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.

Both connective tissue mast cells and mast cells grown in vitro are derived from multipotential hematopoietic stem cells, but these two mast cell populations exhibit many differences in morphology, biochemistry, and function. We investigated whether the phenotype of cultured mast cells or their progeny was altered when the cells were transferred into different locations in vivo. Cultured mast cells were immature by ultrastructure, and stained with alcian blue but with neither safranin or berberine sulfate, a fluorescent dye that binds to the heparin of connective tissue mast cell granules. By contrast, mast cells recovered from the peritoneal cavity of congenitally mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice 10 wk after intraperitoneal injection of cultured WBB6F1-+/+ or C57BL/6-bgJ/bgJ mast cells stained with both safranin and berberine sulfate. Staining with berberine sulfate was prevented by treatment of the cells with heparinase but not chondroitinase ABC, suggesting that the adoptively transferred mast cell population had acquired the ability to synthesize and store heparin. Furthermore, the recovered mast cells were indistinguishable by ultrastructure from the normal mature peritoneal mast cells of WBB6F1-+/+ mice, and contained substantially more histamine than mast cells studied directly from culture. Intravenous injection of cultured mast cells resulted in the development of safranin-and berberine sulfate-positive mast cells in the peritoneal cavity, spleen, skin, and glandular stomach muscularis propria. Mast cells also developed on the glandular stomach mucosa, but these cells stained with alcian blue rather than safranin, and did not stain with berberine sulfate. This result suggests that cultured mast cells can give rise to mast cells of either the connective tissue type or mucosal phenotype, depending on anatomical location. Furthermore, transplantation of cultured mast cells into WBB6F1-W/Wv mice had no measurable effect on the anemia of the recipient mice, suggesting a possible strategy for repairing the mast cell deficiency of WBB6F1-W/Wv mice without affecting other bone marrow-derived populations such as erythrocytes. Intravenous injection of representative connective tissue type mast cells (30-50% pure peritoneal mast cells derived from WBB6F1-+/+ mice) gave results similar to those obtained with cultured mast cells: mast cells developing in the peritoneal cavity, skin, spleen, and glandular stomach muscularis propria of WBB6F1-W/Wv recipients stained with safranin and berberine sulfate, whereas mast cells developing in the mucosa of the glandular stomach stained only with alcian blue.(ABSTRACT TRUNCATED AT 400 WORDS)

Background L-Arginine (L-Arg), one of the essential amino acids, has been reported to have an immunomodulatory effect. The precise mechanism of the L-Arg-induced natural killer (NK) cell activation remains unresolved,and the effect of L-Arg on NK cells in chronic fatigue syndrome (CFS) patients has not been estimated.Methods NK cell function was evaluated in 20 subjects with CFS and compared with that in 21 healthy individuals.Results In healthy control subjects, NK activity was significantly increased after treatment with L-Arg, an NK function enhancer, for 24h, whereas the same treatment failed to enhance NK activity in the CFS patients. We thus focused on L-Arg metabolism, which involves nitric oxide (NO) production through NO synthase (NOS). The expression of inducible NO synthase (iNOS) transcripts in peripheral blood mononuclear cells was not significantly different between healthy control subjects and CFS patients. The L-Arg-mediated NK cell activation was abolished by addition of N-G-monomethyl-L-arginine, an inhibitor for iNOS. Furthermore, incubation with S-nitroso-N-acetyl-penicillamine, an NO donor, stimulated NK activity in healthy control subjects but not in CFS patients.Conclusion These results demonstrate that the L-Arg-induced activation of NK activity is mediated by NO and that a possible dysfunction exists in the NO-mediated NK Cell activation in CFS patients.

