Platelet proteomic signatures reveal neurodegenerative and mitochondrial pathways associated with pain phenotypes in sickle cell disease

Abstract

Abstract Background: Sickle cell disease (SCD) is a complex hematologic disorder marked by chronic pain and neurocognitive impairments. While these complications are well-documented, the biological mechanisms driving them remain elusive. Platelets, known for their role in inflammation and vascular regulation, may serve as a window into systemic dysfunction in SCD. This study explored the platelet proteome in individuals with SCD to expose molecular correlates of pain and indicators of neurological risk. Methods: Peripheral blood samples were obtained from 16 adult volunteers living with sickle cell disease (SCD), all of whom reported experiencing moderate to severe pain (rated >3 on a 0–10 scale) related to SCD within the past 12 months. None of the participants had known degenerative conditions. Informed consent was obtained, and the study was approved by the institutional ethics board. To isolate ultra-pure platelets, a density gradient purification step was employed using OptiPrep™ (iodixanol-based medium). A discontinuous gradient was prepared by layering 6%, 10%, and 14% iodixanol solutions in sterile ultracentrifuge tubes. Platelet proteomes were analyzed using high-resolution mass spectrometry, identifying 4,197 proteins. Of these, 1,047 met statistical significance (FDR < 0.05) and were subjected to unsupervised clustering, pathway enrichment, and protein-protein interaction (PPI) network analyses. Pain phenotypes were assessed using SLANSS scores and pain word descriptors. Results: Unsupervised clustering stratified participants into high and low pain groups. Contrary to our hypothesis, pain-related pathways were not significantly enriched. Instead, 242 pathways showed significant alterations (FDR < 0.05), with robust enrichment in neurodegeneration, cognitive dysfunction, mitochondrial metabolism, ATP regulation, mitophagy, and tRNA aminoacylation. Volcano plot analysis prioritized proteins with FDR < 0.05 and log₂ fold change > 0.56 for downstream analysis. Distinct proteomic profiles emerged between pain groups. High-pain individuals exhibited elevated levels of UBQLN2, AP4B1, and WASHC5—proteins implicated in neurodegeneration and proteostasis. Low-pain individuals showed higher expression of GBA2, ATL1, and SOD1, associated with mitochondrial function and neuroprotection. PPI network analysis revealed tightly connected clusters within neurodegenerative and central nervous system (CNS)-related pathways. Particularly, tRNA aminoacylation proteins formed a distinct cluster, including ERLIN1, REEP2, SPG21, and AP4B1, while mitochondrial and proteostasis proteins such as UBQLN2, VCP, and AFG3L2 formed another. Disease association analysis ranked neurodegenerative and mitochondrial pathways as most strongly associated, surpassing traditional hematologic and pain-related mechanisms. Heatmap analyses confirmed consistent clustering of high-pain individuals with neurodegeneration-associated proteins (e.g., WARS, UBQLN2, COASY), while low-pain individuals aligned with neuroprotective markers (e.g., DNAJC5, SOD1, SCARB2). Conclusions: This study reveals unexpected platelet proteomic signatures in SCD, implicating neurodegenerative and mitochondrial pathways in pain phenotypes. These findings suggest that platelets may serve as accessible biomarkers for peripheral and/or CNS vulnerability and cognitive risk in SCD. The enrichment of tRNA aminoacylation and mitochondrial regulation pathways underscores the metabolic complexity of SCD and opens avenues for biomarker development and targeted intervention. Further longitudinal and functional studies are warranted to validate these findings and explore their clinical utility.