DIFFERENCES IN THE PROTEIN SIGNATURES/PNH PLATELETS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 阵发性睡眠性血红蛋白尿症是一种获得性溶血性贫血，其特征是红细胞对补体的敏感性增加，导致血管内溶血和血红蛋白尿。PNH是由于在X连锁的PIGA基因中获得突变的细胞克隆的扩增。PIGA是合成糖基磷脂酰肌醇（GPI）锚分子所必需的酶亚基。因此，来自突变祖细胞的血细胞缺乏所有GPI锚定分子。我们研究的广泛的长期目标是了解PNH的病理生理学和发病机制。PNH是一种慢性疾病，常伴有大量的发病率和死亡率。血栓形成是最常见的死亡原因。PNH中血栓形成的病理生理机制尚不清楚。我们认为血小板（Plt）缺乏GPI连接蛋白（PNH表型）在PNH血栓形成的发病机制中起主要作用。我们推测，PNH表型的血细胞不仅缺乏所有GPI连接的蛋白质，但也缺乏其他蛋白质，其合成或定位是依赖于正常的GPI锚生产，这些蛋白质的Plt的缺陷可能有助于血栓形成的风险。在拟议的研究中，我们将集中在这些假设的分子方面，通过确定蛋白质和蛋白质修饰的PNH Plt的与PNH。首先，我们将开发可重复的蛋白质组学方法来分离和分析正常和PNH患者的Plt，然后将来自缺乏GPI连接蛋白的Plt的蛋白质谱与正常Pit的蛋白质谱进行比较。最后，我们将开发检测和测量PNH细胞特异性的候选蛋白或蛋白修饰的方法，并验证第二组患者和对照个体中候选蛋白的差异表达。我们提出的调查可能会确定一些意想不到的蛋白质，蛋白质相互作用，和蛋白质修饰，可能会显着推进我们的了解血栓形成的PNH的发病机制，也可能在其他条件下，其中异常克隆造血与血栓形成，例如，真性红细胞增多症或其他骨髓增生综合征。已建立的分析和比较健康和疾病中血小板蛋白质组的方法可能会识别出可用于评估血栓形成风险的新型生物标志物，或识别诊断或药物开发的新靶点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia characterized by the increased sensitivity of red cells to complement leading to intravascular hemolysis and hemoglobinuria. PNH is due to the expansion of a cell clone that has acquired a mutation in the X-linked PIGA gene. PIGA is an enzyme subunit essential for the synthesis of glycosyl phosphatidylinositol (GPI) anchor molecules. Blood cells derived from the mutant progenitor cell are therefore deficient in all GPI-anchored molecules. The broad long-term objective of our research is to understand the pathophysiology and pathogenesis of PNH. PNH is a chronic disease often associated with substantial morbidity and mortality. Thrombosis is the most frequent cause of death. The pathophysiology of thrombosis in PNH is not understood. We propose that platelets (Plt's) deficient in GPI-linked proteins (PNH phenotype) play a major role in the pathogenesis of thrombosis in PNH. We hypothesize that blood cells with the PNH phenotype not only lack all GPI-linked proteins, but are also deficient in other proteins, whose synthesis or localization is dependent on normal GPI anchor production, and that the deficiencies of these proteins on Plt's might contribute to the prothrombotic risk. In the proposed research we will focus on the molecular aspects of these hypotheses by identifying proteins and protein modification in PNH Plt's that are associated with PNH. First, we will develop reproducible proteomic procedures to isolate and analyze Plt's from normal and PNH patients, and then compare the protein profile from Plts' deficient in GPI-linked proteins with the protein profile of normal Pit's. Finally, we will develop assays to detect and measure candidate proteins or protein modifications specific for PNH cells and verify the differential expression of candidate proteins in a second cohort of patient and control individuals. Our proposed investigations are likely to identify a number of unanticipated proteins, protein interactions, and protein modifications that might significantly advance our understanding of the pathogenesis of thrombosis in PNH and possibly also in other conditions, in which an abnormal clonal hematopoiesis is associated with thrombosis, for example, polycythemia vera or other myeloproliferative syndromes. Established methods for analyzing and comparing the platelet proteome in health and disease might lead to the identification of novel biomarkers useful in evaluating the risk of thrombosis or identify new targets for the development of diagnostics or drugs.