Murine reticulated platelets generated during recovery from antibody-induced thrombocytopenia are functionally impaired and transcriptionally rewired

Abstract

Abstract Reticulated platelets (RP) are newly released, RNA-rich platelets that often increase during recovery from thrombocytopenia. Though considered hyperreactive compared to non-RP, their functional and molecular features under immune-mediated stress remain poorly understood. We recently developed a flow cytometric assay for murine RP detection using the nucleic acid dye SYTO 13 and demonstrated a significant increase in RP fraction following antibody-mediated thrombocytopenia (Hamad et al., 2025). In that study, naïve RP showed enhanced P-selectin expression after agonist stimulation compared to non-RP, consistent with a hyperreactive phenotype. However, it remains unclear if this profile is maintained in RP formed during stress-induced megakaryopoiesis. Recent studies suggest RP generated under acute stress may differ functionally from homeostatic RP (Pirabe et al., 2025). These stress-adapted RP may show altered glycoprotein (GP) expression, reduced responsiveness, or changes in granule content, challenging the assumption that elevated RP counts reflect heightened platelet reactivity (Pirabe et al., 2023). The current work expands on these insights by combining functional and transcriptomic profiling of murine RP on day 4 of recovery from antibody-mediated thrombocytopenia. All animal experiments were performed in accordance with German animal protection law. Acute thrombocytopenia was induced in 10- to 14-week-old mice by a single subcutaneous injection of a GPIbα-targeting antibody (1 µg/g body weight, R300, Emfret). Platelet depletion was confirmed by tail vein blood analysis 1 day post-injection. On day 4 of recovery, blood was collected by terminal cardiac puncture, anticoagulated, and diluted 1:10 in Dulbecco's phosphate-buffered saline containing calcium and magnesium (DPBS+/+). RP were identified using SYTO 13 staining and analyzed by flow cytometry for surface GPIbα and P-selectin expression under resting conditions and after stimulation with a thrombin receptor agonist (protease-activated receptor (PAR) 4 peptide, 400 µM, 15 min, 22 °C). Data are presented as mean ± SD. RP from both antibody-treated and naïve mice were sorted by flow cytometry for subsequent bulk RNA sequencing. A single subcutaneous injection of anti-GPIbα antibody induced near-complete platelet depletion by day 1 post-injection (platelet count: 25 ± 8 ×10³/µL, n = 6), followed by full recovery by day 4 (platelet count: 622 ± 95 ×10³/µL, p<0.0001, n = 6). This regenerative phase was accompanied by a significant increase in circulating SYTO 13-high platelets (RP) compared to untreated controls (RP% treated vs. naïve: 35 ± 10 % vs. 10 ± 1 %, p = 0.0026, n = 6). Recovery-phase resting RP displayed markedly reduced GPIbα surface expression, seen as a significant reduction in geometric mean fluorescence intensity (GMFI treated vs. naïve: 3,274 ± 322 vs. 4,440 ± 722, p = 0.0021, n = 6). P-selectin positivity on resting platelets was absent across both groups. Following PAR4 stimulation, only 40 ± 23 % of RP from antibody-treated mice became P-selectin positive, compared to approximately 83 ± 3 % of RP from naïve mice (p = 0.0015, n = 4–6). This was further reflected in a >3-fold difference in P-selectin GMFI (treated vs. naïve: 429 ± 282 vs. 1,341 ± 125, p < 0.0001, n = 4–6). RNA-sequencing revealed a distinct transcriptional signature in recovery-phase RP, with 127 significantly upregulated genes and 41 downregulated genes (log2 fold change >1 or <-1 and q<0.05). Among the significantly upregulated genes were those possibly involved in adhesion and platelet activation (Itga2b, Itgb2, Gp1bb, Ccl5, Adam9), ribosomal and cytoskeletal proteins (Rplp1, Rplp2, Rplp37a, Tubb1, Cfl1, Tpm1, Cnn2), granule components and stress response proteins (Srgn, Thbs1, Clu, S100a11, Tpt1), and mitochondrial regulators (Gpx1, Gpx4, Micu1, Nlrx1, Cyba, Qsox1). In contrast, downregulated genes included regulators of mitochondrial surveillance (Pink1), signaling proteins (Src, Gnb1, Akap7, Slfn5), vesicle trafficking and ubiquitination (Ubash3b, Uba1, Ube2k, Copz1, Copg2), and cytoskeletal motors (Kif3a, Kif5b, Tubb4b).In summary, murine RP generated during recovery from antibody-induced thrombocytopenia exhibit a distinct transcriptional program and altered functional profile. These differences suggest that stress-induced RP may respond differently to hemostatic challenges compared to steady-state RP, with implications for thrombosis risk and platelet-targeted therapies.