P50 Novel Activatory and Inhibitory Platelet Receptors Identified Through Comparative Gene Expression Profiling of Megakaryocytes and Erythroblasts

Summary  Platelets are derived from a polyploid precursor cell, the megakaryocyte (MK). The MK and the precursor of red cells, the erythroblast (EBs) share a common bipotent progenitor and have closely linked differentiation pathways. We hypothesized that the subset of genes that are differentially expressed between MKs and EBs would contain novel, previously uncharacterized, ‘lineage specific’ genes. We have used cDNA microarrays to identify genes differentially expressed between CD34+ Haematopoietic Stem Cell (HSC) derived MKs and EBs. Methods  CD34+ HSCs were purified from Umbilical Cord Blood by magnetic cell separation. For differentiation into MKs, HSCs were cultured for 7 days in serum-free medium containing Thrombopoietin and IL-1β. For differentiation into EBs, HSCs were cultured for 10 days with Erythropoietin, IL-3 and Stem Cell Factor. Differentiated cells were purified by Fluorescence Activated Cell Sorting to >95% purity, RNA isolated and then compared on Sanger Hver2.1.1 15K cDNA arrays. The data was statistically and bioinformatically analysed to identify novel transcripts encoding proteins with transmembrane domains. Taqman Real-time PCR was used to validate the differential expression of both known and novel transcripts and to investigate blood cell distribution. Murine polyclonal antisera were raised against recombinant protein and used to confirm expression of these genes at protein level in platelets and MKs by western blot. Results  Known lineage specific markers, including CD41, CD42a and CD42b were upregulated in MKs relative to EBs. A number of novel transmembrane proteins were also identified as being upregulated in MKs. These include the immunoglobulin fold domain containing proteins c6orf25 (G6b) and c6orf21 (G6f). Transcript profiling of blood cells demonstrated that G6b and G6f expression is restricted to the platelet and megakaryocyte lineages. Western blotting confirmed the presence of G6b and G6f as glycosylated proteins of the platelet plasma membrane. Functional studies have shown that cross-linking of G6b with polyclonal antisera can inhibit platelet aggregation in response to both collagen and ADP, suggesting that this protein has an inhibitory role in vivo. In addition, we have identified a novel G-protein coupled receptor in platelets that may have a co-stimulatory role in platelet aggregation. Conclusions  The comparative analysis of the MK and EB transcriptomes provides an overview of gene expression in these two cells and has identified novel platelet-specific transmembrane proteins. Experiments are underway to characterise the role of these novel proteins in the regulation of platelet function.