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.

免疫血小板减少症（ITP）是最普遍的出血障碍，影响了全球数千万人的生活。在患有ITP的个体中，抗体是针对常见的血小板表面蛋白产生的，并导致免疫系统攻击血小板并将其从血液中取出。 ITP最常使用类固醇或静脉免疫球蛋白（IVIG）的免疫抑制作用治疗。不幸的是，大约有20％的患者对这些治疗是难治性，而较大的百分比只会瞬间做出反应。对IVIG治疗的耐药性表明，还有一种替代血小板清除率的机制，它在耐IVIG ITP患者中起作用。最丰富的血小板表面蛋白之一是杂元GPIB-IX受体复合物。该受体负责与诸如von Willebrand因子等配体的结合，并导致血小板从血液中清除。与IVIG治疗难治性的患者与针对患者血清中GPIB-IX的配体结合结构域（LBD）的抗体之间存在很强的相关性。另外，将抗LBD单克隆抗体（mAb）注射到小鼠中足以引起血小板减少症。诱导血小板减少症的能力使这些mAb独特，因为几乎所有针对GPIB-IX其他部位的抗体都无法诱导血小板减少症。 GPIB-IX信号传导的当前模型不足以解释抗体形结合结构域抗体诱导清除率的独特能力。因此，确定GPIB-IX信号传导的机制对于完全了解血小板清除率和血小板减少症至关重要。最近，GPIB-IX的机理域（MSD）是如何参与GPIB-IX信号传导的方法。该复合物的这个无序区域通常是围绕膜和GPIB-IX的其他亚基的围绕的代理。但是，必须暴露并展开MSD的一部分，以使GPIB-IX复合物的一部分被“脱落”，这是清除率的重要信号。我们假设针对GPIB-IX的配体结合结构域的抗体通过导致MSD的展开和扰动 - 亚基间接口而引起清除。在特定目标1中，我们将确定结合配体结合域并引起清除的抗体是否通过流式细胞仪分析清除信号，并将其与增加与MSD结合的抗体暴露的暴露相关联。在特定的目标2中，我们将研究亚基间接口在GPIB-IX信号传导中的作用。我们将通过利用具有改变残差的突变体在其间隔界面，以及接收器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介导的血小板清除率的基本机制，并促进ITP和其他血小板减少症状的新型诊断方法和疗法的未来开发。