Search

Acid treatment of normal washed platelets prepared by albumin density gradient centrifugation followed by formalin fixation effectively eliminates HLA class I antigens from the platelet surface, without altering the immunoreactivity of platelet membrane glycoproteins Ib and IIb/IIIa complex. Using these platelets, flow cytometric analysis was performed on a total of 20 sera from different patients with chronic ITP, and revealed an inverse linear correlation between the platelet count and the mean fluorescence intensity which represents a relative amount of platelet-bound IgG. These results suggest that acid-treated, formalin-fixed platelets can be used as specimens to detect anti-platelet antibodies to differentiate from those to HLA-class I antigens.

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.

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.

Male (NZW x BXSB) F1 (W/B F1) mice, which develop lupus nephritis, myocardial infarction, and thrombocytopenia, showed reduced platelet lifespan (PLS) and increased platelet-associated antibody (PAA) values. There were statistically significant correlations between the increase in PAA values and either the reduction in PLS or the decrease in platelet counts. This and the results of platelet transfer experiments between old male W/B F1 mice and either female W/B F1 or normal BALB/c mice indicate that PAAs on the platelet surface play a crucial role in the destruction of platelets in W/B F1 mice. The mechanism of thrombocytopenia observed here appears similar to that of human idiopathic thrombocytopenic purpura (ITP). Therefore, we think that W/B F1 mice are a potentially useful animal model for investigating the effectiveness and mode of action of therapeutic agents in human ITP, and that they may provide additional information on the basic mechanisms of this autoimmune phenomenon.

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.

In order to measure platelet-associated IgG (PAIgG), we devised a solid-phase enzyme immunoassay employing a competitive binding of peroxidase-conjugated anti-IgG antiserum between platelets and polystyrene tubes coated with IgG. The amounts of peroxidase bound to the tubes were measured in a spectrophotometer by an enzymatic reaction. This method is highly sensitive, reproducible and can be carried out more simply. the PAIgG values of normal controls averaged 21.6 +/- 6.6 (SD) ng/10(7) platelets. 27 (93%) of 29 patients with idiopathic thrombocytopenic purpura (ITP), who had a platelet count of less than 15 X 10(4)/microliter, had PAIgG values greater than those of controls by 2 SD and averaged 205.5 +/- 323 ng. There was a significant inverse correlation between platelet count and PAIgG value of ITP patients. the PAIgG values of patients with aplastic anemia were within normal range.

40 patients with idiopathic thrombocytopenic purpura (ITP) were studied for T-cell abnormalities using a panel of monoclonal antibodies (McAbs). These patients showed a tendency to have a decreased OKT4+:OKT8+ ratio compared with normal subjects and a significant increase was observed in OKT10-positive cells in ITP patients. Further analyses were made utilizing McAbs of different subclasses in combination (BMA040 mouse IgG1 McAb, helper/inducer T and BMA081 mouse IgG2 McAb, suppressor/cytotoxic T). An increased proportion of cells reactive with both BMA040 and BMA081 McAbs (double-labelled cells) was demonstrated in patients with ITP compared with the normal controls. Furthermore, there was an inverse correlation between the proportion of "double-labelled cells" and the platelet counts. These data suggest that abnormalities of the T-cell subsets in the peripheral blood may play an important role in the pathogenesis of ITP.

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.

High-dose gammaglobulin therapy for patients with idiopathic thrombocytopenic purpura (ITP), introduced by Imbach et al., was applied to 5 adults with chronic refractory ITP to investigate the mechanism of the increase in platelet counts. In 4 of the 5 cases, transient increase in platelet count was observed. Platelet-associated IgG was decreased in 3 cases, increased in 1 case and unchanged in 1 case after treatment. In 4 cases having a variety of autoantibodies, the antibody titers decreased after treatment. No significant changes in antiplatelet antibody titers in serum, or in circulating immune complexes were observed during or after these treatments. No side effects were noted in any of the cases. These results indicate that the treatment is suitable for the treatment of patients prior to surgery and of patients at high risk of intense bleeding and death. It can be reasonably assumed that the increased platelet count is mainly due to a defective removal of antibody-coated platelets.