We investigated the therapeutic efficacy and safety of cefpirome sulfate (CPR) in treatment of hematopoietic disorder-associated infections. A total of 219 patients were admitted to 12 hospitals of Hanshin Study Group of hematopoietic disorders and infections between April 1994 and March 1996 and were enrolled in this study. Most patients received intravenously infused CPR at a dose of 1 or 2 g twice a day for 3 days or more. Twenty nine patients dropped out or were excluded and remaining 190 patients were adopted for the evaluation. A overall response rate was 58.4% (111/190). Among neutropenic patients, the response rate was 50% (8/16) in patients whose peripheral neutrophil counts (PNC) remained less than 100/microliter throughout the observation period and was 53.7% (22/41) in patients with PNC remained less than 500/microliter. In contrast, in patient whose PNC was below 500 before the treatment but exceeded 501/microliter during of at the end of the treatment, the response rate was as high as 78.4% (29/37). When G-CSF was combined, the response rate became significantly (P < 0.05) higher, 68.5% (50/73), as compared with that, 52.1% (61/117), in patients without it. In cases in which the causative organisms could be identified, the organisms were eliminated in 81.8% (9/11) of the patients infected with Gram-positive bacteria, whereas in 100% (12/12) in those infected with Gram-negative bacteria. Skin eruption developed in 6 patients during the treatment with CPR, and vascular pain and parosmia in one each other. These symptoms subsided soon after discontinuation or even without discontinuation of CPR. Abnormal laboratory findings, mainly liver dysfunction, i.e. elevation of slight degree of serum transaminase levels, were observed. The values, however, turned to normal immediately after the cessation or completion of the treatment. In conclusion, CPR is considered to be an antibiotic of value with high efficacy and safety in treatment of hematopoietic disorder-associated infections.

The molecular mechanism underlying the interaction between myeloma cells and stromal cells was investigated by using a human myeloma cell line (OPM-2) and human umbilical vein endothelial cells (HUVECs). Adhesion of OPM-2 cells to HUVECs was found to be significantly augmented with treatment of OPM-2 cells with an alpha-glycosidase inhibitor, castanospermine (CSP). The treatment of OPM-2 cells with CSP resulted in alteration of oligosaccharide structures of cell surface glycoproteins particularly at molecular weight of 220 kD (GP220). To determine if GP220 was involved in the adhesion of OPM-2 cells to HUVECs, cell surface glycoproteins of HUVECs were labeled by biotin and were incubated with the PVDF membrane to which cell surface glycoproteins of OPM-2 cells were blotted. The biotinylated glycoproteins at the plasma membrane of HUVECs specifically bound to GP220 of OPM-2 cells. Purification and partial amino acid sequencing of GP220 revealed that GP220 had a structure homologous to cation-independent mannose 6-phosphate/insulin-like growth factor-II (CIM6P/IGF-II) receptor. Furthermore, an antibody against CIM6P/IGF-II receptor was reactive with GP220, indicating that GP220 was a CIM6P/IGF-II receptor. The adhesion of OPM-2 cells to HUVECs was inhibited by mannose 6-phosphate. Moreover, M6P was found to suppress the adhesion of human myeloma cell lines, OPM-2 and RPMI 8226, to bone marrow stromal cells that was established from the patients with multiple myeloma. In addition, proliferation of OPM-2 was stimulated in response to IGF-II. These results suggest that CIM6P/IGF-II receptor may be functional in terms of supporting cell adhesion and proliferation of myeloma cells.

The c-kit receptor tyrosine kinase (KIT) is activated upon ligand binding, thereby leading to a variety of signaling events that play a fundamental role in hematopoiesis. In addition to ligand-dependent activation, we have previously shown that KIT is constitutively activated in a ligand-independent manner by two point mutations, Val-559-->Gly (G559) mutation in the juxtamembrane domain and Asp-814-->Val (V814) mutation in the phosphotransferase domain. To investigate the biochemical consequence and biologic significance of these mutations, retroviral vectors encoding KITG559 or KITV814 were introduced into murine pro-B-type Ba/F3 cells and myeloid FDC-P1 cells, both of which require interleukin-3 (IL-3) for their growth and survival. In the cells, KITG559 or KITV814 were found to be constitutively phophorylated on tyrosine in the absence of stem cell factor (SCF) that is a ligand for KIT. Chemical cross-linking analysis showed that a substantial fraction of the phosphorylated KITG559 underwent dimerization even in the absence of SCF, whereas the phosphorylated KITV814 did not, suggesting the distinct mechanisms underlying constitutive activation of KIT by G559 and V814 mutations. Furthermore, the cells expressing either KITG559 or KITV814 were found to show a factor-independent growth, whereas the cells expressing wild-type KIT (KITWT) proliferated in response to SCF as well as IL-3. Moreover, subcutaneous injection of Ba/F3 cells expressing KITG559 or KITV814 into nude mice resulted in production of large tumors at all sites of the injection within 2 weeks, and all nude mice quickly succumbed to leukemia and died. These results suggest that, although the mechanisms underlying constitutive activation of KITG559 or KITV814 may be different, both of the activating mutations have a function to induce a factor-independent and tumorigenic phenotype. Also, the data of this study raise the possibility that the constitutively activating mutations of c-kit may play a causal role in development of hematologic malignancies.

