Transcriptomic characterization of platelets in immune thrombocytopenia indicates sustained activation and mitochondrial dysfunction

Abstract Background Immune Thrombocytopenia (ITP) is a rare hematologic autoimmune disease marked by platelet (PLT) destruction and impaired megakaryopoiesis. While most research focuses on B- and T-cell-mediated mechanisms, PLT-intrinsic changes are poorly characterized. Despite lacking nuclei, PLTs preserve a functional transcriptome responsive to external signals and potentially reflecting disease-specific alterations. PLT RNA profiling has revealed mechanistic insights and distinct gene expression patterns in various conditions. However, transcriptome-wide analyses of circulating PLTs in ITP are lacking. We performed RNA sequencing (RNA-seq) of purified PLTs from ITP patients (pts) to assess technical feasibility of PLT transcriptomics in thrombocytopenia and characterize the PLT RNA profile in ITP. Methods Peripheral blood from 6 chronic ITP pts (PLTs 24-45/µl), 6 pts with non-ITP thrombocytopenia matched by PLT count, 6 ITP pts in treatment-free remission, and 8 healthy controls was collected in EDTA tubes and immediately processed. Sampling required ≥ 20/µl PLTs to ensure sufficient RNA yield. PLT-rich plasma was obtained by centrifugation after addition of a PLT inhibition cocktail to prevent ex vivo activation. PLTs were purified by dual leukocyte depletion (filtration and CD45-based magnetic separation). Total RNA was extracted using organic phase separation followed by column purification. Sample purity was confirmed by qPCR for leukocyte- and erythrocyte-specific transcripts. RNA libraries were prepared using Polaris™ rRNA depletion for coding and non-coding transcript detection, and sequenced on a NovaSeq 6000. Reads were aligned to GRCh38 (Ensembl 104) using STAR; only uniquely mapped reads were included. Gene counts were obtained via featureCounts, and differential expression was analyzed using DESeq2 with sex as covariate. Multiple testing correction was applied using Independent Hypothesis Weighting (FDR ≤ 0.05). Results Sufficient RNA was obtained from all samples. Relative to PLT counts, RNA yield per PLT was higher in ITP pts than in controls, indicating increased RNA content. RNA-seq provided high-quality data with low to absent expression of leukocyte- and erythrocyte-specific transcripts. A mean of 60.7 million paired-end reads and a median of 5864 genes per sample (≥1 CPM) were detected. Differential expression analysis between ITP pts and healthy controls identified 3643 upregulated and 3342 downregulated transcripts. Prominently upregulated genes were related to integrin signaling and PLT adhesion (ITGB3, ITGA2B, FLNA, PARVB, P4HB, PPP6R1), as well as granule biogenesis and vesicle trafficking (NBEAL2, PLXNB3, ENO1, MYH9), suggesting a higher proportion of young, activation-primed PLTs. A marked downregulation was observed in numerous mitochondrial transcripts, including components of the respiratory chain (MT-ND1–6, MT-CO1–3, MT-CYB, MT-ATP6/8) and mitochondrial tRNAs. This pattern differs from that of reticulated PLTs described in the literature and may reflect impaired mitochondrial function and increased apoptotic susceptibility. PLT transcriptomes from ITP pts in treatment-free remission resembled healthy controls, consistent with disease resolution and normalized PLT counts. To assess whether the observed transcriptomic changes were ITP-specific or reflected general features of regenerative thrombopoiesis, we included a comparison group of pts with post-chemotherapy thrombocytopenia during hematopoietic recovery. Though PCA revealed clear separation between ITP and post-chemotherapy samples, many transcriptomic features observed in ITP, such as upregulation of cytoskeletal and granule-associated genes and downregulation of mitochondrial transcripts, were also present in the latter group. This overlap suggests that key transcriptomic features in ITP PLTs reflect a general regenerative program triggered by increased PLT demand. Conclusion Our study shows that PLT transcriptome analysis is feasible even in patients with low PLT counts, enabling detailed molecular characterization in this challenging setting. The ITP PLT transcriptome reveals a dominance of young, activation-primed PLTs with signs of impaired maturation. Comparison with post-chemotherapy thrombocytopenia suggests that this profile reflects a general regenerative RNA signature in response to increased PLT demand. In ITP, where this condition is chronically sustained, it may drive persistent PLT activation and functional dysregulation.