Identification of the regulatory elements of human von Willebrand factor for binding to platelet GPlb.

人血管性血友病因子与血小板 GP1b 结合的调节元件的鉴定。

• 批准号: 12670983
• 负责人: MATSUSHITA Tadashi
• 金额: $ 2.11万
• 依托单位: Nagoya 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-12670983/
• 关键词: von Willebrand factor; GPIh; polymerase chain reaction; platelet; thrombosis; coronary heartdisease; alanine scanning mutagencsis; Polymerase chain reaction; von Willebrand因子; ristocetin; botrocetin; mutant; 血小板

In vitro platelet glycoprotein Ib (GPIb) binding of human von Willebrand factor (VWF) increases markedly by exogenous modulators such as ristocetin or botrocetin and the binding does not occur in normal circulation. GPIb binding sites have been assigned in VWF Al domain that consists of a disulfide loop Cysl272 (509l)-Cys1458(695). In contrast, several gain-of-function mutations have been found in two regions comprised of the disulfide loop and its N- and Cterminal flanking regions. In this study, Cysl222(459)-Tyr1271(508), Gin1238(475)Tyrl271 (508), Glu l260(497)-Tyrl27 1(508), and Asp 1459(696)-Asp1472(709) were sequentially deleted of full-length multimeric recombinant VWF. Deletions at either side resulted in normal GPIb binding, indicating that the flanking regions are not GPIb binding sites. However, an additive mutation at Arg1308(545) onto each deletion mutant resulted in the spontaneous GPIb binding without requirements of modulators, suggesting both regions are important for inhibition of GPIb binding. The spontaneous binding was completely inhibited by monoclonal antibodies that recognize the GPIb binding sites. Interestingly, mutations deleted with N-terminal, but not Cterminal flanking regions lost the binding to monoclonal antibodies B328, B710, and 23C7 that selectively inhibit ristocetin-induced GPIb binding and their epitopes were found at His 1268(505) or Asp1269(506). The crystalographic structure of the Al domain suggest that GPIb binding is influenced by the molecular interface between two regions, and the antibody binding to the interface inhibit the binding.

在体外血小板糖蛋白Ib（GPIb）的人血管性血友病因子（VWF）的结合显着增加外源性调节剂，如利托那肽或botrocetin和结合不发生在正常循环。GPIb结合位点已被分配在由二硫环Cys 1272（5091）-Cys 1458（695）组成的VWF Al结构域中。相反，在由二硫环及其N端和C端侧翼区组成的两个区域中发现了几个功能获得性突变。在该研究中，Cysl 222（459）-Tyrl 271（508）、Glnl 238（475）Tyrl 271（508）、Glul 260（497）-Tyrl 271（508）和Aspl 459（696）-Aspl 472（709）依次缺失全长多聚体重组VWF。两侧的缺失导致正常的GPIb结合，表明侧翼区域不是GPIb结合位点。然而，在Arg 1308（545）的添加突变到每个缺失突变体中导致自发GPIb结合而不需要调节剂，这表明这两个区域对于抑制GPIb结合是重要的。识别GPIb结合位点的单克隆抗体完全抑制自发结合。有趣的是，缺失N-末端但不缺失C-末端侧翼区的突变失去了与选择性抑制瑞斯托菌素诱导的GPIb结合的单克隆抗体B328、B710和23 C7的结合，并且在His 1268（505）或Asp 1269（506）处发现了它们的表位。Al结构域的晶体学结构表明GPIb结合受两个区域之间的分子界面的影响，并且与界面结合的抗体抑制结合。

项目成果

K.Ishiguro., T.Matsushita. et al.: "Complete antithrombin deficiency in mice results in embryonic lethality"J Clin Invest. 106. 873-878 (2000)

K.Ishiguro.，T.松下。

K.Ishiguro, T.Matsushita, et al.: "Syndecan-4 deficiency leads to high mortality of lipopolysaccharide-injected mice"J Biol Chem.. 276. 47483-47488 (2001)

K.Ishiguro、T.Matsushita 等人：“Syndecan-4 缺陷导致脂多糖注射小鼠的高死亡率”J Biol Chem.. 276. 47483-47488 (2001)

T.Iwaki, T.Mastushita, et al.: "DNA sequence analysis of protein S deficiency--identification of four point mutations in twelve Japanese subjects"Semin Thromb Hemost. 27. 155-160 (2001)

T.Iwaki、T.Mastushita 等人：“蛋白质 S 缺陷的 DNA 序列分析 - 十二名日本受试者中四个点突变的鉴定”Semin Thromb Hemost。

S. Kunishima, T. Matsushita, etal: "Mutations in the NMMHC-A gene cause autosomal dominant macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly/Sebastian syndrome)"Blood. 97. 1147-1149 (2001)

S. Kunishima、T. Matsushita 等人：“NMMHC-A 基因突变导致常染色体显性巨血小板减少症伴白细胞内含物（May-Hegglin 异常/塞巴斯蒂安综合征）”血液。

T.Sugihara., I.Takahashi., T.Matsushita., et al.: "Identificaion of plasma antibody epitopes and gene abnormalities in Japanese hemophilia A patients with factor VIII inhibitor"Nagoya J Med Sci.. 63. 25-39 (2000)

T.Sugihara.，I.Takahashi.，T.Matsushita.，等：“具有因子 VIII 抑制剂的日本 A 型血友病患者血浆抗体表位和基因异常的鉴定”Nagoya J Med Sci.. 63. 25-39（