Immunodominant epitopes on glycoprotein IIb-IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura.

It was recently reported that autoreactive CD4(+) T cells to glycoprotein IIb-IIIa (GPIIb-IIIa) mediate antiplatelet autoantibody production in patients with immune thrombocytopenic purpura (ITP). To further examine the antigenic specificity of the GPIIb-IIIa-reactive T cells, 6 recombinant fragments encoding different portions of GPIIbalpha or GPIIIa were generated and tested for their ability to stimulate antigen-specific T-cell proliferation and anti-GPIIb-IIIa antibody production in vitro. T cells from the peripheral blood of 25 patients with ITP and 10 healthy donors proliferated in response to recombinant GPIIb-IIIa fragments in various combinations. The amino-terminal portions of both GPIIbalpha and GPIIIa (IIbalpha18-259 and IIIa22-262) were frequently recognized (60% and 64%, respectively) compared with other fragments (4%-28%) in patients with ITP, but this tendency was not detected in healthy donors. In subsequent analyses in patients with ITP, T-cell reactivities to IIbalpha18-259 and IIIa22-262 were consistently detected, whereas those to other fragments were sometimes lost. In vitro antigenic stimulation of peripheral blood mononuclear cells with IIbalpha18-259 or IIIa22-262 promoted the synthesis of anti-GPIIb-IIIa antibodies in patients with ITP, but not in healthy donors. Of 15 CD4(+) T-cell lines specific for platelet-derived GPIIb-IIIa generated from 5 patients with ITP, 13 lines recognized IIbalpha18-259, IIIa22-262, or both. T-cell lines reactive to IIbalpha18-259 or IIIa22-262 promoted the production of anti-GPIIb-IIIa antibodies that were capable of binding to normal platelet surfaces. These results indicate that the immunodominant epitopes recognized by pathogenic CD4(+) T cells in patients with ITP are located within the amino-terminal portions of both GPIIbalpha and GPIIIa.     

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.     

Autoantigenic Epitopes on Platelet Glycoproteins

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by early platelet destruction mediated by antiplatelet autoantibodies. Platelet membrane glycoproteins (GP), especially GPIIb-IIIa and GPIb-IX, contain major autoantigenic determinants in chronic ITP. Recent advances in the localization of autoantigens as well as in the detection of GP-specific antibodies have improved our understanding of the pathophysiology of the disease. The N-terminal globular head of GPIIb-IIIa, particularly the beta-propeller domain in GPIIb, seems to play an important role as a hot spot for autoantigenic epitopes in chronic ITP.     

Increased number of B-cells in the red pulp of the spleen in ITP

Platelets are targeted by autoantibodies and destroyed in the reticuloendothelial system in the spleen, liver and bone marrow in patients with immune thrombocytopenia (ITP). Other mechanisms such as destruction by cytotoxic T-cells and defective production of platelets in the bone marrow also exist. Splenectomy normalizes the platelet count in 70% of ITP patients, however, precious little is known about the spleen in this disease. Our aim was therefore to investigate the splenic morphology and especially the number and localization of splenic leukocytes in patients with ITP and controls and to evaluate factors predicting outcome of splenectomy. Spleen sections from 29 ITP patients and 11 individuals splenectomized due to trauma were analyzed by immunohistochemistry. All except one of the ITP patients had a normalized platelet count 12 months after splenectomy and the platelet count was inversely correlated with age. ITP patients had an increased number of B-cells in the red pulp. The number of white pulp B-cells and number of T-cells in both compartments was unchanged. In conclusion, B-cells are increased in the red pulp of the spleen and together with cytotoxic T-cells, helper T-cells and macrophages line the sinusoids enabling the immunological attack on platelets in ITP.     

B cells expressing CD5 antigen are markedly increased in peripheral blood and spleen lymphocytes from patients with immune thrombocytopenic purpura

