Molecular Characteristics of Platelet Formation Studied by Genetic Profiling.
Authors: Rambhia Suraj H. , Xu Xiao , Gnatenko Dmitri V. , Zhu Wei , Bahou Wadie F. 
Published: October 2019

Abstract
Abstract 4584
Megakaryocyte (Mk) development and proplatelet formation are regulated at multiple levels although terminal stage molecular signals are incompletely delineated. We applied a customized, platelet-restricted oligonucleotide gene chip comprised of 432 genes to characterize genetic changes that occur in CD34+ cells differentiated along the Mk lineage, using a cytokine cocktail containing IL-6 (25 ng/mL), IL-11 (25 ng/mL), FLT3 (50 ng/mL), IL-1β (10 ng/mL), thrombopoietin (TPO, 50 ng/mL), and stem cell factor (SCF, 25 ng/mL). After day (D) 5, >60% of cells remained CD41 (αIIB)-positive by flow cytometric analysis, with 90% CD41-positivity at D20. Positive-selection using a β3 (glycoprotein IIIa) anti-CD61 antibody and magnetic beads was completed daily, and allowed for physical separation and comparative genetic profiles of the CD61+ and CD61- cell fractions. Gene-wise progressive correlation analysis of aggregate CD61+ to CD61- cell fractions demonstrated a correlation distribution centered between -0.2 to 0, confirming the distinct genetic profiles of CD61+ and CD61- cells. Unsupervised hierarchical clustering of the CD61+ cells – in conjunction with 41 normal platelet control profiles – confirmed the presence of two discriminatory dendograms that separated D1-15 profiles from D16-21 and normal platelet profiles, establishing that a genetic switch towards late Mk/proplatelet development occurred around D15/D16. Aggregate gene expression between D1-15 and D16-21 subsets identified 252 differentially-expressed genes (p < 0.05), only 12 of which were upregulated in D16-21 samples (11 of these 12 genes were differentially expressed between day-matched CD61- cell fractions, confirming the specificity of these changes). The conjunction of this 12-gene subset with a common 5-member pathway cluster of progressively induced genes identified three transcripts [transforming growth factor β-2 (TGFB2), CD99 antigen (CD99), and thromboxane A2 receptor (TBXA2R)] that were specifically probed to elucidate transcript and protein expression patterns in circulating platelets. Dual-color quantitative flow cytometry for these three proteins (along with ITGB3, β3, as control for a non-induced gene) was completed in both permeabilized (intracytoplasmic) and non-permeabilized (cell-surface) platelets, in parallel with thiazole orange (TO) as a measure of platelet RNA content; for all analyses, RNA and protein content in individual platelets were normalized by size and volume to adjust for size/volume differences. Despite a wide distribution of platelet size, RNA content, and protein expression, both the RNA and protein concentrations remained relatively constant. Furthermore, the RNA concentration remained tightly restricted within a narrow window, establishing that this parameter is critically maintained for all platelets unrelated to age. For all platelets, there was poor correlation (r <0.15) between RNA and protein concentrations, although correlation coefficients increased considerably in the subset of platelets with the highest (top 3%) RNA content. This was evident for both intracytoplasmic (TGFB2, r=0.41; CD99, r=0.55; TBXA2R, r=0.64) and cell-surface (TGFB2; r=0.34; CD99, r=0.73; TBXA2R, r=0.58) determinations, with smaller correlation coefficients for ITGB3 (0.21 and 0.13, respectively). In summary, these data demonstrate that (1) a genetic switch leading to mature platelet profiles (and presumably proplatelet formation) occurs at D15/D16 of MK maturation; and (2) in late-stage highly expressed transcripts, RNA concentration predicts protein concentration (both intracytoplasmic and cell-surface) in the subset of platelets with highest RNA content. These data provide insights into molecular differences between newly-formed and older platelets, and may have implications for elucidating previously-described functional differences among platelet subsets.

Disclosures:
No relevant conflicts of interest to declare.