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

表达人血小板GPIb/IX受体的稳定细胞系的建立及其在血小板功能中的应用

• 批准号: 12670971
• 负责人: HAYASHI Tomohiro
• 金额: $ 0.58万
• 依托单位: Yamagata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670971/
• 关键词: Bernard-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

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

血小板GPIb/IX复合物是血管性血友病因子（vWF）的表面受体，由GPIb-α、-β和IX三个独立的基因产物组成。这些成分连接在一起，形成vWF的功能性受体。到目前为止，Bernard-Soulier综合征患者的遗传分析表明，GPIb-α中的富含亮氨酸重复序列（LRR）对于复合物的有效表面表达至关重要。LRR是一个短的24个氨基酸的片段，含有保存良好的亮氨酸残基，每个GP具有至少一个这种共有基序。然而，由于缺乏临床病例，关于GPIb-β内LRR的重要性知之甚少。为了评估GPIb-β LRR对GPIb/IX复合物表达的重要性，选择GPIb-β LRR内的4个氨基酸残基，并进行定点诱变以获得一系列突变体：Leu 35、Leu 45和Leu 50 → Ala、瓦尔或Phe、Asn 40 → Ala、Thr或Cys。奥塔伊 ...更多信息 用GPIb-α和GPIX共转染ned质粒，通过流式细胞仪和北方印迹分析GPIb/IX复合物的表达。用突变体质粒转染的GPIb/IX受体的FACS分析：所有Asn 40突变体均显著降低复合物的表面表达。通过使用Leu → Ala或Phe突变体获得类似的结果，然而，当通过FACS分析时，Leu →瓦尔突变体总是显示40-80%的复合物表面表达。II.北方印迹分析：北方印迹分析显示，突变体GPIb-β mRNA水平与对照组基本相同，表明转录后事件是导致GPIb-β受体表达降低的原因。这些数据表明，GPIb-β的LRR内的Leu残基和Asn 40对于GPIb/IX复合物的有效表达是重要的，尽管Leu →瓦尔突变体显示出适度的复合物表达，这可能是由于两种氨基酸的结构相似性。稳定表达人血小板GPIb/IX受体的质粒的建立：我们已经利用上述质粒建立了稳定的转化体。转化子具有类似于人血小板GPIb/IX复合物的血小板聚集能力。但由于长期的栽培，它们逐渐失去了活性。为了保持转化体的聚集潜力，一些修饰是必要的。少

项目成果

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)

Suzuki K.、Hayashi T.、Akiba J.、Yoshino M.、Tajima K.、Satoh S.、Kato T.：“对患有 Bernard-Soulier 综合征的慢性丙型肝炎患者进行成功的静脉注射干扰素 β 治疗” Thromb Res。 . 100. 149-152 (2000)

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

Hayashi T、Suzuki K：“Bernard-Soulier 综合征的分子发病机制。”Semin Thromb Hemost。

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)

Suzuki K、Hayashi T、Akiba J：“对患有 Bernard-Soulier 综合征的慢性丙型肝炎患者进行成功的静脉注射干扰素 B 治疗”Thromb Res.. 100. 149-152 (2000)

Hayashi T., Suzuki K.: "Molecular pathogenesis of Bernard-Soulier syndrome"Sem Thromb Hemost. 26. 53-60 (2000)

Hayashi T.、Suzuki K.：“Bernard-Soulier 综合征的分子发病机制”Sem Thromb Hemost。

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

Hayashi T.、Suzuki K.：“Bernard-Soulier 综合征的分子发病机制”Semin Thromb Hemost。