By two-colour flow cytometric analysis, we examined the proportion of B lymphocytes bearing CD5 cell surface antigen (CD 5+ B cells), which are capable of producing autoantibodies, both in peripheral blood and spleen from patients with chronic immune thrombocytopenic purpura (ITP). The percentage of CD5+ B cells in peripheral blood lymphocytes (PBLs) was significantly increased (P less than 0.005) in patients with ITP (3.7 +/- 2.2%, n = 30) as compared with normal controls (1.7 +/- 0.7%, n = 28). However, there was no correlation between the percentages of circulating CD5+ B cells and platelet counts. The percentage of splenic CD5+ B cells in ITP patients was much more increased (9.0 +/- 4.5%, n = 9), P less than 0.005) compared with that of other disorders (3.2 +/- 0.5%, n = 5). Furthermore, isolated splenic CD5+ B cells from two out of five ITP patients produced high levels of IgM-type, platelet-bindable antibodies (PBIgM) after stimulation with Staphylococcus aureus Cowan I (SAC), while CD5- B cells isolated from the same spleen or splenic CD5+ B cells from other non-autoimmune disorders failed to produce significant amount of PBIgM. In three ITP patients, no increase in PBIgM was detected despite SAC stimulation. The increased proportion of CD5+ B cells in peripheral blood and spleen, and their ability to produce anti-platelet antibodies indicate that they are directly involved in the autoimmune pathogenesis in ITP.     

Thrombopénie immunologique : de la physiopathologie aux traitements

Immune thrombocytopenia (ITP) is a rare autoimmune disease due to an immune peripheral destruction of platelets and an inappropriate platelet production. The pathogenesis of ITP is now better understood: it involves a humoral immune response which dependents on the stimulation of B cells by specific T cells called T follicular helper cells, leading to their differentiation into plasma cells that produce antiplatelet antibodies thus promoting the phagocytosis of platelets mainly by splenic macrophages. The deciphering of ITP pathogenesis has led to a better understanding of the inefficiency of treatments such as rituximab, although it has not provided yet the determination of biological predictive factor of response to treatments. Moreover, new therapeutic perspectives have been opened in the last few years with the development of molecules targeting Fcγ receptor signalling such as Syk inhibitor, or molecules increasing the clearance of pathogenic autoantibodies such as inhibitors of the neonatal Fc receptor (FcRn).     

Antiplatelet antibodies and their correlation with clinical findings in childhood immune thrombocytopenic purpura

The demonstration of antiplatelet antibodies (PAIgG, PAIgM) and decreased detection of platelet surface antigens (CD41, CD61, CD42b) in children with immune thrombocytopenic purpura (ITP) have a diagnostic role. This study was conducted to determine whether these parameters differed in acute and chronic ITP. Chronic ITP was defined as thrombocytopenia persisting for more than 6 months from the onset of illness. A total of 80 subjects were divided into three groups: group 1 included 39 patients with acute ITP; group 2 included 31 patients with chronic ITP, and group 3 included 10 healthy children. At diagnosis, blood samples were obtained for platelet count, mean platelet volume, plateletcrit and platelet distribution width along with platelet surface antigens and antiplatelet immunoglobulins. We found that platelet surface antigens were significantly decreased in both acute and chronic ITP when compared to the control group (p = 0.001). In contrast, PAIgG was increased in acute and chronic ITP patients compared to the control group. PAIgM was significantly higher in acute ITP. We conclude that decreased platelet surface antigens and increased antiplatelet antibodies are observed in both acute and chronic ITP. In patients with chronic progress, a relatively lower level of PAIgM can be identified.     

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.     

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.     

Immunochemical analysis of platelet autoantibodies in HIV-related thrombocytopenic purpura: a study of 68 patients

Serum antiplatelet IgG and platelet-associated IgG (PAIgG) were studied in 68 AIDS-free human immunodeficiency virus (HIV)-infected patients with severe immunologic thrombocytopenic purpura (ITP), for the presence of platelet autoantibodies. Serum IgG with antiplatelet activity was found in 72% of the sera. However, the presence of autoantibodies against platelet surface glycoproteins was not found in these sera by means of Western blot and immunoprecipitation procedures. Nevertheless, an immunoblot immunoassay and an indirect immunofluorescence test against semi-permeabilized platelets demonstrated the presence of antibodies in the patient sera, that reacted with intracytoplasmic platelet components, and which might participate in the elimination of platelet fragments. Direct immunofluorescence tests demonstrated an increased amount of PAIgG in 75% of the patients; the bound antibodies could be eluted with ether in 44% of the cases. These eluates were found to bind to normal platelets but not to Glanzmann type I platelets. Finally, immunoprecipitation procedures demonstrated the presence of platelet autoantibodies in six of the 35 eluates studied. These antibodies recognized GPIIb in two cases, GPIIIa in one case, and an unidentified platelet protein of 150 kDa in the three other cases. The discrepancy between sera and platelet eluates was interpreted as being due to the low titre of the antibodies and to their dilution in polyclonal hypergammaglobulinaemia. The present study provides direct evidence that isolated ITP in some HIV-positive patients is due to the presence of platelet autoantibodies. These results, however, do not exclude either direct or indirect involvement of HIV in the platelet destruction.     

