Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins

Abstract

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein-coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.

Figure 1. Characterization of MK and EB cultures.

(A-B) Megakaryocyte morphology, the MK cultures were predominantly in the megakaryoblast (MB) or promegakaryocyte (pro-MK; horse-shoe-shaped nucleus) stage of differentiation, although some large granular MKs (multilobed nucleus) were observed. (C) Erythroblasts (EBs) were in the proerythroblast stage of differentiation. (D) Flow cytometric analysis of MKs and EBs after sorting. After FACS sorting, 99.3% of the cells were positive for CD41. The parallel EB culture (E) was sorted to a purity of 97.2% (based on CD235a expression). (F) Confirmation of differential expression of lineage-associated markers by TaqMan real-time PCR. Data presented as fold difference in expression (corrected for GAPDH) between paired MK and EB cultures. Error bars represent the SEM of biologic (n = 5) and technical (n = 3) replicates combined. Values above the x-axis indicate genes up-regulated in MKs, whereas values below indicate genes up-regulated in EBs. Cells were stained using modified May-Gruünwald eosine-methylene blue solution (Merck) and 0.5 mM KH₂PO₄, pH 7.4. Images were acquired using a Leica DM 1L microscope (Leica, Mannheim, Germany) equipped with a Leica objective C, plan L 40×/0.50 numerical aperture PH2 with a total magnification of 400×. Images were captured using a Leica DC300 digital camera and Leica imaging software (IM500, version 1.20).

Figure 2. Confirmation of microarray results using TaqMan real-time PCR.

Values for both cDNA array measurements () and real-time PCR () are plotted as Log₂ (fold change). Results are shown as the average for the 5-paired MK/EB comparisons. Although all transcripts were detected by RT-PCR in all 5 of the MK samples, transcripts marked with an asterisk (*) were not detected in all of the EB cultures. Thus, the ratio is based on fewer than 5 comparisons (G6f, n = 2; TMSF15, n = 1; LST1 and SUCNR1, n = 4; all others n = 5). **LRRC32 was identified as differentially expressed in preliminary array experiments and by Taqman real-time PCR. Error bars represent SD of replicates.

Figure 3. Expression of novel transmembrane proteins in platelets.

(A-B) G6b and G6f were detected in platelets. N-glycosidase (lanes marked N), but not O-glycosidase (lanes marked O) treatment of a platelet lysate shows that both G6b and G6f are N-glycosylated platelet proteins. The first lane in each blot is a molecular weight marker (MagicMark; Invitrogen; sizes in kDa). (C-E) Detection of SUCNR1, LRRC32, and LAT2 in platelets by Western blot. (F) Flow cytometric detection of novel proteins in platelets. Dotted line shows fluorescence detection of a matched preimmune serum (or in the case of LAT2, murine IgG1), and solid line shows the fluorescence using antisera/antibodies against the cognate antigen.

Figure 4. Protein distribution in purified populations of blood cells.

G6b, G6f, and LRRC32 are restricted to platelets, whereas LAT2 is also detected in monocytes (CD14⁺) and B cells (CD19⁺). The succinate receptor, SUCNR1, was detected in all cells tested except granulocytes.

Figure 5. Alternative splicing of G6b in platelets.

(A) The G6b (c6orf25) gene consists of 6 exons, with the fifth exon containing 2 potential splice acceptor sites (exon 5a or 5b). (B) Seven splice variants of the G6b transcript are annotated in the Entrez Gene database. Transcripts encoding G6b-F and -G contain the intronic sequences IV and V, respectively. Splice variant-specific primer binding sites are represented by arrows. (C) RT-PCR was used to determine which G6b transcripts are expressed in platelets. The reverse primer G6b-A/C/G potentially amplifies G6b-A, -C, and -G (lane 2); however, only G6b-A is detected because the 457-bp band corresponding to G6b-C is not observed nor is any band detected with the G6b-G–specific primer (lane 7). G6b-B (lane 3) is the only other splice variant detected. Expected sizes of PCR products are shown in Table 4.

Figure 6. Succinate potentiates platelet aggregation in response to low doses of agonists.

(A) Succinate (1 mM) alone has no effect on platelets, whereas a combination of succinate (1 mM) and ADP (1 µM) significantly increases final aggregation. Error bars are calculated based on duplicate measurements of final aggregation in 4 individuals. (B) A representative aggregometry trace from this experiment. (C) Investigation of the dose response to succinate and ADP. Platelets were costimulated with 1 µM ADP and varying concentrations of succinate (0-1000 µM) and final aggregation was measured. Data are shown as the average of triplicate measurements at each concentration for each of 4 donors; error bars show the SEM of the values obtained. (D) A representative aggregometry trace from this experiment. (E-F) Similar concentrations of succinate also potentiate the effect of low doses of TRAP-6 (10 µM) and CRP-XL (0.02 µg) on platelet aggregation.