The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.

Staurosporin, a broad-spectrum kinase inhibitor, induced cell spreading in a human colon cancer cell line, Colo 201. On collagen and laminin, cell spreading was induced in more than 90% of the cells and was dependent on very late activation antigen-3, as shown by an antibody inhibition assay, Cell spreading required divalent cations and showed the order of preference Mn2+ > Mg2+ > Ca2+. On fibronectin, only about 30% of the cells were observed to spread, and spreading occurred via a non-integrin, RGD-independent pathway. Staurosporin-induced spreading was inhibited by treatment with tyrosine kinase inhibitors herbimycin A and methyl 2,5-dihydroxycinnamate. Despite the presence of staurosporin, seven proteins (220, 175, 150, 98, 62, 58, and 45 kDa) showed increased levels of tyrosine phosphorylation in association with cell adhesion. Two of these (58 and 220 kDa) were identified by immunoprecipitation as Src product and tensin, respectively. Flow cytometric analysis showed that the Colo 201 cells expressed the alpha 2, alpha 3, alpha 6, and beta 1 chains of integrin, but expression of these chains was not influenced by staurosporin. Immunofluorescence microscopy revealed that the alpha 3 chain, diffusely expressed on the cell surface in the absence of staurosporin, was concentrated at focal adhesion plaques after staurosporin treatment. Neither alpha 2 nor alpha 6 was focalized by the treatment.

CD36 deficiency is divided into two subgroups: neither platelets nor monocytes express CD36 (type I deficiency), and monocytes express CD36 in spite of the lack of platelet CD36 (type II deficiency). We have already demonstrated that a 478C-->T substitution (proline90-->serine) in platelet CD36 cDNA predominates in type II deficiency (Kashiwagi, H., S. Honda, Y. Tomiyama, H. Mizutani, H. Take, Y. Honda, S. Kosugi, Y. Kanayama, Y. Kurata, and Y. Matsuzawa. 1993. Thromb. Haemostasis. 69:481-484). In this study, we revealed that monocyte CD36 cDNA from two type II deficient subjects was heterozygous for C478 and T478 form, while platelet CD36 cDNA of these subjects consisted of only T478 form. In a type I deficient subject, both platelet and monocyte CD36 cDNA showed only T478 form. Expression assay using C478 or T478 form of CD36 cDNA transfected cells revealed that there was an 81-kD precursor form of CD36, and that the maturation of the 81-kD precursor form to the 88-kD mature form of CD36 was markedly impaired by the substitution. The mutated precursor form of CD36 was subsequently degraded in the cytoplasm. These results indicate that the 478C-->T substitution directly leads to CD36 deficiency via defects in posttranslational modification, and that this substitution is the major defects underlying CD36 deficiency.

We analyzed the molecular genetic defect responsible for type I Glanzmann's thrombasthenia in a Japanese patient. In an immunoblot assay using polyclonal anti-GpIIb-IIIa antibodies, some GPIIIa (15% of normal amount) could be detected in the patient's platelets, whereas GPIIb could not (< 2% of normal amount). Nucleotide sequence analysis of platelet GPIIb mRNA-derived polymerase chain reaction (PCR) products revealed that patient's GPIIb cDNA had a 75-bp deletion in the 3' boundary of exon 17 resulting in an in-frame deletion of 25 amino acids. DNA analysis and family study revealed that the patient was a compound heterozygote of two GPIIb gene defects. One allele derived from her father was not expressed in platelets, and the other allele derived from her mother had a 9644C--> T mutation which was located at the position -3 of the splice donor junction of exon 17 and resulted in a termination codon (TGA). Moreover, quantitative analysis demonstrated that the amount of the abnormal GPIIb transcript in the patient's platelets was markedly reduced. Thus, the C --> T mutation resulting in the abnormal splicing of GPIIb transcript and the reduction in its amount is responsible for Glanzmann's thrombasthenia.

