Specific Inhibition of Ectodomain Shedding of GPIb-alpha

特异性抑制 GPIb-α 的胞外域脱落

• 批准号: 8212483
• 负责人: Renhao Li
• 金额: $ 19.38万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-15 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8212483
• 关键词: Address; Affinity; Binding; Biological; Biological Assay; Blood; Blood Platelets; Cell surface; Cells; Cleaved cell; Detection; Environment; Evolution; Excision; FDA approved; Glycoprotein Ib; Goals; Human; Immunoglobulin Fragments; In Vitro; Investigation; Lead; Libraries; Life; Ligands; Mammalian Cell; Measures; Membrane; Metalloproteases; Methods; Molecular Conformation; Monitor; Mutagenesis; N-terminal; Oligonucleotides; Pharmaceutical Preparations; Platelet Glycoproteins; Play; Process; Property; Proteins; Public Health; RNA; RNA library; Reagent; Recombinant Fusion Proteins; Recombinant Proteins; Recovery; Role; Screening procedure; Site; Stream; Structure; Surface; Testing; Time; Transfusion; Vertebral column; aptamer; base; drug development; glycocalicin; inhibitor/antagonist; interest; novel strategies; public health relevance; receptor; von Willebrand Factor

DESCRIPTION (provided by applicant): The goal of this proposed project is to obtain a macromolecular agent that can block specifically ectodomain shedding of platelet glycoprotein (GP)Ib1. As the primary receptor in platelets for von Willebrand factor (vWF), GPIb1 is proteolyzed or shed by ADAM17 at a juxtamembrane site, resulting in the release of its N-terminal fragment, also known as glycocalicin, from platelet surface. Although the biological significance of ectodomain shedding of GPIb1 and function of glycocalicin remain to be defined, recent evidences suggest that shedding of GPIb1 plays an important role in detection and clearance of pathologically damaged platelets, and that blocking shedding of GPIb1 can hamper clearance of platelets stored in vitro. However, it is not possible at the present time to directly test these hypotheses, because it is not clear whether shedding of GPIb1 is merely an inconsequential indicator for the damaged and to-be-cleared platelets or actually the cause for platelet clearance. Such ambiguity is largely due to the usage of broad-spectrum metalloprotease inhibitors in the investigation and the lack of ability to modulate ectodomain shedding in a substrate-specific manner. Given the compelling need for a shedding inhibitor specific to GPIb1, we propose to screen the scFv phagemid library and the RNA aptamer library for binders that specifically recognize and bind to the juxtamembrane shedding cleavage site in GPIb1. Sufficiently high binding affinity should enable these binders to out-compete ADAM17 for access to, and only to, the GPIb1 shedding cleavage site, and thereby achieving specific inhibition of GPIb1 shedding. Both scFv phagemid and RNA aptamer libraries have been used successfully to produce ligands for various protein targets, some of which have been developed into FDA-approved drugs. Due to their difference in backbone structure, molecule size, folding and amenability to mutagenesis and other related properties, scFv and RNA libraries offer distinct and complementary choices for a shedding inhibitor. For the screening, recombinant proteins that contain the sequence flanking the GPIb1 shedding cleavage site in its native-like conformation will be immobilized as binding targets. Once appropriate binders are identified from the screening, they will be produced in large quantity and tested for their ability to bind the GPIb1 shedding cleavage site and inhibit shedding of GPIb1 in transfected mammalian cells and platelets. With a GPIb1-specific shedding inhibitor, we will be poised to investigate the biological significance of GPIb1 shedding and explore novel strategies to extend the shelf life of stored platelet concentrates. Moreover, our method to develop substrate-specific shedding inhibitors, if successfully developed, can be applied to many other shedding substrates in platelets or cells of interest, which may deliver significantly more membrane receptors as suitable targets for drug development. PUBLIC HEALTH RELEVANCE: Clearance of the stored platelet concentrates may depend on proteolytic removal of GPIb1 from the platelet surface. We plan to produce an agent that specifically inhibits the removal process. This agent is sorely needed to address the mechanism of platelet clearance and it may lead to novel strategies to extend the shelf life of stored platelet concentrates.