In vitro phagocytosis of platelets from patients with idiopathic thrombocytopenic purpura (ITP) (direct assay) and phagocytosis of normal platelets sensitized with patient serum (indirect assay), using normal peripheral blood leukocytes, were studied to find out whether the degree of phagocytosis reflects the severity of ITP. Phagocytosis was measured by the uptake of 51Cr-labeled platelets. Among patients whose platelet count was below 10 X 10(4)/microliters, 92% showed an increase in the phagocytosis ratio by the direct assay and 67% by the indirect assay. There was a highly significant inverse correlation between the direct phagocytosis ratio and the platelet count (p less than 0.001), but not between the indirect phagocytosis ratio and the platelet count. The amount of platelet-associated IgG (PAIgG) measured in patients whose platelets were used for the direct assay also showed an inverse correlation with the platelet count (p less than 0.05). Vincristine administration provoked a reduction in the direct phagocytosis ratio and PAIgG value. We propose that both the direct assay and PAIgG measurement reflect the severity of ITP, and PAIgG might be responsible for platelet phagocytosis.

Chronic ITP is due to antibody-induced destruction of platelets by the reticuloendothelial (RE) system. The role of complement in this process is unclear. We measured platelet-associated complement (PAC) components C3, C3bi, C4 and C9 in 16 patients with chronic ITP, in two of these patients prior to and after splenectomy. Competitive solid-phase radioimmunoassays using monoclonal antibody (anti-C3d, anti-C3bi neoantigen or anti-C9) or affinity-purified heterologous antibody (anti-C4) were used. Mean values (+/- SD) of normal subjects (ng/10(7) plts) were: PAC3d 17.6 +/- 6.8; PAC3bi 11.6 +/- 2.3; PAC4 1.6 +/- 0.5; PAC9 9.9 +/- 2.6. Significantly elevated (greater than 2 SD) PAC3, PAC3bi, PAC4 and PAC9 levels occurred in 12/16, 11/14, 10/14 and 5/9 chronic ITP patients. The PAC3, PAC3bi and PAC9 values correlated inversely with the patients' platelet counts (P less than 0.001); PAC4 levels did not. A positive correlation was also noted between PAC3, PAC3bi and PAC9 while PAC4 values showed no correlation. Two patients with preoperative elevation of all four PAC proteins showed normalization of PAC3, PAC3bi and PAC9 values after a splenectomy-induced remission; PAC4 levels remained elevated for up to 5 months after surgery. We conclude that in vivo C activation occurs in most chronic ITP patients with binding of C3 and C9 to the platelet surface. This in vivo C activation may promote more efficient phagocytosis (C3b) and possibly platelet lysis (C5-9) in some ITP patients.

Peripheral blood T cells from 40 patients with idiopathic thrombocytopenic purpura (ITP) were analysed for the presence of surface Ia antigens using monoclonal antibodies by indirect immunofluorescence. The percentage of Ia-positive (Ia+) T cells was significantly increased in patients with ITP (6.8 +/- 2.9%, P less than 0.005) as compared with normal controls (2.3 +/- 0.9%). There was an inverse correlation between the percentages of Ia+ T cells and platelet counts (r = 0.58, P less than 0.005) and a positive correlation between the percentage of these cells and platelet-associated IgG (PAIgG) values (r = 0.55, P less than 0.01). The percentage of Ia+ T cells was found to decrease within two weeks during therapy with high dose gamma-globulin or corticosteroid. We have previously reported the presence of T cells bearing both helper/inducer (H/I) and suppressor/cytotoxic (S/C) phenotypes (double labelled cells, DLC) in patients with ITP and an inverse correlation between the percentages of DLC and their platelet counts. In the present study, we showed that a major part of Ia+ T cells had both H/I and S/C phenotypes. We also examined the correlation between Ia+ T cells and autologous mixed lymphocyte reaction (AMLR). A defective AMLR was demonstrated in patients with ITP. Furthermore, an inverse correlation was found between the percentages of Ia+ T cells and the proliferative responses to AMLR (r = -0.49, P less than 0.01). These results suggest that increased circulating Ia+ T cells play a role in the abnormalities of the immunoregulatory system of ITP, especially in the regulation of autoantibody production.

