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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.

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.

The clinical significance of high-titer antinuclear antibodies (ANA) and autoantibodies to cellular antigens such as SSA/Ro and nuclear RNP (nRNP) antigens in idiopathic thrombocytopenic purpura (ITP) was examined in a prospective evaluation of 66 adult patients with chronic ITP. ANA were positive in 29 (44%) of 66 patients with chronic ITP. The titers of ANA were high (1:160 or higher) in 14 of 29 ANA-positive patients. Furthermore, 10 of 66 patients had precipitating antibodies to nuclear antigens; seven patients had anti-SSA/Ro antibodies and the other three had anti-nRNP antibodies. None of high-titer ANA- or precipitating antibody-positive patients developed systemic lupus erythematosus (SLE) throughout the follow-up period of 3 years. In addition, we investigated retrospectively precipitating antibodies in stocked sera from 8 patients. These patients had already precipitating antibodies average of 7.7 years before. None of 8 patients developed SLE or Sjogren's syndrome (SS). These data demonstrate that high-titer ANA and antibodies to SSA/Ro or nRNP antigens are often found in patients with ITP, and indicate that the detection of high-titer ANA or the existence of antibodies to SSA/Ro or nRNP antigens by itself is not enough to identify those patients with ITP who are at risk of developing SLE or SS.

We examined anti-neutrophil antibodies in 12 patients with various disorders using enzyme-linked immunosorbent assay (ELISA), and the results obtained by ELISA were compared with those obtained by leukocyte agglutination test (LAT) and granulocyte cytotoxicity test (GCT). IgG anti-neutrophil antibody was positive in 7 of 12 patients, and IgM type antibody was positive in 6 patients. There was a significant correlation between IgG and IgM anti-neutrophil antibodies. The results obtained by ELISA were not in accord with those obtained by LAT or GCT. In addition, we examined anti-neutrophil antibodies in serially collected serum samples from two patients with immune neutropenia. The results obtained by ELISA correlated with their neutrophil counts, suggesting that anti-neutrophil antibodies detected by ELISA have pathological relevance.

An anti-human cytomegalovirus (HCMV) antibody in 330 sera from patients and normal subjects was examined by ELISA. The test results were compared with the results of CF test and there was 99.1% agreement. Specific anti-HCMV IgM antibody was searched for and only 4 sera were positive. This makes it unlikely that screening for IgM anti-HCMV will effectively prevent posttransfusion HCMV infections.

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, alphaIIbbeta3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of alphaIIbbeta3. One clone (AM-1) expressed constitutively active alphaIIbbeta3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antialphaIIbbeta3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of beta3(T562N) was responsible for receptor activation. In fact, T562N also activated alphaVbeta3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated alphaIIbbeta3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of beta3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to alphaIIbbeta3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of beta3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

Platelet activation causes a change in surface expression of several endogenous proteins, such as CD62P, CD63 and CD40L. Therefore, it is possible to analyze the functional in vivo status of the circulating platelet population directly by flow cytometry. In this study we developed the method to be suitable for use in clinical studies. We used EDTA-2K as anticoagulant since the sample anticoagulated with EDTA-2K, sodium citrate or ACD-A showed no difference in the data of activated platelets. We determined whether fixation of sample is necessary. The samples stained before or without fixation showed abnormally high level of activated platelets, indicating that fixation is necessary before staining. It is controversial whether activated platelets circulate in patients with idiopathic thrombocytopenic purpura(ITP). We measured activated platelets in patients with ITP using our optimised method. The percentages of CD62P, CD63 and CD40L positive platelets were significantly high in patients with ITP and 24%, 55% and 36% (respectively) of ITP patients showed elevated level of activated platelets. These data indicate that activated platelets circulate in ITP patients.

Measurements of plasma glycocalicin (GC) and reticulated platelets (RP) have been reported to be useful for classifying thrombocytopenic disorders. However, there have been no reports comparing the clinical usefulness of the two methods. We measured GC and RP levels simultaneously in 39 patients with idiopathic thrombocytopenic purpura (ITP), 15 patients with aplastic anemia (AA), and 17 patients with hypoplastic thrombocytopenia (HypoT) due to chemotherapy. The GC index (GC level normalized for the individual platelet count) and the percentage of RP (%RP), a parameter of platelet life span, were very high (7.5 +/- 11.4 and 20.8 +/- 13.0%, respectively) in patients with ITP as compared with those of healthy subjects (1.3 +/- 0.5 and 7.9 +/- 2.5%, respectively). However, 6 AA patients and 14 HypoT patients, in whom platelet life span is thought to be normal, also had an elevated GC index, suggestive of a false positive result. The RP, a parameter of platelet production, was low in all AA and HypoT patients except for one in each case. However, the GC level, an additional parameter of platelet production, was normal in 4 AA and 8 HypoT patients, indicating that it is not a sensitive indicator. We conclude that the RP and %RP are more feasible markers of thrombopoiesis and platelet life span, respectively, than the GC level and GC index.