Human monoclonal autoantibodies to characterize platelet antigens in immune-mediated thrombocytopenia

Summary Idiopathic thrombocytopenic purpura is characterized by antiplatelet antibodies which mediate the rapid destruction of these cells by the reticuloendothelial cell system. Low serum titers of autoantibodies and the polyclonal nature of human serum make it difficult to identify platelet target antigens with plasma antibodies. To circumvent these problems, we have utilized the techniques of EBV transformation and somatic cell hybridization in order to isolate human monoclonal antibodies from patients with ITP. In this paper we describe the use of human monoclonal autoantibodies to characterize an activation specific antigen on GPIIIa and an autoantigen on the GPIb complex. Ultimately, we hope to determine whether these autoantibodies emerge from a pool of naturally occurring antibodies to activation or senescence antigens, or are triggered by environmental agents such as bacteria or virus, which are comprised of antigens similar to those found on the platelet membrane.Presented at the International Workshop on ITP, August 26 and 27, 1988, Lucerne, Switzerland     

Different Antiplatelet Antibody Specificities in Immune Thrombocytopenic Purpura

It is generally accepted that patients with immune thrombocytopenic purpura (ITP) produce antibody against platelet-associated antigens; however, it is not known if these antiplatelet antibodies are directed towards the same or different antigenic sites. In the present studies, quantities of antiplatelet antibody from different ITP patients, sufficient to saturate platelet antigenic sites, were simultaneously incubated with normal platelets and the quantity of platelet-binding IgG (PBIgG) was determined. In each of the five comparisons made, the amount of PBIgG bound after incubation of normal platelets with saturating quantities of two ITP antibodies approximated to the sum of the PBIgG bound after incubation with the antibodies separately. These data suggest that the antiplatelet antibody from these ITP patients differed in antigenic specificity.     

Two human monoclonal antiplatelet autoantibodies established from patients with chronic idiopathic thrombocytopenic purpura

Two human hybridomas secreting antiplatelet autoantibodies were established by somatic cell fusion using splenocytes from two patients with chronic idiopathic thrombocytopenic purpura (ITP). These monoclonal antibodies, HT7F and HT8C, were of the IgM isotype and reacted with autologous and allogeneic platelets fixed with paraformaldehyde (PFA). They also bound to fresh platelets. An elution study showed that eluates from allogeneic platelets reacted with autologous platelets. These results indicated that HT7F and HT8C were autoantibodies recognizing a site on the platelet surface. Both monoclonal antibodies were able to induce complement activation in vitro. HT7F was demonstrated to bind to a platelet protein having a molecular mass of 105 kDa under both nonreducing and reducing conditions. No human hybridoma synthesizing antibody against 105 kDa platelet protein has been reported to date. These antibodies may play a role in the pathogenesis of thrombocytopenia in some ITP patients.     

A role of autoantibody-mediated platelet destruction in thrombocytopenia in patients with cirrhosis

Thrombocytopenia is a common manifestation in patients with liver cirrhosis (LC), but its underlying mechanism remains controversial. This study examined the role of anti-platelet autoimmunity in cirrhotic thrombocytopenia by determining the autoantibody response to GPIIb-IIIa, a major platelet surface autoantigen recognized by anti-platelet antibodies in patients with idiopathic thrombocytopenic purpura (ITP). Circulating B cells producing anti-GPIIb-IIIa antibodies as well as platelet-associated and plasma anti-GPIIb-IIIa antibodies were examined in 72 patients with LC, 62 patients with ITP, and 52 healthy controls. In vitro anti-GPIIb-IIIa antibody production was induced in cultures of peripheral blood mononuclear cells (PBMCs) by stimulation with GPIIb-IIIa. The frequency of anti-GPIIb-IIIa antibody-producing B cells in patients with LC was significantly greater than in healthy controls (10.9 +/- 6.2 vs. 0.4 +/- 0.3/10(5) PBMCs; P <.0001) and was even higher than the frequency in patients with ITP (8.2 +/- 5.2; P =.007). Anti-GPIIb-IIIa antibodies in the patients with LC and ITP were mainly present on the surfaces of circulating platelets rather than in the plasma in an unbound form. Furthermore, PBMCs from patients with LC and ITP produced anti-GPIIb-IIIa antibodies on antigenic stimulation with GPIIb-IIIa in vitro, and the specific antibodies produced had the capacity to bind normal platelet surfaces. In conclusion, the similar profile of the anti-GPIIb-IIIa autoantibody response in patients with LC and ITP suggests that autoantibody-mediated platelet destruction may contribute at least in part to cirrhotic thrombocytopenia.     

