No clinical or laboratory parameter was found to predict treatment outcome. We conclude that high-dose DXM has a limited effect in patients with chronic ITP. Novel approaches and controlled multicenter trials may help identify new therapeutic strategies for this disease.     

High-dose intravenous IgG for chronic idiopathic thrombocytopenic purpura in adults

Summary Sixteen adult patients of mean age 48 years with chronic ITP were studied for platelet response to high-dose (0.4 g/kg body weight per day for five consecutive days) intravenous polyvalent intact IgG in the absence of any concurrent treatment. The platelet count returned to normal values in nine patients, a partial response (rise in the platelet count between 50 and 150 ×10⁹/l) was observed in three cases. One patient refractory to any other treatment went into a sustained remission. In the other responsive patients the response was only transient. Among seven splenectomised patients only three responded to IgG infusions versus nine in the non-splenectomised group. The length of ITP history appeared as a more critical factor for the response to IgG than previous splenectomy.     

Characterization of autoantibodies against the platelet glycoprotein antigens llb/llla in childhood idiopathic thrombocytopenia purpura

The majority of children with idiopathic thrombocytopenia (ITP) have an acute self-limiting course and no diagnostic test has been identified which will predict the course of thrombocytopenia and detect those with the chronic autoimmune form. The detection of autoantibodies directed against the platelet glycoprotein complex llB/llla, may identify patients with chronic ITP. Serum anti-GP llb/llla antibodies were assessed by the indirect MAIPA assay in 54 children with immune thrompocytopenla at initial presentation along with an additional 7 children previously diagnosed with chronic ITP, to determine if there was a difference in antibody positivity between acute and chronic ITP patients, and whether the identification of antibodies could be used as a predictive test at diagnosis. There was no significant difference in the percentage of antibodies detected in children classified with acute ITP (27/40–68%) compared to children with chronic ITP (13/21-62%, P > 0.05). Patients with acute ITP had significantly lower mean platelet counts at diagnosis compared to the chronic ITP group (16,225/mm³ vs. 32,250/mm³, P < 0.05), though there was no significant difference in the bleeding manifestations between the acute and chronic ITP groups. Serum anti-GP llb/llla antibodies are detected in a high percentage of children with ITP and autoantibodies appear to be involved in the pathogenesis of both acute and chronic ITP. The detection of anti-GP llb/llla antibodies at diagnosis, however, does not appear to be a useful prognostic test in childhood ITP. © 1995 Wiley-Liss, Inc.     

Acute idiopathic thrombocytopenic purpura in children

Acute idiopathic thrombocytopenic purpura (ITP) characteristically follows a viral illness in preschool children. The exact role of viruses in the pathogenesis of this disorder remains uncertain, but the finding of markedly elevated levels of platelet-associated IgG serves to distinguish it from the chronic form of the disease and permits speculation on the mechanisms of platelet destruction. Although the spleen is important in both antibody production and platelet destruction, bone marrow synthesis of IgG has also been shown to be increased. The clinical course may be alarming, but mortality is low and prognosis excellent. Controversy has surrounded the role of steroids in the management of acute childhood ITP in retrospective studies. Controlled studies, however, indicate that thrombocytopenia is reversed sooner in treated patients. New assays for platelet-associated IgG offer new insights into this disorder and will allow delineation of acute and chronic disease at the time of diagnosis.     

Dapsone for chronic idiopathic thrombocytopenic purpura in children and adults--a report on 90 patients

Data on 90 patients (55 adults and 35 children) with chronic idiopathic thrombocytopenic purpura (ITP) and a platelet count of <50 x 10(9)/L treated with dapsone at a dose of 1-2 mg/kg/d are presented. A response was observed in 57 (63.3%) patients. The average time for response was 3.5 months (range 1-9) and the average duration of treatment with dapsone was 10.4 months (range 4-14). Overall response rates of 65.7% and 61.8% were observed in children and adults respectively. Side effects requiring discontinuation of therapy were observed in three (2%) patients. These results demonstrate that dapsone is an effective, inexpensive and well-tolerated treatment for chronic ITP, in both children and adults and could be considered for patients who fail steroid therapy.     

Absence of platelet response after eradication of Helicobacter pylori infection in patients with chronic idiopathic thrombocytopenic purpura

Eradication of Helicobacter pylori infection has been associated with the correction of thrombocytopenia in patients with idiopathic thrombocytopenic purpura (ITP). We have analysed the response to eradication of H. pylori in a series of 56 adult patients with chronic ITP. Forty patients had H. pylori infection (71%) that was eradicated in 23 of 32 evaluable patients (72%). Platelet counts did not significantly vary according to H. pylori treatment outcome. Three of 56 patients (5%) achieved a partial response attributable to H. pylori eradication. Therefore, detection of H. pylori infection should not be routinely included in the initial work-up of ITP.     