描述（由申请人提供）：本项目的目标是获得一种能够特异性阻断血小板糖蛋白（GP）Ib1外结构域脱落的大分子药物。作为血小板中von Willebrand因子（vWF）的主要受体，GPIb1在近膜位点被ADAM17蛋白水解或脱落，导致其n端片段（也称为糖钙素）从血小板表面释放。尽管GPIb1外域脱落的生物学意义和糖钙蛋白的功能仍有待明确，但最近的证据表明，GPIb1的脱落在病理损伤血小板的检测和清除中起着重要作用，阻断GPIb1的脱落会阻碍体外储存的血小板的清除。然而，目前还不可能直接验证这些假设，因为尚不清楚GPIb1的脱落是否仅仅是受损和待清除血小板的无关紧要的指标，还是实际上是血小板清除的原因。这种模糊性主要是由于在研究中使用了广谱金属蛋白酶抑制剂，并且缺乏以底物特异性方式调节外结构域脱落的能力。鉴于对GPIb1特异性脱落抑制剂的迫切需求，我们建议筛选scFv噬菌体文库和RNA适体文库，以寻找特异性识别并结合GPIb1近膜脱落切割位点的结合物。足够高的结合亲和力应该使这些结合物能够与ADAM17竞争，进入且仅进入GPIb1脱落的切割位点，从而实现对GPIb1脱落的特异性抑制。scFv噬菌体和RNA适体文库已经成功地用于生产各种蛋白质靶点的配体，其中一些已被开发成fda批准的药物。由于scFv和RNA文库在主链结构、分子大小、折叠和易变性等相关特性上的差异，它们为脱落抑制剂提供了不同的互补选择。在筛选过程中，将含有GPIb1脱落切割位点两侧天然构象序列的重组蛋白固定为结合靶标。一旦从筛选中确定合适的结合物，它们将被大量生产，并测试它们结合GPIb1脱落切割位点和抑制转染的哺乳动物细胞和血小板中GPIb1脱落的能力。有了GPIb1特异性脱落抑制剂，我们将准备研究GPIb1脱落的生物学意义，并探索延长储存血小板浓缩物保质期的新策略。此外，我们开发底物特异性脱落抑制剂的方法，如果成功开发，可以应用于血小板或感兴趣的细胞中的许多其他脱落底物，这可能会提供更多的膜受体作为药物开发的合适靶点。

项目成果

The organizing principle of the platelet glycoprotein Ib-IX-V complex.

• DOI: 10.1111/jth.12144
• 发表时间: 2013-04
• 期刊: Journal of thrombosis and haemostasis : JTH
• 作者: Li R;Emsley J
• 通讯作者: Emsley J

Specific inhibition of ectodomain shedding of glycoprotein Ibα by targeting its juxtamembrane shedding cleavage site.

• DOI: 10.1111/jth.12425
• 发表时间: 2013-12
• 期刊: Journal of thrombosis and haemostasis : JTH
• 作者: Liang X;Russell SR;Estelle S;Jones LH;Cho S;Kahn ML;Berndt MC;Bunting ST;Ware J;Li R
• 通讯作者: Li R

Transmembrane domains are critical to the interaction between platelet glycoprotein V and glycoprotein Ib-IX complex.

跨膜结构域对于血小板糖蛋白 V 和糖蛋白 Ib-IX 复合物之间的相互作用至关重要。

• DOI: 10.1111/j.1538-7836.2012.04841.x
• 发表时间: 2012-09
• 期刊: Journal of thrombosis and haemostasis : JTH
• 作者: Mo X;Liu L;López JA;Li R
• 通讯作者: Li R