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.

The effect of anti-platelet antibodies, including murine monoclonal antibodies, autoantibodies and alloantibodies, on platelet function was analyzed. The target antigen of these antiplatelet antibodies, investigated in the present study, was a glycoprotein IIb/IIIa, which is a receptor of fibrinogen and plays an important role in platelet aggregation. Some of these antibodies inhibited agonist-induced platelet aggregation. The target antigen of one murine monoclonal antibodies, designated OP-G2, was a glycoprotein IIb/IIIa and interestingly, this antibody induced platelet aggregation, which required divalent cation and fibrinogen. We compared the epitope of these antibodies by inhibition assay and found the epitope of these antibodies to be very close. The binding of OP-G2 to the platelets required Ca2+. These data suggest that OP-G2 recognizes an epitope at or in very close proximity to the fibrinogen binding site of GPIIb/IIIa, as compared with other antibodies.

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.

This study was designed to investigate the pathogenic relevance of platelet-associated autoantibodies in chronic ITP, since the titer of platelet-associated autoantibodies does not appear to correlate with the severity of the disease. Employing a direct immunoprecipitation procedure, we examined platelets from three ITP patients with platelet-associated autoantibodies against GPIIb-IIIa and an unidentified 56 kD protein before and after splenectomy. In two patients, platelet-associated autoantibodies disappeared after splenectomy, and these two patients attained complete remission. In one patient, however, the amount of platelet-associated autoantibodies did not decrease after splenectomy. Although this patient's platelet count transiently increased to 500 x 10(3)/microliters after splenectomy, it decreased to 55 x 10(3)/microliters within a short time. These findings suggest that platelet-associated autoantibodies play a key role in platelet destruction in chronic ITP.

Reticulated platelets retain some residual mRNA in their cytoplasm and are thought to be newly produced platelets. In recent years, it has been reported that the reticulated platelet count (RP) correlates well with platelet production. For that reason, the measurement of RP (%) is considered useful for analyses of platelet kinetics and differential diagnoses of thrombocytopenic disorders. However, certain technical difficulties exist because fluorochrome thiazole orange (TO), which is used for staining purposes, stains platelet granules nonspecifically, and so far, only a few reports have documented the study of precision staining techniques. We evaluated staining criteria precisely in an effort to solve the issue of nonspecific staining by TO, and concluded that the important points for effective staining were (1) fixation of platelets, (2) 1:8 dilution of TO (ReticCount), (3) incubation for 1 to 2 hours, and (4) the capture of platelets using anti-CD42b monoclonal antibody. We stained reticulated platelet samples by the above method and achieved intra-assay reproducibility of 3.4-5.1% RP (%) in normal subjects was 8.7 +/- 2.2%. It was significantly higher (23.6 +/- 13.3%) in patients with idiopathic thrombocytopenic purpura (ITP), and elevated in 87% of all evaluated ITP patients. Our method is sensitive, provides reproducible results, and can be effectively utilized for the analysis of platelet kinetics and differential diagnosis of thrombocytopenia.

The clinical significance of antinuclear antibodies (ANA) in patients with chronic idiopathic thrombocytopenic purpura (ITP) was evaluated. Serum samples of 55 patients with ITP without clinical features of systemic lupus erythematosus (SLE) were examined. The average follow-up period was 5.5 years. Positive ANA was found in 23 of the 55 patients. Twelve of these 23 showed a high-titer of ANA. There was no difference in platelet counts between ANA positive patients and negative patients, and there was no correlation between platelet counts and antibody titers. Ten of the 23 positive sera had precipitating antibodies to nuclear antigens; 7 SS-A and 3 RNP antibodies. The platelet counts of the 7 cases with anti-SS-A antibody positive were slightly low compared with those of the anti-SS-A antibody negative patients. None of the 10 patients developed SLE during the average follow-up period of 8.1 years. These results suggest that ITP patients who have a high-titer of ANA or antibody against SS-A do not always develop SLE. It was concluded that ANA with a high titer or precipitin to nuclear antigens, particularly SS-A, dose not predict a high risk of developing SLE in the future.

Platelet IIbbeta3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbbeta3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbbeta3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbbeta3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbbeta3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and beta3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the beta3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbbeta3. Neither group I nor group II antagonist increased intracellular Ca2+ concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbbeta3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbbeta3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbbeta3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between beta3 LIBS expression and IIbbeta3-mediated intracellular Ca2+ signaling in platelets.