PATHOGENESIS OF THE IMMUNE THROMBOCYTOPENIAS

免疫性血小板减少症通常由自身抗体或同种异体抗体引起。 抗血小板膜糖蛋白，特别是GPIIb/IIIa。我们会 免疫性血小板减少症的两个领域：抗原性评价 抗体特性的光谱和测定。抗原 本地化。我们将确定这两种血小板的表位特异性- 相关抗体和血浆抗体最初集中在抗体上 GPIIb/IIIa，因为这些是最常见的。我们将：(1)显示是否 抗血小板抗体是复合体特异的或蛋白质特异的使用 直接结合试验和抑制试验。(20与抗体结合的测试 一系列跨越糖蛋白分子的多肽。这些多肽 将在大肠杆菌中以与麦芽糖结合蛋白的融合蛋白的形式产生 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进行纯化。 通过直链多糖柱。一旦表位被定位到一个 大多肽的进一步定位将通过确定 抗体与该区域内较小的多肽的结合模式。(3) 测定血小板相关患者慢性ITP的发生率 与GPIIIa胞外部分和血浆反应的自身抗体 针对GPIIIa胞浆部分的自身抗体。致病因素 抗细胞质抗体在体内的作用(如果有的话)将在 这些是狒狒。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 含聚合酶链式反应的载体导入大肠杆菌后的体外培养 从患者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。我们还将研究患者的血小板相关能力 和血浆抗体，以固定补体和结合巨核细胞。这个 以上研究的结果将与患者的临床相关联。 确定哪些参数影响疾病严重程度和 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.