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.     

Identification of anti-platelet autoantibodies by western blot in 45 patients with idiopathic thrombocytopenic purpura (ITP).

BACKGROUND. In sera and platelet eluates of ITP patients, antigen specificity was widely studied by means of sensitive methods including immunoprecipitation, monoclonal antibody immobilization, and immunoblot. These studies indicated that GPIIb-IIIa were the main epitopes of ITP autoantibodies. METHODS. We studied the specificity of antiplatelet autoantibodies in 45 patients with acute and chronic ITP. Patient sera were tested by Western blot on separated platelet proteins in non-reducing conditions; antibody binding was identified using biotinconjugated anti-human IgG and avidin-peroxidase. RESULTS. Two main nonspecific bands of 200 and 125 kD were visible using normal serum; the first referred to platelet IgG, and the second was due to a naturally occurring antibody towards an internal protein. Twenty-five sera (55%) stained one (n = 11), two (n = 7), three (n = 3) or four (n = 4) specific bands. In patients with chronic ITP there was a prevalence of multiple bands. The relative molecular weights of the recognized antigens were in the range of 140-160, 80-100, 50-70 and 40 kd. The 80-100 epitope was recognized as a membrane protein in only 40% of sera, and it was partially characterized as GPIIIa in 4 patients. The other stained epitopes were absorbed by platelet lysate and then identified as internal proteins. CONCLUSIONS. This finding might be related to sensitization to antigens exposed by platelets during immune damage, and may pose an important problem in the identification with the immunoblot technique of target antigens responsible for immune sequestration.     

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.     

Novel approaches to refractory immune thrombocytopenic purpura

Chronic immune thrombocytopenic purpura (ITP) is an organ-specific autoimmune bleeding disorder in which autoantibodies are directed against the individual's own platelets, resulting in increased Fc-mediated platelet destruction by macrophages in the reticuloendothelial system. Although ITP is primarily mediated by IgG autoantibodies, the production of these autoantibodies is regulated by the influence of T lymphocytes and antigen-presenting cells (APC). There is evidence that enhanced T-helper cell/APC interactions in patients with ITP may play an integral role in IgG antiplatelet autoantibody production. New therapies may improve platelet production, decrease platelet antibody production, and decrease monocyte function and/or B-cell and T-cell activities. Understanding these cellular immune responses in ITP may lead to the development of more specific immunoregulatory therapies for the management of this disease.     

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.     

The isolation and characterisation of antiplatelet antibodies

The isolation and characterisation of antiplatelet antibodies in autoimmune thrombocytopenia purpura patients (ITP) is described. Autoimmune thrombocytopenia purpura is an autoimmune disease, clinically defined by low platelet counts, normal or increased megakaryocytopoiesis and antiplatelet antibodies in serum. This study used phage display to isolate Fab antiplatelet antibodies to study the structure-function relationships of pathogenic antibodies in ITP. Out of six randomly selected colonies, four colonies reacted strongly with whole platelets in enzyme-linked immunosorbent assay (ELISA). Sequence analysis showed that all four colonies had the same DNA sequence and were the same antibody. Results of Western blotting against non-reduced human platelet lysate showed that the Fab reacted with platelet proteins with apparent molecular weights of 116, 92 and 39 kD. Furthermore, Western blotting assay against purified membrane glycoprotein IIIa demonstrated reactivity against a band with a molecular weight of 92 kD. Results from Western blotting against platelet lysate and pure platelet glycoprotein confirmed the Fab fragment recognised the platelet glycoprotein IIIa. Three out of the four phage colonies produced soluble Fab, which demonstrated reactivity against platelet autoantigens in ELISA. Further sequence analysis showed that the Fab was somatically mutated suggesting antigen drive and therefore T-cell assistance was important in the development of this antibody. One of the somatic mutations introduced an RSD amino acid sequence in the complementary determining region 1 (CDR1) of the light chain, which may mimic the RGD motif of fibrinogen which binds integrin GPIIb/IIIa. This raises the possibility that somatic mutation and antigen drive have produced a pathogenic autoantibody.