Platelet membrane glycoprotein (GP) IIb carries not only the Baka alloantigen but also an autoantigen in some patients with chronic idiopathic thrombocytopenic purpura (ITP). We previously reported one ITP patient (RY) with anti-GPIIb autoantibody in her plasma. In this report, we investigated whether the epitope of the autoantigen on GPIIb (case RY) is identical to that of the Baka alloantigen or not. The anti-Baka antibody used in this study was obtained from a mother with a child suffering from neonatal alloimmune thrombocytopenic purpura as reported by Okada et al. Immunoblotting showed that the anti-Baka antibody bound to GPIIb from Baka-positive platelets only. In contrast, the anti-GPIIb autoantibody in RY plasma bound to GPIIb, irrespective of Baka phenotype. In addition, after neuraminidase treatment of GPIIb on nitrocellulose paper, the anti-Baka antibody failed to bind to Baka-positive GPIIb, while the autoantibody still bound to GPIIb. From these data, we conclude that the epitope of the autoantigen in RY patient is different from that of the Baka alloantigen.

Circulating immune complexes (CIC) were frequently observed in patients with idiopathic thrombocytopenic purpura (ITP). To analyse the pathogenic role of CIC, we studied the correlation between the disease activity and the CIC level, and whether platelet antigens were involved in CIC from ITP sera. Elevated CIC levels, measured by Clq, anti-C3 and spermatozoa micro-enzyme-linked immunosorbent assay, were found in 37%, 54% and 52% of the patients, respectively. However, there were no significant correlations (r = -0.11, -0.03 and -0.04, respectively) between the platelet count and the CIC level. Platelet antigens in CIC were detected with rabbit anti-human platelet serum. The CIC from 18%, 25% and 25%, respectively, of ITP sera contained platelet antigens, but the CIC from only 3%, 9% and 6% of SLE sera and from 3%, 2% and 5% of the sera of alloimmunized patients, respectively, contained these antigens. There were significant correlations (r = 0.51, 0.80 and 0.65, respectively) between the amount of platelet antigens and the CIC level in ITP sera. However, there were no correlations (r = -0.26, -0.29 and 0.08, respectively) between the platelet count and the amount of platelet antigens in the CIC. We detected platelet antigens in CIC from ITP sera, but surmise that these CIC perhaps do not play an important role in platelet destruction.

Employing an immunoblotting procedure, we have identified and characterized an autoantigen carried on glycoprotein (GP) IIb in a patient with chronic idiopathic thrombocytopenic purpura (ITP), and have compared the location of the autoantigen with that of the platelet-specific alloantigen Baka. Immunoblots, using the partially purified GP IIb/IIIa complex as the target antigen, indicated that GP IIb alpha carried both the ITP autoantigen and the Baka alloantigen. The ITP plasma contained another antibody against a 100 kD protein (P100), a trace contaminant in the GP IIb/IIIa sample, which is probably a proteolytic fragment of an internal 124 kD protein. After chymotrypsin treatment, the auto- and alloantigen were found to be located on 65 kD fragments detectable under reducing conditions. In addition, immunoblots made after two-dimensional nonreduced-reduced SDS-polyacrylamide gel electrophoresis (SDS-PAGE) directly demonstrated that both 65 kD fragments had a molecular weight of 80 kD under nonreducing conditions; this provides evidence that these fragments were one and the same, and were derived from GP IIb alpha. Immunoblots of platelets digested in situ with chymotrypsin indicated that the 65 kD fragment of GP IIb alpha was retained by the platelet membrane. We conclude, therefore, that a 65 kD fragment, which represents the membrane side of the chymotrypsin cleavage site on GP IIb alpha, carries a clinically important determinant(s) recognized not only by the anti-Baka alloantibody, but also by the ITP autoantibody.

T cell subsets of peripheral lymphocytes were studied using monoclonal antibodies (OKT3, OKT4, OKT8) and immunoglobulin (IgG, IgM, IgA) synthesis in vitro by peripheral mononuclear cells stimulated with pokeweed mitogen was studied in adult patients with idiopathic thrombocytopenic purpura before and after high-dose intravenous intact gamma(gamma-)globulin therapy, at a daily dose of 0.4g/kg body weight for 5 consecutive days. A transient increase in platelet count which reached a peak on the 1st to 2nd days after the end of the therapy was observed in four of five patients. Immunoglobulin synthesis in vitro was suppressed remarkably following therapy in all cases: the mean reduction of IgG, IgM and IgA was 78, 66 and 48%, respectively. Titres of various autoantibodies, TGHA, MCHA, ANF and anti-DNA antibody, also decreased following therapy. In correspondence to this, phenotypic analysis in T cell subsets showed a decrease of OKT4+:OKT8+ ratio following therapy, without a change in the proportion of OKT3+ cells. These data indicate that the intravenous gamma-globulin preparations suppressed synthesis of antibodies non-specifically and caused a relative increase of OKT8+ suppressor T cells.

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.