Hemostasis Mediated by the Platelet Glycoprotein (GP)Ib alpha-Ib beta-IX Complex

血小板膜糖蛋白（GP）Ib-V-IX复合物是血管性血友病因子的受体，由四种多肽组成：GPIb α、GPIb β、GPIX和GPV，所有这些多肽都具有富含亮氨酸的重复基序。 这种复合物的定性或定量缺陷导致罕见的人类出血素质Bernard-Soulier综合征（BSS）。 BSS是一种常染色体隐性遗传性状，在婴儿期表现为血小板减少症、循环中的“巨”血小板和婴儿期出血倾向。BSS中的出血比血小板计数预测的严重不成比例，并可解释为原发性止血缺陷。我们发现了一个新的突变（P96 S）在GPIb β基因位点的婴儿单倍型不足，由于杂合缺失染色体22 q11（Velocardiofacial综合征）。当用野生型GPIb β转染时，稳定表面表达人GPIb α和GPIX（CHO α-IX）的转染中国仓鼠卵巢细胞的流式细胞术和共聚焦成像表明，P96 S GPIb β消除了血小板vWF受体复合物的表面组装。 基于与nogo-66神经元受体（也是一种富含亮氨酸的重复蛋白，其晶体结构已被表征）的氨基酸同源性，我们提出了一种GPib β蛋白结构模型，该模型支持P96和其他残基的重要性。先前报道为GPib β构象中的错义突变及其与GPIX的相互作用。GPIb β代表了这种最近表征的血小板粘附复合物的最关键组分。GPIb β及其在血小板粘附和止血中的关键作用的进一步研究正在取得进展，以更精确地阐明其与GPIX和GPIb α的相互作用，并希望为BSS患者提供新的治疗方法。在过去的一年中，我们成功地在HEK-293细胞中表达了FLAG标记的GPIb β cDNA，并进行了部分氨基酸测序，证实了其身份。 我们的努力，以纯化天然蛋白质的结晶分析，以及产生针对它的（GP）Ib-V-IX复合物的未来研究的特异性抗体正在继续。

The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor and is composed of four polypeptides: GPIb alpha, GPIb beta, GPIX, and GPV, all of which feature leucine-rich repeat motifs. A qualitative or quantitative deficiency in this complex causes the rare human bleeding diathesis Bernard-Soulier syndrome (BSS). BSS is an autosomal recessive trait presenting in infancy with thrombocytopenia, circulating "giant" platelets and bleeding tendency in infancy. Bleeding in BSS is disproportionately more severe than predicted by platelet count, and is explained by a defect in primary hemostasis. We identified a novel mutation (P96S) at the GPIb beta locus in an infant haploinsufficient for this gene, due to heterozygous deletion of chromosome 22q11 (Velocardiofacial syndrome). Flow cytometry and confocal imaging of transfected Chinese hamster ovary cells that stably surface-express human GPIb alpha and GPIX (CHO alpha-IX) when transfected with wild-type GPIb beta demonstrated that P96S GPIb beta abrogates surface assembly of the platelet vWF receptor complex. Based on amino acid homology to the nogo-66 neuronal receptor (also a leucine-rich repeat protein, the crystal structure for which has been characterized), we proposed a model of GPIb beta protein structure that supports the importance of P96 and other residues previously reported as missense mutations in the conformation of GPIb beta, and its interaction with GPIX. GPIb beta represents the most critical component of this recently characterized platelet adhesion complex. Further study of GPIb beta and its critical role in platelet adhesion and hemostasis is progress to illuminate more precisely its interaction with GPIX and GPIb alpha and in hope of novel therapeutic approaches for BSS patients. In the past year, we successfully expressed the a FLAG-tagged GPIb beta cDNA in HEK-293 cells and performed partial amino acid sequencing that confirmed its identity. Our efforts to purify the native protein for crystallographic analysis as well as to generate specific antibodies against it for future studies of the (GP)Ib-V-IX complex are continuing.