Perrault Christelle, Mangin Pierre, Santer Martine, Baas Marie-Jeanne, Moog Sylvie, Cranmer Susan L, Pikovski Inna, Williamson David, Jackson Shaun P, Cazenave Jean-Pierre, Lanza François
INSERM U311, Etablissement Français du Sang, Alsace, Strasbourg, France.
Blood. 2003 May 1;101(9):3477-84. doi: 10.1182/blood-2002-06-1847. Epub 2003 Jan 9.
Glycoprotein (GP) Ib/V/IX complex-dependent platelet adhesion to von Willebrand factor (VWF) is supported by the 45-kd N-terminal extracellular domain of the GPIb alpha subunit. Recent results with an adhesion blocking antibody (RAM.1) against GPIb beta, which is disulfide linked to GPIb alpha, have suggested a novel function of this subunit in regulating VWF-mediated platelet adhesion, possibly involving its intracellular face. A putative cooperation between the GPIb alpha and GPIb beta cytoplasmic domains was investigated by measuring the adhesion under flow to immobilized VWF of K562 and Chinese hamster ovary (CHO) cells transfected with GPIb/(V)/IX containing mutations in this region. Adhesion of cells carrying a glycine substitution of the GPIb beta Ser166 phosphorylation site was 50% lower than normal and became insensitive to inhibition by RAM.1. In contrast, forskolin or PGE(1) treatment increased both the phosphorylation of GPIb beta and adhesion of control cells, both effects being reversed by RAM.1, but had no influence on cells expressing the Ser166Gly mutation. A role of the GPIb alpha intracellular domain was also apparent as the VWF-dependent adhesion of cells containing deletions of the entire (Delta 518-610) or portions (Delta 535-568, Delta 569-610) of the GPIb alpha cytoplasmic tail was insensitive to RAM.1 inhibition. Cells carrying progressive 11 amino acid deletions spanning the GPIb alpha 535-590 region were equally unresponsive to RAM.1, with the exception of those containing GPIb alpha Delta 569-579, which behaved like control cells. These findings support a role of the GPIb beta intracellular domain in controlling the adhesive properties of the GPIb/V/IX complex through phosphorylation of GPIb beta Ser166 and point to the existence of cross-talk between the GPIb beta and GPIb alpha intracellular domains.
糖蛋白（GP）Ib/V/IX复合物依赖的血小板与血管性血友病因子（VWF）的黏附由GPIbα亚基45-kd的N端细胞外结构域支持。最近针对与GPIbα通过二硫键相连的GPIbβ的黏附阻断抗体（RAM.1）的研究结果表明，该亚基在调节VWF介导的血小板黏附方面具有新功能，可能涉及其细胞内表面。通过测量转染了该区域含有突变的GPIb/（V）/IX的K562细胞和中国仓鼠卵巢（CHO）细胞在流动状态下与固定化VWF的黏附，研究了GPIbα和GPIbβ细胞质结构域之间可能的协同作用。携带GPIbβ丝氨酸166磷酸化位点甘氨酸替代的细胞黏附比正常情况低50%，并且对RAM.1的抑制不敏感。相反，福斯可林或前列腺素E1（PGE1）处理增加了GPIbβ的磷酸化以及对照细胞的黏附，这两种效应都被RAM.1逆转，但对表达Ser166Gly突变的细胞没有影响。GPIbα细胞内结构域的作用也很明显，因为含有GPIbα细胞质尾巴全部缺失（Δ518 - 610）或部分缺失（Δ535 - 568、Δ569 - 610）的细胞对VWF的依赖性黏附对RAM.1抑制不敏感。携带跨越GPIbα 535 - 590区域逐步缺失11个氨基酸的细胞对RAM.1同样无反应，除了含有GPIbα Δ569 - 579的细胞，其表现与对照细胞相似。这些发现支持GPIbβ细胞内结构域通过GPIbβ丝氨酸166磷酸化在控制GPIb/V/IX复合物黏附特性方面的作用，并指出GPIbβ和GPIbα细胞内结构域之间存在相互作用。
