Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ibα using canine-human chimeras
Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ibα using canine-human chimeras
复制标题
DOI：
10.1182/blood.v99.1.145
复制
发表时间：
2002-01-01
期刊：
影响因子：
20.3
通讯作者：
Andrews, RK
中科院分区：
文献类型：
--
作者：
Shen, Y;Dong, JF;Andrews, RK
血小板糖蛋白Ib-IX-V（GPIb-IX-V）介导（病理性）生理血栓形成中与血管性血友病因子（vWF）的粘附。vWF结合GPIb α的N-末端282个残基，由N-末端侧翼（His 1-lle 35）、7个富含亮氨酸的重复序列（Leu 36-Ala 200）、C-末端侧翼（Phe 201-Gly 268）和硫酸化酪氨酸序列（Asp 269-Glu 282）组成。通过在中国仓鼠卵巢细胞上表达GPIb的犬-人嵌合体，先前绘制了功能性抗GPIb α抗体与各个结构域的结合位点，并且显示富含亮氨酸的重复序列2至4是静态或流动条件下最佳vWF识别所需的。使用新的犬-人嵌合体解剖C-末端侧翼，现在证明了（1）Phe 201-Glu 225含有AP 1的表位，AP 1是一种抗GPIb α单克隆抗体，其抑制瑞斯托霉素和肉毒杆菌素依赖性vWF结合;（2）VM 116 d，一种优先抑制Botrocetin依赖性vWF结合的抗体，识别Cys 248周围的序列Val 226-Gly 268，与Cys 209形成二硫键;（3）vWF与嵌合GPIb α的结合与2个嵌合体中的野生型相当，其中第六个富含亮氨酸的重复序列与第一个二硫环属于同一物种（Phe 201-Cys 248），表明这些结构域之间的相互作用对于最佳vWF结合可能是重要的;和（4）用相应的犬序列替换人GPIba中C-末端侧翼第二个二硫环（Asp 249-Gly 268），在静态和流动条件下增强vWF结合，提供了与C-末端侧翼第二个环相关的功能获得表型的第一个证据。
Platelet glycoprotein Ib-IX-V (GPIb-IX-V) mediates adhesion to von Willebrand factor (vWF) in (patho)physiological thrombus formation. vWF binds the N-terminal 282 residues of GPIb alpha, consisting of an N-terminal flank (His1-lle35), 7 leucine-rich repeats (Leu36-Ala200), a C-terminal flank (Phe201-Gly268), and a sulfated tyrosine sequence (Asp269-Glu282). By expressing canine-human chimeras of GPIba on Chinese hamster ovary cells, binding sites for functional anti-GPIb alpha antibodies to individual domains were previously mapped, and it was shown that leucine-rich repeats 2 to 4 were required for optimal vWF recognition tinder static or flow conditions. Using novel canine-human chimeras dissecting the C-terminal flank, it is now demonstrated that (1) Phe201-Glu225 contains the epitope for AP1, an anti-GPIb alpha monoclonal antibody that inhibits both ristocetin- and botrocetin-dependent vWF binding; (2) VM116d, an antibody that preferentially inhibits botrocetin-dependent vWF binding, recognizes the sequence Val226-Gly268, surrounding Cys248, which forms a disulfide-bond with Cys209; (3) vWF binding to chimeric GPIb alpha is comparable to wild-type in 2 chimeras in which the sixth leucine-rich repeat was of the same species as the first disulfide loop (Phe201-Cys248) of the C-terminal flank, suggesting an interaction between these domains may be important for optimal vWF binding; and (4) replacing the C-terminal flank second disulfide loop (Asp249-Gly268) in human GPIba with the corresponding canine sequence enhanced vWF binding under static and flow conditions, providing the first evidence fora gain-of-function phenotype associated with the second loop of the C-terminal flank.