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.     

Investigation of megakaryocyte apoptosis in children with acute and chronic idiopathic thrombocytopenic purpura

OBJECTIVE: Although the platelet destruction shows a primary role in the thrombocytopenia of idiopathic thrombocytopenic purpura (ITP), it has been demonstrated that impaired platelet production may also contribute to the severity of thrombocytopenia in ITP. The present study examined megakaryocyte apoptosis in bone marrow aspirates of children with acute and chronic ITP and investigated the role of megakaryocyte apoptosis in ITP pathophysiology. METHODS: Thirteen children diagnosed with acute ITP and eight children diagnosed with chronic ITP comprised the study group. Ten children, who were hospitalized for scoliosis operation but healthy otherwise, comprised the control group. In all children, megakaryocytes were isolated from the same amount of bone marrow aspirate samples using MACS CD61 MicroBeads (Miltenyl Biotec, Auburn, CA, USA). Megakaryocyte apoptosis was studied with transferase-mediated d-UTP-bitin nick end-labeling method. RESULTS: Isolated megakaryocyte counts did not differ significantly between acute ITP, chronic ITP and control groups. The percentage of apoptotic megakaryocytes did not differ significantly between acute ITP group and control group and between chronic ITP group and control group. The percentage of apoptotic megakaryocytes in patients with chronic ITP was significantly lower than the patients with acute ITP. There was no correlation between the percentage of apoptotic megakaryocytes and platelet counts of the cases. CONCLUSIONS: Increased megakaryocytic apoptosis does not play a role in the pathogenesis of dysmegakaryopoiesis and impaired platelet production in children with ITP. Decreased megakaryocyte apoptosis in cases with chronic ITP may be due to suppression of megakaryocyte maturation, as the terminal phase of the megakaryocyte lifespan is characterized by the onset of apoptosis.     

Analysis of outcome of laparoscopic splenectomy for idiopathic thrombocytopenic purpura by platelet count

Laparoscopic splenectomy (LS) is now performed routinely in patients with idiopathic thrombocytopenic purpura (ITP) refractory to the medical treatment. Low preoperative platelet count was deemed to be a contraindication for a laparoscopic approach; however, there is no data reporting the outcome in those patients. We aimed to evaluate the influence of the preoperative platelet count on the operative and postoperative course and complication rate. Retrospective cohort study that was conducted in tertiary care university-affiliated medical center and included 110 consecutive patients who underwent LS. All patients were divided into three groups by their preoperative platelet counts: 50 x 10(9)/L (n = 80). The outcome and the influence of preoperative factors predictive of complications, blood transfusion, and length of stay were compared between the groups. Patients with a platelet count of 20 x 10(9)/L before surgery. Patients with counts >20 x 10(9)/L can safely undergo LS.     

Incidence of autoantibodies to GPIIb/IIIa in chronic autoimmune thrombocytopenic purpura may be overestimated by the MAIPA

Summary Twenty-one serum samples from patients with thrombocytopenia and five from normal individuals were analysed by the MAIPA test to determine specificity of autoantibody reactivity against platelet glycoprotein IIb/IIIa using murine monoclonal antibodies. The sera were also analysed by dot blot to determine their anti-murine IgG activity. Two of the three patient samples positive in the MAIPA test were positive in the dot blot test for anti-murine IgG activity, and on repeating the MAIPA following preabsorption of the serum with mouse ascites proteins they gave negative results. Reactivity of the sample which did not correct by this procedure was shown to be due to an anti-HPA1a alloantibody. The potential problem of human heterophile antibodies reacting with mouse antibodies in the MAIPA has been identified and a modification which corrects this has been demonstrated.     

High-dose intravenous methylprednisolone for acute childhood idiopathic thrombocytopenic purpura

49 children with acute idiopathic thrombocytopenic purpura (ITP) were divided into non-treatment, oral prednisone (2 mg/kg), and high-dose intravenous methylprednisolone (HIVMP) treatment groups which consisted of 17, 16 and 16 children respectively. Platelet counts rose above 150,000/microliters over a 2-week period in 5 (29.4%) children in the first group, 5 (31.2%) in the second group and 15 (93.7%) children in the third group. Platelet counts reached the normal level in only 3 days in 11 (68.7%) children treated with HIVMP. Initially, antiplatelet antibodies (APA) were shown by the Handin and Stossel method in every patient. With normalization of platelet counts, the antibodies decreased but could still be detected in every case; antibody decrease was greater in the HIVMP group. With the exception of mild cushingoid appearance, none of the major corticosteroid side effects was observed in the treated children.