Hajime Mizutani

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.

Demonstration of the heterogeneity of epitopes of the platelet-specific alloantigen, Baka

It is well known that the platelet‐specific alloantigen, Baka is carried on glycoprotein (GP) IIb, but little is known about the biochemical characteristics of its epitopes. To clarify the characteristics of the epitopes, we examined the interaction of four anti‐Baka sera (Yam, Lin, Kl and MO) with their epitopes, either with or without modifications by sodium dodecyl sulphate (SDS) and/or neuraminidase. By immunoprecipitation, all four antisera bound to the intact GP IIb/IIIa complex from a Baka‐positive subject. In contrast, immunoblotting demonstrated that Yam, Lin and Kl bound to SDS‐denatured GP IIb, while MO did not. When blotted GP IIb was treated with neuraminidase, Yam and Lin did not bind to desialylated GP IIb, while Kl still did. When the purified GP IIb/IIIa complex or washed platelets were treated first with neuraminidase followed by immunoblotting, the molecular weight of GP IIb decreased from 145 kD to 138 kD; Yam did not bind to desialylated GP IIb, but Kl did. Furthermore, to eliminate the effect of SDS, we examined the interaction of Yam and Lin with neuraminidase‐treated platelets using flow cytometry. The results were the same as those obtained using immunoblotting. Our results thus demonstrate that the expression of the Baka epitopes is not uniform and that sialic acid contributes to the expression of some actual allogenic epitopes.

Immunochemical characterization of an autoantigen on platelet glycoprotein IIb in chronic ITP : comparison with the Baka alloantigen

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 IIIα 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 IIbα. Immunoblots of platelets digested in situ with chymotrypsin indicated that the 65 kD fragment of GP IIbα 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 IIbα, carries a clinically important determinant(s) recognized not only by the anti‐Baka alloantibody, but also by the ITP autoantibody.

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

Summary 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<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<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.

Formation of Tubuloreticular Inclusions in Mitogen-Stimulated Human Lymphocyte Cultures by Endogenous or Exogenous Alpha Interferon

Tubuloreticular inclusions (TRI) were induced in normal blood lymphocytes after incubation with Staphylococcus aureus Cowan 1 (STA), but they were not induced by pokeweed mitogen (PWM), as we reported previously. TRI were also induced in Raji cells when grown in the medium of STA culture. Alpha-interferon (alpha IFN) was detected only in the medium of STA culture and not in PWM culture. The cells of PWM cultures formed TRI when exposed to various concentrations of human leukocyte alpha IFN. The incidences of TRI-positive cells in the presence of 50-500 IU/ml of alpha IFN were 3-5% on day 2 and increased to 10% on day 7. On days 5-7 of the PWM cultures, plasmacytoid cells containing TRI were seen not infrequently. In the presence of a high concentration of alpha IFN (10,000 IU/ml), which was sufficient to inhibit cell growth and differentiation, the growth of the TRI region was not altered and the incidence of TRI-positive cells was 9% on day 2 and increased to 15% on day 7. Our observations suggest that the TRI formation in STA culture is attributable to the alpha IFN produced endogenously by STA-stimulated cells and that some relationship might exist between the incidences of TRI-positive cells in these mitogen-stimulated cultures and the biologic functions of IFN.

Expression of GPIV and Naka antigen on monocytes in Naka‐negative subjects whose platelets lack GPIV

Summary. The platelet antigen Naka was once considered to be a platelet‐specific alloantigen and is carried on platelet membrane glycoprotein (GP) IV. Recent studies suggest that Naka‐negative subjects lack platelet GPIV. GPIV is an important adhesive receptor and expressed on the surface of monocytes as well as of platelets. In the present study, flow cytometry was used to detect GPIV and Naka antigen on the surface of monocytes. Naka antigen was expressed on monocytes as well as on platelets in Naka‐positive subjects (n= 6) (P‐GPIV‐positive subjects). To our surprise, monocytes of Naka‐negative subjects (n= 7) (P‐GPIV‐negative subjects) having no anti‐Naka antibody in their serum expressed GPIV and Naka antigen to almost the same degree as did the monocytes of P‐GPIV‐positive subjects. Competitive experiments using OKM5 (a monoclonal antibody against GPIV) and anti‐Naka antibody showed that the epitope of anti‐Naka antibody on monocytes was very close to that of OKM5. In two P‐GPIV‐negative subjects having anti‐Naka antibody in their serum, GPIV and Naka antigen were not expressed on the surface of either monocytes or platelets. These results indicate that the GPIV molecules and Naka antigen are expressed on the surface of monocytes in the majority of P‐GPIV‐negative subjects, but that in a very few P‐GPIV‐negative subjects neither GPIV nor Naka antigen is expressed on the surface of their monocytes. We hypothesize that P‐GPIV‐negative subjects who carry neither GPIV nor Naka antigen on their monocytes produce anti‐Naka antibody as a result of transfusion or pregnancy.

Demonstration of platelet antigens that bind platelet-associated autoantibodies in chronic ITP by direct immunoprecipitation procedure

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.

Acid Treatment of Platelets as a Simple Procedure for Distinguishing Platelet-Specific Antibodies from Anti-HLA Antibodies: Comparison with Chloroquine Treatment

Abstract. The identification of antibodies to platelet‐specific antigens is important for correctly diagnosing neonatal alloimmune thrombocytopenia, posttransfusion purpura and refractoriness due to platelet‐specific antibodies. However, the serologic identification of these platelet‐specific antibodies is complicated by the presence of anti‐HLA antibodies. We examined and compared the diagnostic usefulness of acid‐treated and chloroquine‐treated platelets for the discrimination of platelet‐specific antibodies from anti‐HLA antibodies. The viability of acid‐treated platelets is 83.4%, which is better than that of chloroquine‐treated platelets (52.6%). The antigenicity of HLA class I antigens of acid‐treated platelets was significantly reduced compared with that of PBS‐ or chloroquine‐treated platelets. On the other hand, platelet surface glycoprotein Ib and glycoprotein IIb/IIIa, and platelet‐specific antigens were stable following acid or chloroquine treatment. Chloroquine‐treated platelets were not suitable targets for analysis by immunofluorescence flow cytometry because of nonspecific fluorescence derived from platelet damage. We conclude that acid‐treated platelets are more suitable targets than chloroquine‐treated platelets for screening for platelet‐specific antibodies and also for analyses of the specificity of platelet‐specific antibodies.

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.

Impaired suppressor function of T cells induced by autologous mixed lymphocyte reaction in patients with idiopathic thrombocytopenic purpura.

Autologous mixed lymphocyte reaction (AMLR)-induced suppressor function was studied in 12 patients with chronic idiopathic thrombocytopenic purpura (ITP). The function was found to be significantly impaired (20 +/- 55%; p less than 0.005) compared with normal subjects (69 +/- 25%). AMLRs in these patients were significantly decreased (p less than 0.05) compared with normal subjects. There was no significant correlation between AMLR-induced suppressor function and platelet counts. Nine patients were studied for AMLR-induced suppressor function before and after splenectomy. The platelet counts increased significantly as a result of splenectomy, but the AMLR-induced suppressor function showed no significant improvement. The results of this study suggest that suppressor dysfunction in ITP may be an immunologic defect irrespective of disease activity. We consider that this abnormality may reflect in vivo failure of the immunoregulatory (feedback) mechanism in ITP.