Summary. In this study employing a sensitive immunoblot assay, we have characterized GPIIb and GPIIIa in thrombasthenic platelets from seven type II and four type I patients from 10 unrelated families. The amounts of GPIIb and GPIIIa were both markedly reduced in all these patients, and abnormal molecular weight GPIIb or GPIIIa was not detected. In all of four type I patients the amount of GPIIb was much lower than that of GPIIIa. In this study, however, we found that the amount of GPIIb was also lower even in six out of seven type II patients. Immunodepletion of patients’platelets with AP2 (a monoclonal antibody specific for the GPIIb‐IIIa complex), AP3 (specific for GPIIIa) or AMF7 (specific for αv) further confirmed that GPIIIa existed in excess, and demonstrated that excess GPIIIa were mostly in free form and not associated with GPIIb or αv. The reduction of GPIIb may represent an abnormality in GPIIb processing in these type II and type I thrombasthenic platelets. It remains unclear whether these two subgroups represent distinct categories.

We report a 28-year-old Japanese with Klinefelter's syndrome who developed mixed connective disease (MCTD) and Sjögren syndrome. Previously being well, he presented with Raynaud's phenomenon, dry eye, fever and polyarthralgia. Clinical examinations revealed anti-nRNP autoantibody, leukopenia and lung fibrosis. Then he was found to have Klinefelter's syndrome. Flow cytometric analysis showed a relative increase of peripheral CD8+ T lymphocytes carrying either HLA-DR or CD57. Lymphocyte IL-2 production induced in vitro by concanavalin A was decreased. Such T cell abnormalities may be implicated in the development of autoimmune disease in Klinefelter's syndrome.

Arbekacin (ABK) was administered to 17 patients with MRSA infections that complicated underlying hematopoietic disorders, and the efficacy and safety were evaluated. The underlying diseases included acute myelocytic leukemia (8 cases), acute lymphocytic leukemia (1) myelodysplastic syndrome (3), chronic myelocytic leukemia (1), non-Hodgkin's lymphoma (2), Hodgkin's disease (1) and adult T cell leukemia (1). The infections consisted of septicemia (5 cases), pneumonia (4), upper respiratory tract infections (6) and urinary tract infections (2). ABK was administered by i.v. drip infusion in daily doses of 150-200 mg, given in two divided dosages. The therapeutic efficacies were: excellent in 2 (2 septicemias), good in 7 (1 septicemia, 4 upper respiratory infections, 2 urinary tract infections), fair in 2 (septicemia and pneumonia) and poor in 6 (1 septicemia, 3 pneumonias, 2 upper respiratory infections). As a side effect, reversible renal dysfunction was detected in four cases. Causative bacteria were isolated from six cases. They were all coagulase type II and MIC's of ABK were from 0.25 microgram/ml to 4.0 micrograms/ml. Arbekacin therapy was found to be effective even in patients with hematopoietic disorders accompanied by MRSA infections.

We performed a molecular analysis of a subject whose platelets and monocytes did not express any cell surface CD36 (designated as a type I CD36 deficiency). Amplification of the 5′ half of platelet and monocyte CD36cDNA (corresponding to nucleotide [nt] 191–1009 of the published CD36 cDNA sequence [Oquendo et al, Cell, 58:95, 1989]) showed that two different-sized CD36 cDNAs existed. One cDNA was of predicted normal size, whereas the other was about 150 bp smaller than that predicted for normal CD36 cDNA. Amplification of the 3′ region of CD36 cDNA (nt 962–1714) in this subject showed only normal-sized CD36 cDNA. Cloning and nt sequence analysis of the cDNAs showed that the smaller sized CD36 cDNA had 161-bp deletion (from nt 331 to 491), and a dinucleotide deletion starting at nt position 539. The same dinucleotide deletion was also detected in the normal sized CD36 cDNA. Both deletions caused a frameshift leading to the appearance of a translation stop codon. RNA blot analysis and quantitative assay using the reverse transcription- polymerase chain reaction (RT-PCR) showed that the CD36 transcripts in both platelets and monocytes were greatly reduced. Comparison of the determined cDNA sequences with the genomic DNA sequence for the human CD36 gene showed that the dinucleotide deletion was located in exon 5, and that the 161-bp deletion corresponded to a loss of exon 4. PCR- based analysis using genomic DNA showed that this subject was homozygous for the dinucleotide deletion in exon 5. Except for the dinucleotide deletion, we could not find any abnormalities around exon 3, 4, and 5 including the splice junctions. These results suggested that the deletions in CD36 mRNA were likely to be responsible for instability of the transcripts, and the dinucleotide deletion in exon 5 might affect the splicing of exon 4.

