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Prepublished online as a Blood First Edition Paper on November 14, 2002; DOI 10.1182/blood-2002-09-2797.
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Blood, 15 March 2003, Vol. 101, No. 6, pp. 2285-2293
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Transcript profiling of human platelets using microarray and
serial analysis of gene expression
Dmitri V. Gnatenko,
John J. Dunn,
Sean R. McCorkle,
David Weissmann,
Peter L. Perrotta, and
Wadie F. Bahou
From the Department of Medicine, Department of
Pathology, and Program in Genetics, State University of New York, Stony
Brook; Biology Department, Brookhaven National Laboratory, Upton, NY;
and Department of Pathology, Robert Wood Johnson Medical Center, New
Brunswick, NJ.
Human platelets are anucleate blood cells that retain cytoplasmic
mRNA and maintain functionally intact protein translational capabilities. We have adapted complementary techniques of microarray and serial analysis of gene expression (SAGE) for genetic profiling of
highly purified human blood platelets. Microarray analysis using the
Affymetrix HG-U95Av2 approximately 12 600-probe set maximally
identified the expression of 2147 (range, 13%-17%) platelet-expressed transcripts, with approximately 22% collectively involved in
metabolism and receptor/signaling, and an overrepresentation of genes
with unassigned function (32%). In contrast, a modified SAGE protocol using the Type IIS restriction enzyme MmeI (generating
21-base pair [bp] or 22-bp tags) demonstrated that 89% of tags
represented mitochondrial (mt) transcripts (enriched in 16S and 12S
ribosomal RNAs), presumably related to persistent mt-transcription in
the absence of nuclear-derived transcripts. The frequency of non-mt SAGE tags paralleled average difference values (relative expression) for the most "abundant" transcripts as determined by microarray analysis, establishing the concordance of both techniques for platelet
profiling. Quantitative reverse transcription-polymerase chain reaction
(PCR) confirmed the highest frequency of mt-derived transcripts, along
with the mRNAs for neurogranin (NGN, a protein kinase C substrate) and
the complement lysis inhibitor clusterin among the top 5 most abundant
transcripts. For confirmatory characterization, immunoblots and flow
cytometric analyses were performed, establishing abundant cell-surface
expression of clusterin and intracellular expression of NGN. These
observations demonstrate a strong correlation between high transcript
abundance and protein expression, and they establish the validity of
transcript analysis as a tool for identifying novel platelet proteins
that may regulate normal and pathologic platelet (and/or
megakaryocyte) functions.
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