Search

We investigated whether thrombocytopenia in patients with chronic hepatitis C is due to anti-platelet autoantibodies. Platelet-associated IgG (PAIgG) and platelet-associated IgM (PAIgM) were measured by direct immunofluorescent flow cytometric analysis. Elevation of PAIgM level was detected in 70% of chronic hepatitis C patients, while only a mild elevation of PAIgG level was detected in 32% of the cases. The elevation of PAIgM values in these patients was comparable to that in patients with chronic immune thrombocytopenic purpura (ITP). However, elevated PAIgM was also found in both patients with and without thrombocytopenia, and no correlation was found between PAIgM and platelet count. Eluted PAIgM did not react with normal platelets in all cases with a positive PAIgM value, indicating that eluted PAIgM contained no detectable anti-platelet antibodies. During alpha-interferon therapy, the level of PAIgM increased in association with the decrease in platelet counts in 75% of the cases; however, eluted PAIgM at any day point never reacted with platelets from normal donors. PAIgM was elevated in patients with chronic hepatitis C, but contained no detectable anti-platelet autoantibodies. Thrombocytopenia in these patients is not due to anti-platelet autoantibodies.

Effects of splenectomy on platelet kinetics and production of anti-platelet antibodies were studied in male (NZW x BXSB) F1 (W/B F1) mice, which are known as the animal model of immune thrombocytopenic purpura (ITP). Studies on organ localization of radiolabeled platelets revealed that splenic uptake significantly increases in W/B F1 mice in comparison with that of normal controls. W/B F1 mice showed a significant increase in platelet counts and, in contrast with sham-operated controls, high levels of platelet counts were maintained up to 6 weeks after splenectomy. Platelet lifespans (PLSs) did not reach normal levels, although prolonged PLSs were observed. In addition, platelet-associated antibody (PAA) values showed a tendency towards transient decrease, but there was no change in platelet-bindable serum antibodies (PBAs). These findings indicate that the suppression of anti-platelet antibody production is essential to the treatment of ITP; splenectomy may not be effective in treating severely affected ITP patients because, although the spleen is one of the major sites of platelet sequestration and antibody production, reticulo-endothelial systems (RESs) (liver, bone marrow, lymphnodes, etc.) other than the spleen are also responsible for the destruction of platelets. We therefore consider the W/B F1 mouse to be a useful model of human ITP, and believe that it provides valuable information for the development of new therapeutic agents in patients with ITP, especially those who do not respond to splenectomy.

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.

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.

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.

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.

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.

A new method was studied for eliminating HLA class I antigens from the surface of platelets without damaging the cells. Platelets were exposed to an acid solution (pH 3.0) to eliminate the antigenicity of HLA class I antigens. The reduction in antigenicities of HLA class I common antigen and individual HLA class I antigens by acid treatment was marked. Patients' sera which contained multispecific HLA antibodies reacted with PBS-treated platelets, but not with acid-treated platelets. No changes were observed in the antigenicities of glycoprotein Ib or glycoprotein IIb/IIIa. The viability of acid-treated platelets was 83%. Ultrastructural investigations revealed no significant difference between the PBS-treated platelets and acid-treated platelets. The platelet function studies showed that the aggregation of acid-treated platelets induced by various agonists was only slightly reduced compared with PBS-treated platelets. We propose that acid-treated platelets are promising for clinical use in patients refractory to platelet transfusions and may be superior to chloroquine-treated platelets for analysis of the specificity of antiplatelet antibodies.

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.

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.

We investigated the location of platelet-specific alloantigen Baka on platelet membrane glycoproteins. In indirect immunoprecipitation experiments, the anti-Baka antibody precipitated glycoprotein (GP) II b and a small amount of GP III a. The immunoblots using partially purified GP II b/III a complex as the target antigen indicated that GP II b alpha carried the Baka alloantigen. When the partially purified GP II b/III a complex digested with chymotrypsin was examined, the Baka alloantigen was found on a 65 kD fragment derived from GP II b alpha under reducing conditions. In addition, the immunoblots after two-dimensional nonreduced-reduced SDS-PAGE directly indicated that the 65 kD fragment had a mol. wt. of 80 kD under nonreducing conditions. The immunoblots using platelets digested in situ with chymotrypsin indicated that the 65 kD fragment of GP II b alpha was retained by the platelet membrane. We conclude, therefore, that the Baka alloantigen is located on a 65 kD fragment that represents the membrane side of the cleavage site of chymotrypsin on GP II b alpha.