AUTOIMMUNE THROMBOCYTOPENIA

描述 （改编自申请人摘要）自身免疫性血小板减少症通常是由于 对于与血小板糖蛋白（GP）复合物反应的抗体， 尤其是GPIIb/IIIa。 自身表位通常是二价阳离子依赖性的。 研究人员将集中在两个研究领域：自身抗体 表征和抗原定位。 自身抗体表征。 他们将：1）证明存在血小板相关和血浆 抗体，并侧重于抗GPIIb/IIIa的抗体，因为这些是最常见的 常见; 2）通过检查轻链和IgG评价抗体克隆性 研究自身抗体与巨核细胞的结合 在体外并确定结合抗体是否影响巨核细胞成熟， 激活补体或诱导吞噬作用。 抗原定位。 他们 将：1）确定兔抗体是否与GPIIb/IIIa钙结合 位点将抑制自身抗体结合; 2）测试自身抗体与 一系列大的重组肽，跨越糖蛋白IIb和IIIa。 他们将特定的PCR产生的cDNA片段插入载体中， 结果在E.大肠杆菌作为融合蛋白， 6X-组氨酸标签，然后可以使用镍亲和纯化 次氮基三乙酸树脂柱 抗体结合到其中一个 大肽将通过确定结合模式进一步研究， 该区域内较小的肽; 3）评估表位，未通过 这项技术，与随机肽库和GPIIb/IIIa嵌合体。 将抗体与表达几种蛋白质的fUSE 5丝状噬菌体一起孵育。 百万个随机肽序列。 噬菌体表达序列，其结合 通过连续的“计划”将抗体与非特异性噬菌体分离， 葡萄球菌-A蛋白琼脂糖，然后酸洗脱。 的序列 确定特定的肽。 此外，他们还将开发 人/爪蟾和人/禽GPIIb/IIIa嵌合体，他们将使用这些嵌合体来 检测人类抗血小板自身抗体的结合模式。 结果 这些研究将与患者的临床病程相关， 血小板生成素水平。 此外，免疫性血小板减少症的效果 对患者生活质量的影响。

DESCRIPTION (Adapted from applicant's abstract) Autoimmune thrombocytopenia is often due to antibodies reactive with platelet glycoprotein (GP) complexes, particularly GPIIb/IIIa. Autoepitopes are often divalent cation-dependent. The investigators will focus on two research areas: autoantibody characterization and antigenic localization. Autoantibody characterization. They will: 1) Demonstrate the presence of platelet-associated and plasma antibodies and focus on antibodies against GPIIb/IIIa, since these are most common; 2) Evaluate antibody clonality by examining light chain and IgG subtype characteristics and 3) Study autoantibody binding to megakaryocytes in vitro and determine if bound antibody effects megakaryocyte maturation, activates complement or induces phagocytosis. Antigen localization. They will: 1) Determine if rabbit antibody to the GPIIb/IIIa calcium-bindings sites will inhibit autoantibody binding; 2) Test autoantibody binding to a series of large recombinant peptides, spanning glycoproteins Iib and IIIa. They will insert specific PCR-generated cDNA fragments into a vector which results in their expression by E. Coli as fusion proteins with a 6X-histidine tag which can then be affinity-purified using a nickel nitrilo-tricacetic acid resin column. Antibodies which bind to one of the large peptides will be studied further by determining binding patterns to smaller peptides within this region; 3) Evaluate epitopes, not identified by this technique, with random peptide libraries and GPIIb/IIIa chimeras. Antibodies are incubated with fUSE5 filamentous phage expressing several million random peptide sequences. Phage expressing sequences which bind to antibodies are separated from non-specific phage by serial 'planning' with staphylococcal-A protein agarose followed by acid elution. The sequence of the specific peptide(s) is determined. In addition, they will develop human/xenopus and human/avian GPIIb/IIIa chimeras which they will use to examine the binding patterns of human antiplatelet autoantibodies. Results of these studies will be correlated with the patients' clinical course and thrombopoietin levels. In addition, the effect of immune thrombocytopenia on the patients' quality of life will be studied.