Taisuke Kanaji

Convulxin Binds to Native, Human Glycoprotein Ibα

Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIbα. First, CVX binds to human GPIbα expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIbα but more strongly to human platelet GPIbα, as evidenced by comparative binding to wild-type, GPVI(–/–), FcRγ (–/–), and human GPIb transgenic mice. Third, the binding of CVX to human GPIbα is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIbα is disrupted by phenylalanine substitutions at GPIbα tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and α-thrombin binding to GPIbα. Fifth, CVX binding to GPIbα on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIbα antibodies. Lastly, CVX fails to bind to denatured GPIbα in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIbα and GPVI. Furthermore, the binding site on GPIbα for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIbα to CVX-induced platelet responses needs to be carefully re-evaluated.

Periostin and bone marrow fibrosis

Periostin is a secreted protein that shares structural homology with the insect axon guidance protein fasciclin 1. Periostin is expressed predominantly in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses. We have shown previously that periostin is a novel component of subepithelial fibrosis in bronchial asthma. Here, we investigated the relationship between periostin and bone marrow (BM) fibrosis. Periostin was expressed in the stroma and stromal cells of BM fibrosis specimens and to a great extent its expression levels correlated closely to the grade of fibrosis, as estimated by silver staining. However, in the present study, we found no relationship between plasma periostin levels and the extent of BM fibrosis. We also demonstrated that periostin is secreted by human BM hTERT stromal cells and that its secretion is enhanced by TGF-β, a cytokine produced by clonal proliferation of megakaryocytes and/or monocytes. These results indicate that periostin is a component of BM fibrosis and that it may play a role in the disease progression.

Platelet Dysfunction and a High Bone Mass Phenotype in a Murine Model of Platelet-Type von Willebrand Disease

The platelet glycoprotein Ib-IX receptor binds surface-bound von Willebrand factor and supports platelet adhesion to damaged vascular surfaces. A limited number of mutations within the glycoprotein Ib-IX complex have been described that permit a structurally altered receptor to interact with soluble von Willebrand factor, and this is the molecular basis of platelet-type von Willebrand disease. We have developed and characterized a mouse model of platelet-type von Willebrand disease (G233V) and have confirmed a platelet phenotype mimicking the human disorder. The mice have a dramatic increase in splenic megakaryocytes and splenomegaly. Recent studies have demonstrated that hematopoetic cells can influence the differentiation of osteogenic cells. Thus, we examined the skeletal phenotype of mice expressing the G233V variant complex. At 6 months of age, G233V mice exhibit a high bone mass phenotype with an approximate doubling of trabecular bone volume in both the tibia and femur. Serum measures of bone resorption were significantly decreased in G233V animals. With decreased bone resorption, cortical thickness was increased, medullary area decreased, and consequently, the mechanical strength of the femur was significantly increased. Using ex vivo bone marrow cultures, osteoclast-specific staining in the G233V mutant marrow was diminished, whereas osteoblastogenesis was unaffected. These studies provide new insights into the relationship between the regulation of megakaryocytopoiesis and bone mass.

IL-13: A Promising Therapeutic Target for Bronchial Asthma

The incidence of allergic diseases has dramatically increased in recent decades, especially in urban and industrialized areas. It is important socially as well as medically to establish more useful strategies to overcome allergic disorders. Bronchial asthma is a complex disease characterized by airway inflammation involving a Th2-cytokine, interleukin (IL)-13. A substantial body of evidence has accumulated pointing to the pivotal role of IL-13 in the pathogenesis of bronchial asthma, based on mainly analyses of mouse models. In addition to such analyses, the high expression of IL-13 in lesions and genetic association of several genes coding IL-13 signaling molecules with bronchial asthma have raised the possibility that IL-13 plays a pivotal role in the onset or exacerbation of human bronchial asthma. Therefore, IL-13 and its signal pathway are thought to be promising targets to develop a therapeutic agent for bronchial asthma. In this article, we describe how IL-13 is involved in the pathogenesis of bronchial asthma and then how therapeutic agents to block IL-13 signals are developed for bronchial asthma.

Convulxin binds to native, human glycoprotein Ibα (GPIbα)

Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIbα. First, CVX binds to human GPIbα expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIbα but more strongly to human platelet GPIbα, as evidenced by comparative binding to wild-type, GPVI(–/–), FcRγ (–/–), and human GPIb transgenic mice. Third, the binding of CVX to human GPIbα is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIbα is disrupted by phenylalanine substitutions at GPIbα tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and α-thrombin binding to GPIbα. Fifth, CVX binding to GPIbα on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIbα antibodies. Lastly, CVX fails to bind to denatured GPIbα in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIbα and GPVI. Furthermore, the binding site on GPIbα for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIbα to CVX-induced platelet responses needs to be carefully re-evaluated.

Quantitative analysis of AML1/ETO transcripts in peripheral blood stem cell harvests from patients with t(8;21) acute myelogenous leukaemia.

