A transgenic mouse model for reticulated platelet detection reveals expansion after myocardial ischemia/reperfusion

Figures

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  Figure 1.. RiboTag-based identification and phenotypic characterization of RP in naïve mice. (A) Schematic of the Pf4-Cre:RiboTag transgenic mouse model, enabling lineage-specific HA tagging of ribosomes in megakaryocytes and their progeny. (B) Gating strategy for platelet identification in whole blood, based on singlet selection and CD42b positivity. (C-E) Representative flow cytometry plots showing RP detection using HA-RiboTag (RiboTag-RP) (C), SYTO 13 nucleic acid staining (SYTO-RP) (D), and TO staining (TO-RP) (E). (F) Expression of platelet surface markers in RiboTag-RP vs non-RP under resting and activated (PAR-4 agonist, 400 μM) conditions. Resting RiboTag-RP displayed higher expression of GPIbα (CD42b), and higher levels of activated GPIIb/IIIa (JON/A binding) and P-selectin (CD62P) following agonist-induced activation. Values from 6 mice are shown as GMFI ± standard deviation (SD) (n = 6). Statistical comparisons were made by 1-way analysis of variance (ANOVA) with Bonferroni post hoc test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. rpl22, ribosomal protein l22.

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  Figure 2.. Myocardial I/R injury triggers a time-dependent increase in circulating RP. (A) Schematic overview of the experimental workflow. Myocardial I/R injury was induced by 30-minute ligation of the LAD coronary artery followed by reperfusion for 1 or 2 days. Sham-operated mice underwent the same procedure without LAD ligation. RP were quantified at baseline and postsurgery using 3 independent flow-cytometric detection methods. (B) RiboTag-based detection of RP in whole blood revealed a modest increase in RiboTag-RP percentage on day 1 after I/R injury and a significant elevation by day 2, compared to sham controls. (C) SYTO 13 nucleic acid staining confirmed the I/R-induced increase in SYTO-RP levels at day 2, with no significant change on day 1. (D) TO staining yielded similar increase in TO-RP levels at day 2, supporting a robust thrombopoietic response after I/R injury. Data from 6 to 7 mice per group are presented as mean ± SD. Statistical significance was assessed by 1-way ANOVA with the Bonferroni post hoc test. ∗P < .05, ∗∗∗P < .001, ∗∗∗∗P < .0001.

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  Figure 3.. RP display higher surface receptor expression and enhanced agonist responsiveness compared to non-RP, with altered phenotype after I/R injury. Flow-cytometric analysis was performed on whole blood from Pf4-Cre:RiboTag mice 1 and 2 days after sham or myocardial I/R injury. (A) Surface expression of GPIbα (CD42b) was significantly higher in RiboTag-RP compared to non-RP under all conditions. RiboTag-RP retained significantly elevated levels at rest and after PAR-4 agonist stimulation. (B) GPIIb/IIIa activation (JON/A binding) was significantly greater in RiboTag-RP vs non-RP in both sham and I/R groups, although overall activation was modestly reduced after I/R injury. (C) Surface P-selectin (CD62P) expression was negligible at rest and markedly increased after PAR-4 stimulation, with RiboTag-RP consistently showing higher levels than non-RP. Data represent GMFI ± SD (n = 7). Statistical testing: 1-way ANOVA with Bonferroni post hoc test. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.

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  Figure 4.. RNA sequencing of RP and non-RP after experimental myocardial I/R injury. (A) Principal component analysis of gene expression in mouse RP and non-RP isolated by flow cytometry on day 2 after I/R injury based on SYTO 13 intensity (brightest vs dimmest 20% sorted in lysis buffer; n = 4-6 per group). (B) Expression of Pf4, Odc1, and Itga2b in SYTO-RP and non-RP. (C) Differential gene expression analysis comparing SYTO-RP and non-RP after myocardial I/R injury. (D) Top 10 biological processes enriched (with q values) among genes upregulated in SYTO-RP vs non-RP as derived from Gene Ontology. (E) Predicted cell-cell communication between RP and cardiac cells after I/R injury. Receptor expression in endothelial cells, fibroblasts, macrophages, and cardiomyocytes was derived from Molenaar et al.30 MI, myocardial infarction.