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 against platelet membrane glycoproteins in children with acute and chronic immune thrombocytopenic purpura

The autoimmune nature of chronic immune thrombocytopenic purpura (ITP) in adults is widely accepted. In contrast, the pathogenetic mechanism in acute and chronic ITP in children is not known. In this report, we studied 39 children with destructive thrombocytopenia, 15 patients with acute ITP and 24 patients with chronic ITP. Platelet autoantibodies to platelet glycoprotein IIb/IIIa were detected in 14 of 24 patients (58.3%) in the chronic ITP group and in four of 15 (26.7%) with acute ITP. Binding ratios (+/- SD) of positive patients were significantly greater (P = .01) in chronic ITP (8.0 +/- 9.1) when compared with those of acute ITP where the binding ratios were only slightly above the normal range (1.9 +/- 0.4). The results show that autoantibodies against platelet glycoproteins are present in the majority of children with chronic ITP confirming the autoimmune nature of this disorder. The minimal elevation seen in the positive children with acute ITP suggests a different pathogenetic mechanism. These data suggest that this approach may be useful in differentiating acute from chronic ITP patients.     

Autoantibodies to the presumptive cytoplasmic domain of platelet glycoprotein IIIa in patients with chronic immune thrombocytopenic purpura.

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to autoantibodies against platelets that result in their destruction. In some patients, these autoantibodies bind to platelet glycoprotein (GP) IIIa. With the aim of better defining the antigenic epitopes, plasma from 13 selected patients with chronic ITP known to have anti-GPIIb/IIIa autoantibodies was tested for reactivity with nine synthetic peptides corresponding to different regions of the GP IIIa molecule. Of these plasmas, five bound significantly (P less than .001) with either peptide 8 (amino acids 721-744) or peptide 9 (amino acids 742-762), which together form most of the carboxyterminal region presumed to be the cytoplasmic domain. Three of these positive plasmas, were tested further. In two of these positive plasmas, the anti-peptide antibodies represented greater than 80% of the detectable circulating autoantibody. To further evaluate the importance of the carboxyterminal region as an antigenic site, the chronic ITP plasmas were tested against Chinese hamster ovary cells transfected with GPIIb and either whole GPIIIa or GPIIIa lacking amino acids 728 to 762. Ten of the 13 plasmas required the presence of this region for significant autoantibody binding. We conclude that the carboxyterminal region is an important area for stimulating antiplatelet autoantibody formation in some patients with chronic ITP. It is not known whether these autoantibodies to the presumed cytoplasmic domain play an important role in the pathogenesis of the disease or occur as a secondary phenomenon during the course of platelet destruction.     

Extracellular epitopes of platelet glycoprotein Ib alpha reactive with serum antibodies from patients with chronic idiopathic thrombocytopenic purpura

Glycoproteins (GPs) IIb/IIIa and Ib/IX are principal targets of autoantibodies (autoAbs) in idiopathic thrombocytopenic purpura (ITP). Platelet-associated Abs against GPIIb/IIIa primarily recognize discontinuous or nonlinear epitopes (Fujisawa et al, Blood 81:1284, 1993). This study focused on whether Abs against the extracellular domain of GPIb/IX might react with short linear amino acid (aa) sequences of GPIb alpha. Complementary DNAs (cDNAs) coding for two overlapping fragments of GPIb alpha were amplified, cloned into pFLAG.2 plasmids, and expressed in Escherichia coli DH5 alpha competent cells as FLAG fusion proteins, which were purified by anti-FLAG immunoaffinity chromatography. Of 16 selected ITP sera containing anti- GPIb/IX, 6 reacted in microtiter radioimmunoassays (RIAs) with recombinant protein fragment 2 (aas 240 to 485); 1 also with fragment 1 (aas 1 to 247). When synthetic peptides corresponding to 4 segments of fragment 2 with high antigenic indices (P1 to P4) were used as targets in RIAs, all 6 sera reacted with P2 (aas 326 to 346); 1 also reacted with P4 (aas 389 to 412). P2 was shown to be present on the surface of intact platelets by adsorption studies, and anti-P2 was detected in direct eluates of platelets from ITP patients. Glycocalicin in solution effectively competed with immobilized P2 for anti-P2; P2 in solution was a less effective competitor. Epitope scanning with a panel of synthetic 15-mer peptides localized the P2 epitope to the sequence, TKEQTTFPP. Epitope definition may offer insight into the pathophysiology of and more specific treatments for 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 the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP

Chronic idiopathic thrombocytopenic purpura (ITP) is caused by an antibody reactive with platelet-associated antigens. The present studies provide direct evidence that some patients with chronic ITP have autoantibodies against the platelet glycoprotein (GP) IIb/IIIa complex. Microtiter wells, coated with a monoclonal antibody (2G12) specific for GPIIb/GPIIIa were reacted with GPIIb/GPIIIa contained in a platelet extract. Control wells containing the same antibody were reacted with a cell extract containing no GPIIb/GPIIIa. After washing, the wells were reacted with patient or control plasma, and IgG binding was detected using 125I-Fab2-anti-human IgG. Assay values were expressed as binding ratios (cpm GPIIb/GPIIIa wells/cpm control wells). Plasma from 5 of 56 patients with chronic ITP had ratios (1.36-3.14) greater than 3 standard deviations above the mean (+/- SD) of control plasmas--0.93 +/- 0.12. Elevated values were also noted in two patients with anti-P1A1 antibody (ratios greater than 30) and in one patient with Hodgkin's disease and an ITP-like syndrome (ratio 1.53). Normal values were noted in 34 patients with a variety of immune and nonimmune diseases. Plasma from two of the positive ITP patients was reacted with 125I-surface-labeled platelets and, after solubilization, the IgG and bound antigen were precipitated with Staph-A. Autoradiographs from SDS- PAGE electrophoresis of the Staph-A-bound proteins shows two radioactive bands consistent in size with GPIIb and GPIIIa.     

Helicobacter pylori eradication in patients with chronic immune thrombocytopenic purpura

AIM:To assess the effect of Helicobacter pylori(H.pylori)eradication on platelet counts in patients with chronic immune thrombocytopenic purpura(cITP).METHODS:A total of 36 cITP patients were included in the study.The diagnosis of H.pylori was done by rapid urease test and Giemsa staining of the gastric biopsy specimen.All H.pylori positive patients received standard triple therapy for 14 d and were subjected for repeat endoscopy at 6 wk.Patients who continued to be positive for H.pylori on second endoscopyreceived second line salvage therapy.All the patients were assessed for platelet response at 6 wk,3rd and 6th months.RESULTS:Of the 36 patients,17 were positive for H.pylori infection and eradication was achieved in16 patients.The mean baseline platelet count in the eradicated patients was 88615.38±30117.93/mm3and platelet count after eradication at 6 wk,3 mo and6 mo was 143230.77±52437.51/mm3(P=0.003),152562.50±52892.3/mm3(P=0.0001),150187.50±41796.68/mm3(P=0.0001)respectively and in the negative patients,the mean baseline count was71000.00±33216.46/mm3 and at 6 wk,3rd and 6th month follow up was 137631.58±74364.13/mm3(P=0.001),125578.95±71472.1/mm3(P=0.005),77210.53±56892.28/mm3(P=0.684)respectively.CONCLUSION:Eradication of H.pylori leads to increase in platelet counts in patients with cITP and can be recommended as a complementary treatment with conventional therapy.     

Autoantibodies to platelet glycoprotein IIb/IIIa and to the acetylcholine receptor in a patient with chronic idiopathic thrombocytopenic purpura and myasthenia gravis

The coexistence of chronic idiopathic thrombocytopenic purpura and myasthenia gravis has been infrequently reported. We report another case and show the coexistence of autoantibodies to the platelet glycoprotein IIb and IIIa complex and the acetylcholine receptor. Autoantibody levels were followed during 8 weeks of treatment with cyclophosphamide, vincristine, and prednisone; the concentration of both autoantibodies fell during treatment but without measureable clinical improvement.     

