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）：该提议的项目的目的是获得一个大分子代理，该代理可以阻止特定的血小板糖蛋白（GP）IB1的异构域脱落。作为Von Willebrand因子（VWF）血小板中的主要受体，GPIB1在近膜膜部位被ADAM17蛋白水解或脱落，导致其N末端片段释放，也称为糖蛋白，也称为糖质蛋白，从血小板表面释放。 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.但是，目前不可能直接检验这些假设，因为尚不清楚GPIB1的脱落仅仅是损坏和已被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。这种歧义在很大程度上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生域脱落的能力。鉴于对GPIB1具有特异性抑制剂的迫切需求，我们建议筛选SCFV phagemid库和RNA适体库，用于在GPIB1中专门识别并与叶膜脱落裂解位点并结合的粘合剂。足够高的结合亲和力应使这些粘合剂能够超出竞争ADAM17，以便访问，并且只能访问GPIB1脱落裂解位点，从而获得特定的GPIB1脱落的抑制作用。 SCFV吞噬和RNA适体库都已成功地用于生产各种蛋白质靶标的配体，其中一些已发展为FDA批准的药物。由于它们在主链结构，分子大小，折叠和对诱变和其他相关特性的差异的差异，SCFV和RNA文库为脱落抑制剂提供了明显的互补选择。为了进行筛选，包含在其天然样构象中GPIB1脱落裂解位点两侧序列的重组蛋白将被固定为结合靶标。一旦从筛选中鉴定出适当的粘合剂，它们就会大量生产，并测试其结合GPIB1脱落裂解位点并抑制GPIB1在转染的哺乳动物细胞和血小板中脱落的能力。借助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，并探索新的策略，以延长储存的血小板浓缩物的保质期。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血小板或细胞中的许多其他脱落底物，这可能会给膜受体带来更多的膜受体作为药物开发的合适靶标。 公共卫生相关性：清除储存的血小板浓缩物可能取决于从血小板表面的GPIB1的蛋白水解去除。我们计划生产一种特异性抑制去除过程的代理。迫切需要该试剂来解决血小板清除的机理，并可能导致延长储存血小板浓缩物的保质期的新型策略。

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