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

• 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
• 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

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命。在ITP患者中，针对常见的血小板表面蛋白产生抗体，导致免疫系统攻击血小板并将其从血液中清除。ITP最常用的治疗方法是免疫抑制治疗，如类固醇或静脉注射免疫球蛋白（IVIG）。不幸的是，大约20%的患者对这些治疗是难治的，更大比例的患者只是短暂的反应。对IVIG治疗的耐药性表明，在IVIG耐药的ITP患者中，存在另一种血小板清除机制。血小板表面最丰富的蛋白之一是异四聚体GPIb-IX受体复合物。该受体负责结合配体，如血管性血友病因子，并导致血小板从血液中清除。IVIG治疗难治性患者与患者血清中抗GPIb-IX配体结合域（LBD）的抗体有很强的相关性。此外，小鼠体内注射抗lbd单克隆抗体（mab）足以引起血小板减少症。诱导血小板减少的能力使这些单克隆抗体独一无二，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。目前的GPIb-IX信号传导模型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理解血小板清除和血小板减少症至关重要。最近，GPIb-IX的机械感觉结构域（MSD）被发现参与GPIb-IX信号传导。这个紊乱的复合物区域通常盘绕在膜和GPIb-IX的其他亚基的近端。然而，为了使GPIb-IX复合体的一部分“脱落”，必须暴露和展开MSD的一段，这是清除的重要信号。我们假设针对GPIb-IX配体结合域的抗体通过引起MSD的展开和亚基间界面的干扰来诱导清除。在具体目标1中，我们将通过流式细胞术分析清除信号，确定结合配体结合结构域并引起清除的抗体是否也会展开MSD，并将其与结合MSD的抗体的表位暴露增加联系起来。在具体目标2中，我们将研究亚基间接口在GPIb-IX信令中的作用。我们将利用亚基间界面残基改变的突变体，以及受体信号的抗体激动剂和拮抗剂来做到这一点。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ITP和其他血小板减少性疾病的新诊断方法和治疗的未来发展。