Low prevalence of a polymorphism of platelet membrane glycoprotein Ib beta associated with neonatal alloimmune thrombocytopenic purpura in Asian populations

Iy alloantigen system is the first polymorphism of platelet glycoprotein Ib beta reported to cause neonatal alloimmune thrombocytopenic purpura. We investigated the allelic frequency of Iy alloantigen among Japanese and Korean populations by polymerase chain reaction-restriction fragment length method to determine the possibility of alloimmunization against Iy. Two hundred and nine Japanese and 97 Korean subjects were examined. All 306 individuals were homozygous for glycine at amino acid position 15 and negative for Iy. The allelic frequency of Iy in these populations was calculated to be less than 0.0016. Alloimmunization associated with Iy antigen in Asian populations seems unlikely from these results.     

Mapping helper T-cell epitopes on platelet membrane glycoprotein IIIa in chronic autoimmune thrombocytopenic purpura

Chronic autoimmune thrombocytopenic purpura (AITP) is associated with autoantibodies specific for platelet membrane components, often including glycoprotein GPIIIa. T helper (Th) cells reactive with GPIIIa, which are capable of driving the autoantibody response, are activated in AITP, and the aim here was to map the epitopes that they recognize. Peripheral blood mononuclear cells (PBMCs) were obtained from 31 patients with AITP and 30 control donors and stimulated with a panel of 86 overlapping synthetic 15-mer peptides spanning the complete sequence of GPIIIa. One or more peptides elicited recall proliferation by PBMCs from 28 of the patients, and, typically, multiple sequences were stimulatory. In contrast, responses in healthy control donors were rare (chi-square test = 115.967; P 相似文献   

Autoantibodies against platelet glycoproteins in autoimmune thrombocytopenic purpura: their clinical significance and response to treatment

Autoantibodies against platelet glycoproteins (GP) have been demonstrated in patients with autoimmune thrombocytopenic purpura (ATP). However, their clinical and pathogenetic significance as well as their response to immunosuppressive treatment is unknown. Using an immunobead assay capable of measuring autoantibodies against GPIIb-IIIa and GPIb-IX, we studied 58 adult patients with active ATP (platelet count < 150 x 10(9)/L) and 26 patients with ATP in remission (platelet count > 150 x 10(9)/L and without any therapy at time of investigation). Platelet-associated autoantibodies were detected in 39 of 53 patients with active ATP (73.6%) and in 2 of 26 patients in remission (7.7%). Circulating plasma autoantibodies were noted in 17 of 58 patients in the group with active disease (29.3%) and in none of the patients in remission. Twelve patients with active ATP and autoantibodies against GPIIb-IIIa were studied prospectively during treatment with corticosteroids. Of eight patients whose platelet count normalized during treatment, platelet-associated and plasma antibodies decreased significantly in two or became undetectable in six. In contrast, of four patients whose platelet counts were unchanged or increased moderately, we noted no significant change in antibodies. Moreover, autoantibodies reappeared in two responding patients at the time of relapse. The effect of high-dose intravenous immunoglobulin was studied in six active ATP patients with antiglycoprotein autoantibodies and refractoriness to prednisone. In one patient who developed a sustained remission after IvIgG, platelet-associated and plasma antibodies to GPIIb-IIIa decreased and became undetectable. In contrast, two patients who had only a transient rise of the platelet count after IvIgG showed no significant change in autoantibody. In three unresponsive patients, autoantibodies were without change in two and decreased transiently in one patient. We conclude that in ATP the presence of autoantibodies to GPIIb-IIIa and GPIb-IX is related to the activity of the disease. Corticosteroids may inhibit autoantibody formation in some ATP patients, whereas during the early response to IvIgG, autoantibody production may not be affected.     

