Essential thrombocythemia (ET) is a myeloproliferative disease (MPD) characterized by a chronically elevated platelet count and an increased risk of thrombosis. Although known to be a clonal disorder of platelet production, no protein or chromosomal marker has been identified to serve as a diagnostic test; there is no understanding of any underlying pathways that are affected; and although an effective therapy (anagrelide) exists, its mode of action is not well understood. Our understanding of ET pales in contrast to the much less common but well-studied MPD, chronic myeloid leukemia (CML), for which there is a known diagnostic chromosomal translocation, a clear understanding of its pathophysiology, and a targeted effective therapy. To address this lack of knowledge about ET we have performed preliminary DMA microarray and proteomic analysis of the platelets from patients with ET and normal subjects and have found major differences in their expression patterns. Microarray analysis showed 272 gene sequences significantly increased and 419 gene sequences decreased in ET compared with normal platelets. Proteomics analysis showed about 15 proteins altered in amount or mobility. Preliminary analysis of a few of these has shown that c-mpl, ribosomal protein S6, and Talin 1 are highly altered in ET platelets. In this proposal we seek to define the proteome of normal and ET platelets using 2-D gel electrophoresis, high-resolution image analysis, and bioinformatics methods. In addition we will use several novel functional proteomics approaches to study the phosphoproteins, protein kinases, and membrane proteins in ET platelets. Specificity of these proteins for ET will be assessed by comparison with platelets from patients with other MPD and reactive thrombocytosis. Using multivariant analysis of these proteins, diagnostic and prognostic tests will be created for ET. Finally, a ranking of proteins that may be relevant to the pathophysiology of ET will be made based on confirmatory testing in ET megakaryocytes and inhibition of their effect on cultures of CD34-derived megakaryocytes. Completion of this work should improve the public health of those with ET, a common MPD, by providing a diagnostic test for ET, predicting its clinical complications of thrombosis, bleeding, or fibrosis, and helping us understand how common treatments work. Moreover, this work should identify genes that provide insight into the etiology of ET and suggest new targets for therapy.
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