PATHOGENESIS OF THE IMMUNE THROMBOCYTOPENIAS

免疫性血小板减少症通常是由于自身抗体或同种抗体 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两个重点领域： 光谱和抗体特性测定。 抗原 本地化 我们将确定血小板- 相关抗体和血浆抗体最初集中在抗体， GPIIb/IIIa，因为它们是最常见的。 我们将：（1）显示是否 抗血小板抗体是复合物特异性的或蛋白质特异性的， 直接结合和抑制测定。 (20抗体结合试验 跨越糖蛋白分子的一系列肽。 这些肽 将在E.杆菌 通过插入含有cDNA片段的载体， 聚合酶链反应 将通过传代纯化融合蛋白， 穿过直链淀粉柱 一旦表位定位于其中一个表位， 大的肽，进一步的定位将通过确定 抗体与该区域内较小肽的结合模式。 （三） 确定血小板相关性紫癜患者慢性ITP的发病率 与GPIIIa和血浆的细胞外部分反应的自身抗体 对GPIIIa的胞质部分特异的自身抗体。 发病 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。 我们将确定人类是否 模拟患者抗体的抗血小板抗体可以在 离体培养E.在引入含有PCR产生的 从患者RNA产生的重链和轻链组合文库。 将阳性克隆的抗体特异性与患者进行比较 抗体的 我们还将研究患者血小板相关的 和血浆抗体以固定补体并结合巨核细胞。 的 上述研究的结果将与患者的临床 确定哪些参数影响疾病严重程度的过程， 对治疗的反应。

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.