Pathogenesis of chronic immune thrombocytopenic purpura

PURPOSE OF REVIEW: This article summarizes recent insights into the pathophysiology of immune thrombocytopenic purpura, a disorder in which autoantibodies against cell-specific glycoproteins (GPIIb-IIIa, GPIb-IX and others) accelerate platelet destruction. RECENT FINDINGS: Autoantibodies are produced by a limited number of B-cell clones. Platelet antibodies may also impair megakaryocyte development and platelet turnover, thromobopoietin levels are normal or only modestly increased and a compensatory increase in platelet production is not effective in many patients. Patients may show impaired immune regulation manifested by increased proliferation of helper T lymphocytes. Cytotoxic T lymphocytes from patients can lyse platelets in vitro. If cytotoxic T lymphocytes are also capable of perturbing megakaryocyte function, this mechanism may contribute to impaired platelet production. Polymorphisms in the Fcgamma-RIIIa gene may correlate with response to certain forms of therapy and similar genetic approaches may help to identify subsets of patients that differ in their natural history and response to various interventions. SUMMARY: Better understanding of autoantibody development, inhibition of thrombopoiesis and Fcgamma receptor and other polymorphisms will assume increased importance in elucidating the pathogenesis and targeting treatment of chronic immune thrombocytopenic purpura.     

Different specificities of platelet-associated and plasma autoantibodies to platelet GPIIb-IIIa in patients with chronic immune thrombocytopenic purpura.

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to antiplatelet autoantibodies, many of which are directed against platelet membrane glycoprotein (GP) IIb-IIIa or GPIb-IX. In a recent study, we described plasma autoantibodies from 13 selected ITP patients, which required the presence of the putative GPIIIa cytoplasmic region for antibody binding. Since this region may not be available for antibody binding under physiologic conditions, we evaluated the frequency of binding to this or other regions of GPIIb-IIIa by platelet-associated and plasma autoantibody from a group of chronic ITP patients. We studied platelet-associated autoantibodies in 27 patients and plasma antibodies in 21 patients; in 15 patients, both were studied. To determine if autoantibodies were directed to the cytoplasmic portion of GPIIIa or to another portion of the GPIIb-IIIa molecule, antibody eluted from patient platelets or plasma antibody was tested in an antigen capture assay for binding to GPIIb-IIIa obtained from Chinese hamster ovary (CHO) cells transfected with GPIIb and either intact GPIIIa or GPIIIa lacking the carboxy terminal 35 residues. Of the 21 plasma autoantibodies tested, 13 bound primarily to the carboxy terminus of GPIIIa and eight to other epitopes. Conversely, all 26 platelet-associated autoantibodies, including eight of the 13 with anti-carboxy terminus antibodies, bound to epitopes in other regions of GPIIb-IIIa. Comparison of the degree of antibody adsorption by intact or lysed platelets indicated that epitopes on the c-terminal region of GPIIIa are relatively inaccessible on the surface of intact washed platelets when compared with other epitopes. We conclude that the importance of plasma autoantibodies in chronic ITP patients should be interpreted cautiously, since their specificity may differ from that of antibodies bound to the platelet. Whether antibodies against the c-terminus of GPIIIa are of pathogenetic importance remains to be determined, although patients with these antibodies have particularly severe disease.     

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.     

Rituximab is effective for selected patients with chronic steroid-refractory immune thrombocytopenic purpura

We report results of Rituximab therapy in four patients with chronic immune thrombocytopenic purpura (ITP) refractory to 3-8 prior therapeutic regimens. Rituximab was administered at a dose of 375 mg/m2 once weekly for 4-6 weeks. Three out of four patients achieved a complete remission (rise to platelet count above 100,000/microl). Response duration was 4, 16+, and 11+ months. Rituximab was well tolerated but one patient (a 77 year-old male) developed two serious infections, pneumonia and a hepatic abscess, at 2 and 4 months. We conclude that Rituximab is effective in patients with refractory ITP; nevertheless, careful patient selection is mandatory.     

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.     

Danazol therapy in refractory chronic immune thrombocytopenic purpura

We report our experience with danazol in the treatment of patients with refractory immune thrombocytopenic purpura (ITP). The effects of this drug were investigated in 10 patients, 6 males and 4 females, aged from 40 to 85 years, (median 58 years), with a platelet count below 50 X 10(9)/l. The patients had previously been treated with steroids; one of them had also been unsuccessfully splenectomized. Danazol was administered at a dosage of 600 mg/day for 3 months. Before and after treatment, detection of antiplatelet antibodies was performed. Seven patients were treated for 3 months. One of them showed a transient increase of platelet count, in the others, no significant rise was noted. Six patients experienced side effects during treatment. We think that danazol does not appear to be an alternative therapeutical approach in refractory ITP.