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Establishment of the stable cell line expressing human platelet GPIb/IX receptor and its utilization for platelet function

Research Project

Project/Area Number 12670971
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Hematology
Research InstitutionYamagata University

Principal Investigator

HAYASHI Tomohiro  Yamagata University, School of Medicine, Assistant Professor, 医学部, 講師 (90228586)

Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
KeywordsBernard-Soulier syndrome / GPIb / IX receptor / leucine rich repeat / trangent gene expression / flow cytometry / Northern blotting / stable transformant / ristocetin aggregation / 先天性血小板機能異常症 / 遺伝子発現 / stable cell line
Research Abstract

The platelet GPIb/IX complex, a surface receptor for von Willebrand factor (vWF), is composed of three-independent gene products of GPIb-alpha, -beta and IX. Those components linked together and form a functional receptor for vWF. So far, genetic analyses of the patients with Bernard-Soulier syndrome have shown that the leucine rich repeat (LRR) in the GPIb-alpha is essential for an efficient surface expression of the complex. LRR is a short 24 amino acid stretch containing well preserved leucine residues, and each GP possesses at least one of this consensus motif. Little is known, however, about the importance of the LRR within GPIb-beta because of the absence of the clinical case. To assess the importance of the GPIb-beta LRR for the expression of GPIb/IX complex, four amino acid residues within the LRR of the GPIb-beta have been chosen and site-directed mutagenesis was performed to obtain series of mutants; Leu35, Leu45 and Leu50 → Ala, Val or Phe, Asn40 → Ala, Thr or Cys. The obtai … More ned plasmids were co-transfected with GPIb-alpha and GPIX, and the expression of GPIb/IX complex was analyzed by FACS and Northern blotting.
I. FACS analyses of the GPIb/IX receptor transfected with the mutant plasmids: All Asn40-mutants drastically decreased the surface expression of the complex. Similar results were obtained by using Leu → Ala or Phe mutants, however, Leu → Val mutants always showed 40-80% surface expression of the complex when analyzed by FACS.
II. Northern blot analyses of the mutants: Northern blotting analyses showed that GPIb-beta mRNA level of the mutants were almost the same as control, indicating post-transcriptional events were causative for the decreased receptor expression. These data have suggested that Leu residues and Asn40 within the LRR of the GPIb-beta are important for the efficient expression of the GPIb/IX complex, although Leu → Val mutants showed moderate complex expression probably due to the structural similarity of both amino acids.
III. Establishment of stable transformant expressing human platelet GPIb/IX receptor: We have utilized the above described plasmids to establish the stable transformants. The transformants possess the platelet-like aggregatory potential by ristocetin, mimic the human platelet GPIb/IX complex. However, they gradually lost the activity by long-term cultivation. Some modifications are necessary to maintain the aggregatory potential of the transformants. Less

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (7 results)

All Other

All Publications (7 results)

  • [Publications] Hayashi T, Suzuki K: "Molecular pathogenesis of Bernard-Soulier syndrome"Semin Thromb Hemost. 26. 53-60 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Suzuki K, Hayashi T, Akiba J: "Successful intravenous interferon-b treatment for a chronic hepatitis C patient with Bernard-Soulier syndrome"Thromb Res.. 100. 149-152 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Hayashi T., Suzuki K.: "Molecular pathogenesis of Bernard-Soulier syndrome"Sem Thromb Hemost. 26. 53-60 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Suzuki K., Hayashi T., Akiba J., Yoshino M., Tajima K., Satoh S., Kato T.: "Successful intravenous interferon-β treatment for a chronic hepatitis C patient with Bernard-Soulier syndrome"Thromb Res.. 100. 149-152 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Hayashi T., Suzuki K.: "Molecular pathogenesis of Bernard-Soulier syndrome"Semin Thromb Hemost. 26. 53-60 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Suzuki K., Hayashi T., Akiba J., et al.: "Successful intravenous interferon-b treatment for a chronic hepatitis C patient with Bernard-Soulier syndrome"Thromb Res. 100. 149-152 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Hayashi T,Suzuki K: "Molecular pathogenesis of Bernard-Soulier syndrome."Semin Thromb Hemost. 26. 53-60 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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