Human blood platelets play critical roles in normal hemostatic processes and pathologic conditions such as thrombosis, vascular remodeling, inflammation, and wound repair. Generated as cytoplasmic buds from precursor bone marrow megakaryocytes, platelets are enucleate and lack nuclear DNA, although they retain megakaryocyte-derived mRNAs. Towards the goal of defining the molecular anatomy of the platelet transcriptome, we have adapted complementary techniques of microarray and serial analysis of gene expression (SAGE) for genetic profiling of highly purified human blood platelets (Blood 101:2285-2293), and demonstrated the potential applicability of this approach for the molecular analysis of a rare platelet disorder (essential thrombocythemia). While these observations established the initial proof-of-principle supporting this research direction, current platelet isolation procedures require plateletpheresis and relatively cumbersome purification methods for optimal determinations, limiting wider applicability. During the tenure of this grant, we propose to develop a miniaturized system for high-throughput platelet transcriptome analysis.
In specific aim 1, we will adapt and develop mechanical shear as an efficient methodology for separation of ultra-pure platelets from whole blood (1 mL), followed by mRNA isolation and representative transcript amplification uniquely adapted for small RNA yields.
In specific aim 2, we will develop a customized platelet cDNA chip for confirmatory studies of amplified platelet mRNA fidelity as established by microarray analysis. If successful, this project will develop the appropriate infrastructure and methodologies for more comprehensive profiling of larger data sets, a major long-term goal of this area of investigation. Given the importance of platelets in cardiovascular disease and stroke, these studies will have considerable implications for novel gene discovery, and for molecular diagnostics targeted at large patient populations.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Exploratory/Developmental Grants (R21)
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Hemostasis and Thrombosis Study Section (HT)
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Ganguly, Pankaj
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State University New York Stony Brook
Internal Medicine/Medicine
Schools of Medicine
Stony Brook
United States
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Gnatenko, Dmitri V; Zhu, Wei; Xu, Xiao et al. (2010) Class prediction models of thrombocytosis using genetic biomarkers. Blood 115:7-14
Gnatenko, Dmitri V; Dunn, John J; Schwedes, John et al. (2009) Transcript profiling of human platelets using microarray and serial analysis of gene expression (SAGE). Methods Mol Biol 496:245-72
Gnatenko, Dmitri V; Perrotta, Peter L; Bahou, Wadie F (2006) Proteomic approaches to dissect platelet function: Half the story. Blood 108:3983-91
Gnatenko, Dmitri V; Cupit, Lisa D; Huang, Emily C et al. (2005) Platelets express steroidogenic 17beta-hydroxysteroid dehydrogenases. Distinct profiles predict the essential thrombocythemic phenotype. Thromb Haemost 94:412-21