Tetsuro-Takahiro Fujimoto

Is eradication therapy useful as the first line of treatment in Helicobacter pylori-positive idiopathic thrombocytopenic purpura? Analysis of 207 eradicated chronic ITP cases in Japan.

A retrospective study was performed to determine the prevalence of Helicobacter pylori (H pylori) infection, the effect of H pylori eradication on platelet counts, and the characteristic clinical features of chronic immune or idiopathic thrombocytopenic purpura (ITP) with H pylori infection. H pylori infection was found in 300 patients, a group that was significantly older (P < .005) and had more cases of hyperplastic megakaryocytes in the bone marrow (P = .01) than patients without H pylori infection.H pylori eradication therapy was performed in 207 H pylori-positive ITP cases, and the platelet count response was observed in 63% of the successful eradication group and in 33% of the unsuccessful eradication group (P < .005). In the successful group, the complete remission and partial remission rates were 23% and 42%, respectively, 12 months after eradication. In the majority of responders, the platelet count response occurred 1 month after eradication therapy, and the increased platelet count continued without ITP treatment for more than 12 months. H pylori eradication therapy was effective even in refractory cases, which were unresponsive to splenectomy. In conclusion, H pylori infection was involved in most ITP patients older than 40 years in Japan, and eradication therapy should be the first line of treatment in H pylori-positive ITP patients.

A New Member of the LIM Protein Family Binds to Filamin B and Localizes at Stress Fibers

Human filamins are 280-kDa proteins containing an N-terminal actin-binding domain followed by 24 characteristic repeats. They also interact with a number of other cellular proteins. All of those identified to date, with the exception of actin, bind to the C-terminal third of a filamin. In a yeast two-hybrid search of a human placental library, using as bait repeats 10–18 of filamin B, we isolated a cDNA coding for a novel 374 amino acid protein containing a proline-rich domain near its N terminus and two LIM domains at its C terminus. We term this protein filamin-binding LIM protein-1, FBLP-1. Yeast two-hybrid studies with deletion mutants localized the areas of interaction in FBLP-1 to its N-terminal domain and in filamin B to repeats 10–13. FBLP-1 mRNA was detected in a variety of tissues and cells including platelets and endothelial cells. We also have identified two FBLP-1 variants. Both contain three C-terminal LIM domains, but one lacks the N-terminal proline-rich domain. Transfection of FBLP-1 into 293A cells promoted stress fiber formation, and both FBLP-1 and filamin B localized to stress fibers in the transfected cells. The association between filamin B and FBLP-1 may play a hitherto unknown role in cytoskeletal function, cell adhesion, and cell motility.

Involvement of Fcγ receptor polymorphism in the therapeutic response of idiopathic thrombocytopenic purpura

Clearance of autoantibody‐sensitized platelets through Fcγ receptors on phagocytic cells is one of the main mechanisms of thrombocytopenia in idiopathic thrombocytopenic purpura (ITP). We examined the FcγRIIA‐131R/H and FcγRIIIA‐158V/F polymorphisms in 104 adult chronic ITP patients, and in 59 healthy control subjects using polymerase chain reaction‐based allele‐specific restriction analysis. The frequency of FcγRIIA genotypes (131H/H, H/R, R/R) was not significantly different between patients and controls, and did not correlate with the responsiveness to treatment. In contrast, among FcγRIIIA genotypes, frequency of 158F/F homotype was smaller in ITP (P < 0·05). Furthermore, in FcγRIIIA‐158V/V homotype, the complete remission (CR) rate with medication (treatment with corticosteroid or other immunosuppressive agents) was significantly higher (60%) than that in 158V/F (10%) or 158V/F plus 158F/F, (P < 0·01, P < 0·05). Conversely, the CR rate after splenectomy in 158F/F and 158V/F types (64·3% and 54·6%) was higher than in 158V/V (25%). Our results indicate that the polymorphism of FcγRIIIA, but not FcγRIIA, influences the response to treatment in ITP.

Glanzmann thrombasthenia with acute myeloid leukemia successfully treated by bone marrow transplantation.

We report successful treatment by bone marrow transplantation (BMT) in an acute myeloid leukemia (AML) patient with Glanzmann thrombasthenia (GT). Genetic analysis revealed that a novel point mutation in exon 3 of the GPIIb gene led to abnormal splicing resulting in an amino acid substitution and an in-frame deletion of 3 amino acid residues. Expression studies suggested a rapid degradation of the uncomplexed protein within the cells. Induction therapy for AML was performed with frequent platelet transfusions because of the patient’s severe hemorrhagic manifestations. In the second remission, the patient was successfully treated by BMT from an HLA-matched unrelated donor. Platelet function returned to normal, and the GT phenotype completely disappeared. Our experience suggests that BMT is a curative therapeutic strategy for GT. Furthermore, we believe this study is the first to demonstrate that engraftment after BMT for AML can be determined by monitoring the congenital genetic defect of GT.

Glanzmann thrombasthenia associated with a 21-amino acid deletion (Leu817-Gln837) in glycoprotein IIb due to abnormal splicing in exon 25

We report a novel genetic defect in a Japanese patient with type I Glanzmann thrombasthenia.The glycoprotein (GP) IIb complementary DNA (cDNA) from platelet messenger RNA had a 63-base pair deletion in the 5′ boundary of exon 25, resulting in an in-frame deletion of 21 amino acid residues (Leu817-Gln837) in the calf-2 domain.The deleted region was present in the genomic DNA, but the splice acceptor site (AG) of exon 25 was mutated to AC, leading to the use of an AG sequence in the middle of exon 25 as an abnormal cryptic splice acceptor site. The effect of this deletion on protein synthesis was further analyzed. Mutant GPIIb-IIIa complexes were not detected on the surfaces of cells cotransfected with cDNAs of mutant GPIIb and normal GPIIIa. Mutant pro-GPIIb was detected in cell lysates and was coimmunoprecipitated with an anti-GPIIb-IIIa complex antibody. Immunostaining demonstrated that the mutant pro-GPIIb colocalized with an endoplasmic reticulum protein, calnexin, within the cells. These results indicate that complex formation was not completely prevented and that impairment of the subsequent transport was the major reason for the defect in cell surface expression. The data suggest that the GPIIb calf-2 domain is important for intracellular transport of GPIIb-IIIa complexes.

Novel alternatively spliced form of β3-endonexin

beta(3)-Endonexin is a binding protein to the cytoplasmic tail of beta(3) integrin and can activate alpha(IIb)beta(3) in Chinese hamster ovary (CHO) cells. Initially, two forms were identified, and only the shorter form showed the function. However, it localized mainly to the nucleus because of a nuclear localization signal (K(62)RKK). We identified two additional forms of beta(3)-endonexin. One encoded 177 amino acids and was identical to the protein previously reported as a thyroid hormone receptor-binding protein. The other is a novel shortest form encoding 62 amino acids. Although the novel form lacked nuclear localization signal and was observed diffusely in the cytoplasm of transfected cells, this form did not show interaction with beta(3) integrin. Then, the ideal form as an integrin modulator was not found among these isoforms. Nevertheless, when the nuclear localization signal of the shorter form was disrupted, beta(3)-endonexin was localized near the cell surface and modulated the affinity of alpha(IIb)beta(3) more intensively. These results suggest the presence of various isoforms and the relationship between subcellular localization and integrin-activating function of beta(3)-endonexin.