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

血小板膜糖蛋白（GP）Ib-V-IX复合物是血管性血液病因子的受体，由四种多肽组成：GPIb α、GPIb β、GPIX和GPV，它们都具有富含亮氨酸的重复基序。这种复合物在质或量上的缺陷导致罕见的人类出血性素质伯纳德-苏利尔综合征（BSS）。BSS是一种常染色体隐性性状，在婴儿期表现为血小板减少，循环“巨大”血小板和婴儿期出血倾向。BSS的出血比血小板计数预测的严重得多，这可以用原发性止血缺陷来解释。由于22q11染色体杂合缺失（心面快速综合征），我们在婴儿GPIb β位点发现了一个新的突变（P96S）。流式细胞术和共聚焦成像显示，转染野生型GPIb β后，表面稳定表达人GPIb α和GPIX （CHO α - ix）的中国仓鼠卵巢细胞的P96S 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，我们提出了一个GPIb β蛋白结构模型，该模型支持P96和其他残基在GPIb β构象中的重要性，以及它与GPIX的相互作用。GPIb β是最近发现的血小板粘附复合物中最关键的成分。进一步研究GPIb β及其在血小板粘附和止血中的关键作用，以更准确地阐明其与GPIX和GPIb α的相互作用，并希望为BSS患者提供新的治疗方法。在过去的一年里，我们成功地在HEK-293细胞中表达了a flag标记的GPIb β cDNA，并进行了部分氨基酸测序，证实了其身份。我们正在继续努力纯化天然蛋白以进行晶体学分析，并为（GP）Ib-V-IX复合物的未来研究生成针对它的特异性抗体。

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.