Summary Peripheral blood stem cells (PBSC) have been used increasingly for haemopoietic reconstitution after marrow‐ablative chemotherapy in patients with acute leukaemia because of the possibility that there is a lower risk of leukaemic contamination. We have developed a titration assay using a competitive reverse transcriptase polymerase chain reaction (RT‐PCR) which is able to estimate the number of AML1/ETO transcripts so that minimal residual disease (MRD) can be monitored quantitatively in patients with t(8;21) acute myelogenous leukaemia (AML). Using a qualitative RT‐PCR method, AML1/ETO transcripts could be detected in all samples from 15 first PBSC harvests and 11 second PBSC harvests obtained from 15 patients with t(8;21) AML. With our competitive RT‐PCR assay, the number of AML1/ETO transcripts was found to be lower in the second PBSC harvest than that in the first in every individual. Furthermore, MRD in PBSC harvests was less than that in the corresponding bone marrow obtained on the day of PBSC collection in the individual patients studied. In 10 patients who received autologous blood stem cell transplantation (ABSCT), we could not find a relationship between the number of AML1/ETO transcripts in the infused PBSC harvests and the clinical outcome after ABSCT. The present study clearly indicates that although PBSC harvests collected after consolidation chemotherapy are contaminated by leukaemic cells, the degree of leukaemic contamination may decrease as chemotherapy is repeated. The mobilization of PBSC by repeated chemotherapy may provide an advantageous source of haemopoietic stem cells for ABSCT.

GPIbα regulates platelet size by controlling the subcellular localization of filamin

Interaction between the cytoplasmic domain of GPIbα with its cytoskeletal binding partner, filamin, is a major determinant of platelet size, and deficiency of either protein results in macrothrombocytopenia. To clarify the mechanism by which GPIbα-filamin interactions regulate platelet production, we manipulated the expression levels of filamin and GPIb in cultured embryonic stem cells (ESCs) that were subsequently differentiated into platelets. Knocking down filamins A and B resulted in the production of ESC-derived proplatelets with abnormally large swellings and proplatelet shafts that generated giant platelets in culture. Large platelets could also be generated by overexpressing GPIbα in ESCs, or by overexpressing in vivo a transgene encoding a chimeric protein containing the cytoplasmic domain of GPIbα. To identify the mechanism by which the GPIb:filamin ratio regulates platelet size, we manipulated filamin and GPIbα levels in HEK293T cells and examined the effects of overexpressing either protein on their ability to traffic to the cell periphery. Accumulation of either protein within the endoplasmic reticulum resulted in trapping of the other. Taken together, these data demonstrate that coordinated expression of GPIbα and filamin is required for efficient trafficking of either protein to the cell surface, and for production of normal-sized platelets.

Effects of granulocyte colony-stimulating factor on the hemostatic system in healthy volunteers

We previously identified a receptor for granulocyte colony-stimulating factor (G-CSFR) on platelet membranes, and reported that G-CSF enhanced ADP-induced platelet aggregation. Here, we investigated the priming effect of G-CSF on the hemostatic system in healthy volunteers given G-CSF. Following the administration of rhG-CSF (10 micrograms/kg for 30 min div) to 10 healthy volunteers, we found a significant elevation in the maximum platelet aggregation rate induced by ADP or collagen, thromboxane B2 level and amount of thrombin-antithrombin III complex. The D-D dimer and plasminogen activator inhibitor-1 showed no significant changes. These observations indicate that G-CSF administration may induce hypercoagulability in susceptible subjects. Therefore, patients or donors at risk of thrombosis or hypercoagulable state should be followed carefully after G-CSF administration.

Platelet hyperreactivity explains the bleeding abnormality and macrothrombocytopenia in a murine model of sitosterolemia

Sitosterolemia is a rare, autosomal recessive disease caused by mutations in the adenosine triphosphate-binding cassette transporter genes ABCG5 or ABCG8 that result in accumulation of xenosterols in the body. Clinical manifestations include tendon xanthomas, premature coronary artery disease, hemolytic anemia, macrothrombocytopenia, and bleeding. Although the effect of sterol accumulation on the predisposition for atherosclerosis is evident, how xenosterol accumulation leads to defects in platelet physiology is unknown. Sitosterolemia induced in Abcg5- and Abcg8-deficient mice fed a high plant sterol diet resulted in accumulation of free sterols in platelet plasma membranes, leading to hyperactivatable platelets characterized by constitutive binding of fibrinogen to its αIIbβ3 integrin receptor, internalization of the αIIbβ3 complex, generation of platelet-derived microparticles, and changes in the quantity and subcellular localization of filamin. The latter was associated with macrothrombocytopenia, shedding of GPIbα, impaired platelet adhesion to von Willebrand factor, and inability to form stable thrombi. Plasma levels of soluble GPIbα were strongly correlated with plasma sitosterol levels in samples from human sitosterolemic patients, implicating a similar mechanism of sterol-induced platelet passivation in the human disease. Intercalation of plant sterols into the plasma membrane therefore results in dysregulation of multiple platelet activation pathways, leading to macrothrombocytopenia and bleeding.

Characterization of the light chain-restricted clonal B cells in peripheral blood of HCV-positive patients.

To investigate the association between hepatitis C virus (HCV) and B cell proliferation, we searched for the clonal B cells by flow cytometric analysis of the surface immunoglobulin kappa (κ):lambda (λ) light chain ratios of the circulating B (CD19+) cells in 240 HCV-positive patients and 150 negative controls with liver diseases. Clonal B cells with light chain restriction (κ:λ ratio >3:1 or <1:2) were analyzed for CD5 expression and the presence of monoclonal immunoglobulin heavy-chain (IGH) gene rearrangements and the t(14;18) chromosomal translocation. Clonal B cells were detected in 7 cases with HCV (2.9%), but was never detected in the controls (p < 0.05). Of the 7 cases, all had monoclonal IGH gene rearrangements and one had the t(14;18) chromosomal translocation. These HCV-related clonal B cells are not uniform in the intensity of CD5 expression and showed no increase in the frequencies of CD5+ population compared with non-clonal B cells. No “chronic lymphocytic leukemia-phenotype” cells were found. The loss of clonality was observed in 2 cases treated with interferon and in one case treated with splenectomy. The longitudinal study is required to determine whether these circulating clonal B cells progress to lymphoproliferative disorders in future or not.