MEGAprofiles provide big insightsinto platelet function

This issue of Blood contains reports from 2 groups that have used microarray profiling of CD34⁺-differentiated hematopoietic stem cells to dissect molecular mechanisms regulating megakaryocytopoiesis and/or for identification of functionally novel platelet receptors. Both reports provide new insights into proplatelet formation and platelet function.

Hematologists are intrigued by megakaryocytes (MKs), hematopoietic cells readily distinguishable by their abundant cytoplasm and large size (∼ 35-160 μm), nuclear poly-ploidy (up to 64N), and relative paucity (0.02%-0.05% of total nucleated bone marrow cells). While discovery of the existence and hemostatic functions of blood platelets is generally assigned to Bizzozero based on pioneering observations made during the 1880s, linking megakaryocytes to platelet biogenesis is credited to James Wright in 1906, who made conclusions based on histomorphometric similarities of granular and cytoplasmic content between both cell types.¹  In the subsequent century, considerable insight has been achieved in identifying receptors and signaling pathways that regulate platelet and megakaryocyte biology. Nonetheless, the genetic machinery controlling megakaryocyte transition into the distinctive endomitotic switch that precedes the epiphany of cytoplasmic maturation, membrane demarcation, proplatelet formation, and platelet release remains incompletely understood. Similarly, the application of genomic technologies for identification of novel, functionally-important platelet genes and proteins remains a high priority.² 

Raslova and colleagues describe cellular mRNA profiling to specifically dissect genetic changes occurring during an in vitro model of megakaryocyte differentiation and ploidization. Cells were flow-sorted by modal ploidy, and gene changes were compared between 2 cellular subsets: aggregate 2N+4N MKs versus aggregate 8N+16N MKs. Of interest, transcript changes were limited to approximately 350 genes across all the subsets, 106 of which were consistently down-regulated and 248 of which were consistently up-regulated between the 2 groups. Further analysis highlighted additional differences between the up-regulated and down-regulated subsets; specifically, members of the latter subset not infrequently (24/105) corresponded to genes involved in DNA replication (arrest) and recombination repair, while a majority of the former corresponded to genes important to platelet biogenesis, viability, and function (ie, actin and microtubule cytoskeleton, glycoproteins, and signaling/transport proteins). It is important to note, however, that when the gene subsets are carefully analyzed by gene ontology functional classification, considerable overlap exists between these 2 groups, confounding detailed interpretations. Nonetheless, the data do support a role of ploidization in modulating gene expression, although a direct, regulatory role in platelet biogenesis remains speculative.

The study by Macaulay and colleagues adapts a nearly-identical in vitro strategy of MK differentiation, coupled with a bioinformatic strategy to specifically identify novel transmembrane domain–containing MK receptor proteins. An initial gene list of 151 transcripts was assembled using paired, com-parative expression profiling with CD34⁺-differentiated erythroblasts, and the list further pared using strict criteria to identify putative, functionally-relevant platelet proteins. Five of 8 highly-selected genes were characterized by transcript and protein expression studies, 3 of which were shown to be platelet restricted (G6b, G6f, and LRRC32), and another of which (SUCNR1) encoded the G protein–coupled succinate receptor.³  More detailed functional studies established that succinate (a key component of the citric acid cycle) exhibited costimulatory effects on platelet aggregation induced by various platelet agonists (adenosine diphosphate, thrombin receptor activating peptide, and a glycoprotein VI–specific collagen peptide). The latter functional data are especially insightful in that they identify a novel, cocoupling signal transduction pathway in platelets, while opening new avenues of research linked to platelet hyperreactivity.⁴ 

While both study designs overlap in their initial in vitro differentiation strategies using megakaryocytes, the conclusions, future directions, and ability to compare data sets are distinct and limited. One restriction inherent in cross-experimental microarray data-sharing is the disparate platforms used among investigators, well exemplified in these studies, that used nonoverlapping oligonucleotide or cDNA probe sets for their analyses.^5,6  This limitation does not minimize results, although it emphasizes the importance of validation strategies of transcript differences initially identified by microarray. The identification of a costimulatory succinate receptor on platelets represents a discrete end product of integrated MK transcriptomic studies, coupled with a concrete hypothesis and sophisticated experimental design to characterize novel functional receptors. Likewise, the application of microarray technology to dissect MK ploidization is highly novel, and although the results are less focused in scope, they are likely to yield broader implications in the foreseeable future. Finally, such unique data sets open up exciting opportunities for sophisticated data mining likely to provide unexpected insights into molecular mechanisms of MK and platelet function.⁷