Abstract WP375: Age-related red blood cell alterations in stroke patients

Background: During human aging, red blood cells (RBCs) undergo a series of changes, including reduced deformability, increased density, and elevated distribution width. These changes not only compromise their function, but also contribute to various age-related diseases including stroke. However, the underlying molecular mechanism of how aging RBCs contribute to ischemic brain injury remains largely unknown. Growing evidence has revealed that abnormal RBCs are an independent predictor of stroke risk and poor stroke outcome. Here, we systematically profile age-related RBC changes in ischemic stroke patients. Method: We prospectively recruited 218 consecutive ischemic stroke patients under IRB-approved protocol. RBCs were collected from study participants. RBC proteome and phosphoproteome were profiled by mass spectrometry. To study the impact of RBC protein phosphorylation, isolated RBCs were cultured in RPMI 1640 media supplemented with 10% FBS, and were treated with broad-spectrum protein kinase inhibitor staurosporin (0, 2, 5 µM) for 24 hr. Result: Aging was significantly correlated with increased RBC distribution width (RDW) (r = 0.33, p < 0.001) ( Figure 1A ). Proteomics analysis revealed age-related changes in RBC kinases ( Figure 1B ), with oxidative stress-responsive kinase 1 (OXSR1) showing the most significant decline in older patients ( Figure 1C ). Reduced OXSR1 levels were associated with greater stroke severity. ( Figure 1D ). Phosphoproteomic profiling further showed that age-dependent phosphorylation changes were mainly enriched in RBC membrane and cytoskeletal proteins ( Figure 1E ). Inhibiting RBC phosphorylation with staurosporin resulted in marked disruption of RBC structure ( Figure 1F ), underscoring the importance of kinase signaling in maintaining RBC integrity. Conclusion: RBC structural changes are frequently observed during aging. Our study showed that this process may be linked to alterations in RBC kinases and associated protein phosphorylation, particularly in membrane and cytoskeletal components. And these changes in RBC kinases are associated with stroke severity, suggesting a mechanistic link between RBC aging and cerebrovascular disease. Further investigations are ongoing to delineate specific RBC kinase signaling pathways that drive age-related changes and their connections with stroke and other age-related neurodegenerative disease.