Dissection of the thrombopoietic transcriptome using a platelet specific microarray

Human blood platelets function in wound healing, inflammation, and clot formation. Despite significant progress molecular mechanisms of megakaryocyte maturation remain poorly characterized. We have applied a customized, platelet-specific oligonucleotide gene chip to dissect the molecular events of megakaryopoiesis. CD34 hematopoietic stem cells obtained from 2 distinct adult human donors were differentiated in vitro for 21 days along the megakaryocyte lineage Cell samples were fixed and screened each day for one-color FACS analysis of GPIIB megakaryocyte-specific events (CD41 marker). In parallel, a daily sample was taken for MK-isolation using magnetically labeled CD61 antibodies (MK GPIllA-specific). Both CD61 and CD61^- cells were processed for total RNA purification and samples were hybridized to platelet microarrays. Filtering/analysis of microarray data revealed six genes that respond to administration of cytokines and are differentially expressed between early and late stages of the differentiation experiment (2 fold change, p <0.05). Of these 6 genes, MA , surfactant (pulmonary associated protein B), and integrin alpha2B transcripts were significantly upregulated between early and late stages of differentiation. While integrin alpha2B (GPIIB) is a known marker on platelets and mature MKs, possible roles of the MA protein and surfactant in MK maturation and proplatelet formation are being investigated.