AUTOIMMUNE THROMBOCYTOPENIA

描述 (摘自申请者的摘要)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)复合体反应的抗体， 特别是GPIIb/IIIa。自体表位通常是二价阳离子依赖的。 研究人员将专注于两个研究领域：自身抗体 鉴定和抗原定位。自身抗体的特征。 他们将：1)证明存在与血小板相关的和血浆 抗体和重点放在针对GPIIb/IIIa的抗体上，因为这些是最 2)通过检测轻链和免疫球蛋白来评价抗体的克隆性 亚型特征和3)自身抗体与巨核细胞结合的研究 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)测定兔抗GPIIb/IIIa钙结合抗体 部位将抑制自身抗体结合；2)检测自身抗体与 一系列大的重组多肽，跨越糖蛋白IIb和IIIa。 他们将把特定的聚合酶链式反应产生的cdna片段插入到载体中， 导致它们在大肠杆菌中以融合蛋白的形式表达 6x-组氨酸标签，然后可以使用镍进行亲和纯化 氮三乙酸树脂柱。与其中一个细胞结合的抗体 大肽将通过确定结合模式来进一步研究 该区域内较小的多肽；3)评估表位，未经鉴定 该技术利用随机多肽文库和GPIIb/IIIa嵌合体。 抗体与fUSE5丝状噬菌体孵育，表达几个 百万个随机的多肽序列。与之结合的噬菌体表达序列 抗体与非特异性噬菌体的分离是通过与 葡萄球菌-A蛋白琼脂糖酸洗脱。按顺序排列 确定了特定的多肽(S)。此外，他们还将发展 人/非洲爪哇和人/鸟GPIIb/IIIa嵌合体，它们将用来 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和 血小板生成素水平。此外，免疫性血小板减少症的影响 将对患者的生活质量进行研究。

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.