A multidisciplinary approach to elucidate the pathophysiology of thrombocytopenia

阐明血小板减少症病理生理学的多学科方法

基本信息

批准号：
9358329
负责人：
Michael Ian Quach
金额：
$ 4.4万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9358329
关键词：
Adult Affect Agonist Amino Acids Animal Model Antibodies Binding Binding Sites Biological Assay Blood Blood Circulation Blood Coagulation Disorders Blood Platelets Cell Line Child Clinical Common Epitope Complex Data Deuterium Development Disease Epitopes Fc Receptor Flow Cytometry Functional disorder Future Glycoproteins Goals Hematology Hemorrhage Human Hydrogen Immune Immune system Immunosuppression Immunosuppressive Agents Immunotherapy Individual Injectable Injection of therapeutic agent Intravenous Immunoglobulins Ligand Binding Ligand Binding Domain Ligands Link Mass Spectrum Analysis Mediating Membrane Membrane Proteins Microscopy Modeling Molecular Conformation Monoclonal Antibodies Mus Mutagenesis Outcome Pathway interactions Patients Play Receptor Signaling Refractory Resistance Role Serum Severities Signal Transduction Steroids Surface Testing Thrombocytopenia Work antigen binding glycoprotein receptor GPIb-IX improved interdisciplinary approach macrophage mutant novel diagnostics receptor tool von Willebrand Factor

项目摘要

Immune thrombocytopenia (ITP) is the most prevalent bleeding disorder, affecting the lives of tens of millions of people worldwide. In individuals with ITP, antibodies are developed against common platelet surface proteins and cause the immune system to attack platelets and remove them from the bloodstream. ITP is most commonly treated using immunosuppressive treatments like steroids or intravenous immunoglobulin (IVIG). Unfortunately, approximately 20% of patients are refractory to these treatments, and a larger percentage only respond transiently. Resistance to IVIG treatment suggests that there is an alternative mechanism of platelet clearance which plays a role in IVIG-resistant ITP patients. One of the most abundant platelet surface proteins is the heterotetrameric GPIb-IX receptor complex. The receptor is responsible for binding to ligands such as von Willebrand factor, and leading to clearance of the platelet from the blood. There is a strong correlation between patients who are refractory to IVIG treatment and antibodies against the ligand binding domain (LBD) of GPIb-IX in patient serum. Additionally, injection of anti-LBD monoclonal antibodies (MAbs) into mice is sufficient to cause thrombocytopenia. The ability to induce thrombocytopenia makes these MAbs unique, since almost all antibodies against other parts of GPIb-IX cannot induce thrombocytopenia. Current models of GPIb-IX signaling are not sufficient to explain the unique ability of anti-ligand binding domain antibodies to induce clearance. Therefore, determining the mechanism of GPIb-IX signaling is vital for complete understanding of platelet clearance and thrombocytopenia. Recently, the mechanosensory domain (MSD) of GPIb-IX has been hown to be involved in GPIb-IX signaling. This disordered region of the complex is normally coiled proximal to the membrane and the other subunits of GPIb-IX. However, a segment of the MSD must be exposed and unfolded in order for part of the GPIb-IX complex to be “shed”, which is an important signal for clearance. We hypothesize that antibodies against the ligand binding domain of GPIb-IX induce clearance by causing the unfolding of the MSD and perturbing inter-subunit interfaces. In specific aim 1, we will determine whether antibodies which bind the ligand-binding domain and cause clearance also unfold the MSD by analyzing clearance signals via flow cytometry, and correlating this to increased exposure of the epitope for an antibody which binds the MSD. In specific aim 2, we will investigate the role of inter-subunit interfaces in GPIb-IX signaling. We will do this by making use of mutants with altered residues in their inter-subunit interfaces, as well as antibody agonists and antagonists of receptor signaling. My proposed studies will elucidate fundamental mechanisms of GPIb-IX mediated platelet clearance and facilitate future development of novel diagnostic approaches and therapies for ITP and other thrombocytopenic conditions.

免疫性血小板减少症 (ITP) 是最常见的出血性疾病，影响着全世界数千万人的生活。在患有 ITP 的个体中，会产生针对常见血小板表面蛋白的抗体，导致免疫系统攻击血小板并将其从血液中清除。 ITP 最常见的治疗方法是使用类固醇或静脉注射免疫球蛋白 (IVIG) 等免疫抑制疗法，但不幸的是，大约 20% 的患者对这些疗法无效，而且更大比例的患者仅产生短暂反应。对 IVIG 治疗的抵抗表明，存在另一种血小板清除机制，该机制在 IVIG 抵抗性 ITP 患者中发挥作用。最丰富的血小板表面蛋白之一是异四聚体 GPIb-IX 受体复合物，该受体负责与配体结合。 IVIG 治疗难治的患者与抗 GPIb-IX 配体结合域 (LBD) 的抗体之间存在很强的相关性。此外，将抗LBD单克隆抗体（MAb）注射到小鼠体内足以引起血小板减少症，这些MAbs具有独特的能力，因为几乎所有针对GPIb-IX其他部分的抗体都不能诱导血小板减少症。 GPIb-IX 信号传导的机制不足以解释抗配体结合域抗体诱导清除的独特能力，因此，确定 GPIb-IX 信号传导的机制对于完全了解血小板清除至关重要。最近，GPIb-IX 的机械感觉结构域 (MSD) 已被证明参与 GPIb-IX 信号传导。，MSD 的一部分必须暴露并展开，以便部分 GPIb-IX 复合物“脱落”，这是我们针对配体截获的重要抗体信号。 GPIb-IX 的结合结构域通过引起 MSD 的展开和扰动亚基间界面来诱导清除。在具体目标 1 中，我们将通过分析清除信号来确定结合配体结合结构域并引起清除的抗体是否也展开 MSD。通过流式细胞术，并将其与结合 MSD 的抗体表位的增加相关联。在具体目标 2 中，我们将研究亚基间界面在 GPIb-IX 信号传导中的作用。通过利用亚基间界面残基发生改变的突变体以及受体信号传导的抗体激动剂和拮抗剂，我提出的研究将阐明 GPIb-IX 介导的血小板清除的基本机制，并促进新诊断方法和疗法的未来发展。用于 ITP 和其他血小板减少性疾病。