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.     

Platelet glycoprotein IIb as a target antigen in two patients with chronic idiopathic thrombocytopenic purpura

We have investigated the target antigens recognized by anti-platelet antibodies in patients with chronic idiopathic thrombocytopenic purpura (ITP) using an immunoblot procedure which could electrically separate the glycoprotein (GP) IIb/IIIa complex into GPIIb and GPIIIa. Various platelet proteins, having molecular weights of 167, 160, 145, 135, 124, 102, 92 and 80 kD, were recognized by circulating antibodies in 11 of 40 ITP patients. We identified the 145 kD antigen band, seen in two ITP patients, as GPIIb using thrombasthenic platelets as a source of target antigens. In one patient the anti-GPIIb antibody reacted with autologous GPIIb. These studies provide direct evidence for the presence of autoantibodies against GPIIb in some ITP patients.     

Anti-GMP140 (CD62) autoantibody in a patient with autoimmune thrombocytopenic purpura

The presence of platelet GMP140 (CD62) antibodies was analysed by the MAIPA test in 57 sera from patients with AITP and on platelets from 54 patients with thrombocytopenia of suspected immune origin. A CD62 antibody was found in only one serum. Its specificity was confirmed by an ELISA and a radioimmunoprecipitation procedure using total intact platelets and immuno-purified GMP140. An increased amount of platelet-associated (PA) IgG, due to in vivo fixation of GMP140 and GpIIb/IIIa antibodies, was also found on the patient platelet membrane, suggesting that GMP140 autoantibody may contribute to immune platelet destruction. No increase in PAGMP140 antibody was found on the other 54 platelet suspensions.     

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.     

Idiopathic (autoimmune) thrombocytopenic purpura with a complement-fixing autoantibody and response to plasma exchange

A 69-year-old male presented with severe, life-threatening thrombocytopenia, subsequently diagnosed as autoimmune thrombocytopenic purpura (ATP). A potent complement-activating platelet-specific autoantibody was present in the patient's serum. The IgG autoantibody induced lysis of autologous and isologous platelets but was nonreactive with red cells and lymphocytes. It reacted in an indirect immunofluorescence test with platelets from 50 control subjects and with thrombasthenic platelets but failed to react with platelets from a patient with the Bernard-Soulier syndrome (BSS), suggesting that it recognizes a glycoprotein absent from BSS platelets. Treatment by exchange transfusion and plasmapheresis was followed by disappearance of the autoantibody and sustained remission.     

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.     

Mixed-type autoimmune hemolytic anemia after splenectomy for idiopathic thrombocytopenic purpura

A 30-year-old woman was admitted because of anemia and jaundice in 1999. She had been diagnosed as having idiopathic thrombocytopenic purpura (ITP) in 1996, but had shown no response to prednisolone (PSL). Subsequent splenectomy in 1998, followed by further treatment with PSL, had also been ineffective. On admission in 1999, the direct and indirect Coombs tests gave positive results, the cold agglutinin titer was high with anti-I specificity (IgM), and warm-type autoantibody (IgG) was positive. Autoagglutination of the patient's red blood cells was 3+ at 37 degrees C. A diagnosis of mixed-type autoimmune hemolytic anemia (AIHA) associated with ITP was made. Mixed-type AIHA occurring after splenectomy for ITP is very rare, only one case having been reported to date.     

Autoantibodies against platelet glycoprotein Ib in patients with chronic immune thrombocytopenic purpura

The present studies provide direct evidence that some patients with chronic immune thrombocytopenic purpura (ITP) have autoantibodies reactive with platelet glycoprotein Ib ( GPIb ). Microtiter wells coated with a monoclonal antibody that recognized GPIb were reacted with either platelet extract or a control cell extract. After washing and incubating with test plasma, well-bound IgG was quantitated using radioactive anti-IgG. When compared to plasma from normal subjects, plasma from 3 of 106 patients with chronic ITP had significantly increased quantities of IgG bound to microtiter wells reacted with platelet extracts. Negative results were obtained with the remaining 103 patients with chronic ITP and 59 patients with a variety of other platelet disorders. Plasma from two of the three positive patients precipitated a protein from 125I-surface-labeled platelet extract with a molecular weight similar to GPlb . One of the three patients with anti- GPlb antibody also had demonstrable autoantibodies to the platelet glycoprotein llb / llla complex.     

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.     

改良间接MAIPA法对免疫性与非免疫性血小板减少的鉴别诊断价值

目的:检测慢性特发性血小板减少性紫癜(CITP)患者及非免疫性血小板减少症患者的抗血小板特异性抗体,评价其诊断价值。方法:建立单克隆抗体特异性俘获血小板抗原技术并加以改进(改良MAIPA法)借以检测CITP患者、慢性再生障碍性贫血(CAA)患者、肿瘤化疗后血小板减少患者抗血小板GPⅡb/Ⅲa、GPⅠb/Ⅸ的特异性抗体。结果:CITP组血小板糖GPⅡb/Ⅲa、GPⅠb/Ⅸ抗体的总阳性率高于CAA组和肿瘤化疗组,且差异有统计学意义(χ2=15.56,P<0.01和χ2=13.02,P<0.01)。结论:在CITP患者中血小板特异性抗体有较高的特异性,具有较高诊断价值,且2种抗体的功效相同。     

Acute immune (idiopathic) thrombocytopenic purpura in childhood

Acute immune (idiopathic) thrombocytopenic purpura (ITP) in childhood is most commonly a self-limiting condition with unexplained onset and resolution. In cases of severe thrombocytopenia, or situations where the condition persists beyond 6 months, treatment may be required to minimize the danger of life-threatening intracranial hemorrhage. Nonsurgical treatment options include corticosteroids, intravenous gammaglobulin (i.v.Ig), or anti-D. Specific indications, benefits, and limitations of these modalities are discussed, with recommendations for future directions in therapy.     

Dapsone for autoimmune thrombocytopenic purpura

Twenty-one human immunodeficiency virus (HIV)-free and six HIV-infected adults with autoimmune thrombocytopenic purpura (AITP) were treated with dapsone (100 mg/day). A response was observed in 13 patients (median platelet count before 25 × 10⁹/L, range 3–49; after 109 × 10⁹/L, range 69–241). Thrombocytopenia recurred in four of the responders in whom dapsone was discontinued. No response was observed in 12 patients. Dapsone had to be withdrawn after two weeks of treatment in the remaining two patients and after six to eight weeks in three other patients due to intolerance. No serious hematological complications were observed. These results confirm that dapsone is a safe, inexpensive, and effective treatment of AITP. © 1993 Wiley-Liss, Inc.     

Platelet autoantibody in a case of infectious mononucleosis presenting as thrombocytopenic purpura

In this unusual case acute thrombocytopenic purpura was the sole clinical manifestation of infectious mononucleosis. An antiplatelet antibody was demonstrated in the serum by two newly described technics: release of radioactive serotonin from platelets and inhibition of platelet aggregation following exposure of platelets to serum containing antiplatelet activity. The antiplatelet activity was completely inactivated by adsorption to washed platelets and was located in the immunoglobulin G (IgG) gamma globulin fraction of serum. The patient's serum also reacted against her own platelets indicating that the antiplatelet factor behaved as a true autoantibody.