PATHOGENESIS OF THE IMMUNE THROMBOCYTOPENIAS

免疫性血小板减少症的发病机制

• 批准号: 3353927
• 负责人: ROBERT MCMILLAN
• 金额: $ 19.27万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3353927
• 关键词: Escherichia coli; antibody specificity; antigen antibody reaction; autoantibody; baboons; binding proteins; chimeric proteins; complement fixation tests; epitope mapping; glycoprotein structure; glycoproteins; human subject; immunoglobulin structure; immunohematology; immunopathology; isoantibody; maltose; megakaryocytes; monoclonal antibody; platelets; polymerase chain reaction; protein purification; thrombocytopenia

Immune thrombocytopenia is often due to autoantibodies or alloantibodies against platelet glycoproteins (GP), particularly GPIIb/IIIa. We will focus on two areas in immune thrombocytopenia: evaluation of the antigenic spectrum and determination of antibody characteristics. Antigen localization. We will determine the epitope specificity of both platelet- associated and plasma antibodies concentrating initially on antibodies to GPIIb/IIIa, since these are the most common. We will: (1) Show whether antiplatelet antibodies are complex-specific or protein-specific using direct binding and inhibition assays. (20 Test for antibody binding to a series of peptides which span the glycoprotein molecule. These peptides will be produced as fusion proteins with maltose binding protein in E. Coli by insertion of vectors containing CDNA fragments generated by the polymerase chain reaction. The fusion proteins will be purified by passage through an amylose column. Once the epitope is localized to one of the large peptides, further localization will be achieved by determining the antibody binding pattern to smaller peptides within this region. (3) Determine the incidence in chronic ITP of patients with platelet-associated autoantibody reactive with extracellular portions of GPIIIa and plasma autoantibody specific for the cytoplasmic part of GPIIIa. The pathogenetic role in vivo (if any) of the anti-cytoplasmic antibodies will be studied in baboons. Autoantibody characteristics. We will determine whether human antiplatelet antibody, which mimics patient antibody, can be synthesized in vitro by E. Coli after introduction of vectors containing PCR-generated heavy and light chain combinatorial libraries produced from patient RNA. Antibody specificity of positive clones will be compared with patient antibody. We will also study the ability of patient platelet-associated and plasma antibody to fix complement and bind to megakaryocytes. The results of the above studies will be correlated with the patients' clinical course to determine which of the parameters influence disease severity and response to therapy.

免疫性血小板减少症通常是由自身抗体或同种抗体引起 对抗血小板糖蛋白 (GP)，特别是 GPIIb/IIIa。 我们将 免疫性血小板减少症的重点是两个领域：抗原评估 谱和抗体特性的测定。 抗原 本土化。 我们将确定两种血小板的表位特异性 相关抗体和血浆抗体最初集中于抗体 GPIIb/IIIa，因为它们是最常见的。 我们将： (1) 显示是否 抗血小板抗体具有复合物特异性或蛋白质特异性，使用 直接结合和抑制测定。 （20 测试抗体与 跨越糖蛋白分子的一系列肽。 这些肽 将在大肠杆菌中与麦芽糖结合蛋白形成融合蛋白 通过插入含有 cDNA 片段的载体 聚合酶链式反应。 融合蛋白将通过传代纯化 通过直链淀粉柱。 一旦表位定位于其中之一 大肽，将通过确定来实现进一步的定位 抗体与该区域内较小肽的结合模式。 (3) 确定血小板相关患者慢性 ITP 的发生率 与 GPIIIa 和血浆的细胞外部分发生反应的自身抗体 GPIIIa 细胞质部分特异性的自身抗体。 发病机理 将研究抗细胞质抗体的体内作用（如果有的话） 狒狒。 自身抗体特征。 我们将确定是否人类 模拟患者抗体的抗血小板抗体可以在 引入含有 PCR 生成的载体后，通过大肠杆菌进行体外培养 由患者 RNA 产生的重链和轻链组合文库。 阳性克隆的抗体特异性将与患者进行比较 抗体。 我们还将研究患者血小板相关的能力 血浆抗体固定补体并与巨核细胞结合。 这 上述研究结果将与患者的临床情况相关 确定哪些参数影响疾病的严重程度和 对治疗的反应。