DIFFERENCES IN THE PROTEIN SIGNATURES/PNH PLATELETS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 阵发性睡眠性血红蛋白尿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是由于一个X连锁PIGA基因突变的细胞克隆的扩张所致。PIGA是合成糖基磷脂酰肌醇(GPI)锚定分子所必需的酶亚基。因此，来自突变的祖细胞的血细胞缺乏所有GPI锚定的分子。我们研究的长期目标是了解PNH的病理生理学和发病机制。PNH是一种慢性疾病，通常与相当大的发病率和死亡率有关。血栓形成是最常见的死亡原因。PNH血栓形成的病理生理学机制尚不清楚。我们认为血小板(Plt‘s)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，具有PNH表型的血细胞不仅缺乏所有与GPI相关的蛋白质，而且还缺乏其他蛋白质，这些蛋白质的合成或定位依赖于正常的GPI锚点产生，这些蛋白质在Plt上的缺乏可能导致血栓前风险。在拟议的研究中，我们将通过识别PNH Plt中与PNH相关的蛋白质和蛋白质修饰来关注这些假说的分子方面。首先，我们将开发可重复的蛋白质组学程序来分离和分析正常和PNH患者的Plt，然后将缺乏GPI连接蛋白的Plt的蛋白质图谱与正常Pit的蛋白质图谱进行比较。最后，我们将开发检测和测量PNH细胞特有的候选蛋白质或蛋白质修饰的方法，并验证候选蛋白质在第二组患者和对照个体中的差异表达。我们拟议的研究可能会发现一些意想不到的蛋白质、蛋白质相互作用和蛋白质修饰，这些可能显著提高我们对PNH和其他情况下血栓形成机制的理解，在其他情况下，异常克隆性造血与血栓形成有关，例如真性红细胞增多症或其他骨髓增生性综合征。建立分析和比较健康和疾病中的血小板蛋白质组的方法可能会导致识别新的生物标志物，这些生物标志物有助于评估血栓形成的风险，或者为诊断或药物的开发确定新的靶点。

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.