Extracellular matrix (ECM) molecules such as fibronectin (FN), collagens, and laminin have important roles in hematopoiesis. However, little is known about the precise mechanisms by which ECM molecules regulate proliferation of human hematopoietic progenitor cells. In this study, we have investigated the effects of ECM molecules, particularly of FN, on the proliferation of a myeloid leukemia cell line, M07E, which proliferates in response to either human granulocyte/macrophage colony-stimulating factor (GM-CSF) or stem cell factor (SCF). The [3H]thymidine incorporation and cell enumeration assays showed that FN strikingly inhibited GM-CSF- or SCF-induced proliferation of M07E cells in a dose-dependent manner, whereas little or no inhibition was induced by collagen types I and IV. The growth suppression of M07E cells was not due to the inhibitory effect of FN on ligand binding or very early events in the signal transduction pathways from the GM-CSF or SCF receptors. DNA content analysis using flow cytometry after staining with propidium iodide revealed that the treatment of M07E cells with FN did not block the entry of the cells into the cell cycle after stimulation with GM-CSF or SCF, whereas the treatment resulted in the appearance of subdiploid peak. Furthermore, FN was found to induce oligonucleosomal DNA fragmentation and chromatin condensation in the cells even in the presence of GM-CSF or SCF, suggesting the involvement of programmed cell death (apoptosis) in the FN-induced growth suppression. The growth suppression or apoptosis induced by FN was rescued by the addition of either anti-FN antibody, anti-very late antigen 5 monoclonal antibody (anti-VLA5 mAb), or GRGDSP peptide, but not by that of anti-VLA4 mAb or GRGESP peptide, suggesting that the FN effects on M07E cells were mediated through VLA5. In addition, the FN-induced apoptosis was detectable in VLA5-positive human hematopoietic cell lines other than M07E cells, but not in any of the VLA5-negative cell lines. These results suggest that FN is capable of inducing apoptosis via its interaction with VLA5, and also raise the possibility that the FN-VLA5 interaction may contribute, at least in part, to negative regulation of hematopoiesis.

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.

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is known to play a crucial role in mast cell growth and differentiation. In a human mast cell leukemia cell line (HMC-1), KitR was found to be constitutively phosphorylated on tyrosine, activated and associated with phosphatidylinositol 3-kinase (P13K) in the absence of autocrine production of SCF. Sequencing of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations in codon 560 and codon 816, resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp, respectively. Murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in cells of a human embryonic kidney cell line (293T). In the transfected cells, KitR (Gly-559 + Val-814) and KitR (Val-814) were strikingly phosphorylated on tyrosine and activated in the absence of SCF, whereas tyrosine phosphorylation and activation of KitR (Gly-559) or wild-type KitR was modest or little, respectively. These results suggest that constitutive activation of KitR in HMC-1 results from the activating mutations of c-kit gene, and raise the possibility that the activating mutations, particularly at codon 814 of murine c-kit or at codon 816 of human c-kit, may participate in oncogenesis of mast cells.

Summary. We studied a female patient with cyclic fluctuation in platelet count following splenectomy for autoimmune thrombocytopenia. The cyclical fluctuation appeared to be in phase with her menstrual cycle and her platelet count was low during menses. Bone marrow examinations performed at the peak as well as the bottom of the platelet count showed normal or increased numbers of megakaryocytes. The patient's platelet count increased rapidly after intravenous gamma‐globulin (IVIgG) therapy, suggesting that a failure of platelet production is unlikely to account for the cycle. Platelet‐associated IgM (PAIgM) was markedly elevated, whereas PAIgG was normal at any stage of the cycle. MACE assay demonstrated that PAIgM contained IgM anti‐glycoprotein (GP) IIb‐IIIa autoantibodies. Comparison between MACE assay using untreated and EDTA‐treated platelets at 3 7°C demonstrated that the platelet‐associated IgM autoantibodies mainly recognized divalent cation‐dependent conformation(s) of GPUb‐IIIa. No antibodies were, however, detected in her serum. The levels of IgM anti‐GPIIb‐IIIa showed an inverse relationship with the platelet count. In spite of the marked increase in platelet count after IVIgG, however, the levels of IgM anti‐GPIIb‐IIIa remained elevated. These findings suggest that plateletassociated IgM anti‐GPIIb‐IIIa autoantibodies are of pathogenic significance in this patient.