Multiparametric Extracellular Vesicle Profiling Identifies a Distinct Circulating Signature of Bicuspid Aortic Valve Disease

Background: Calcific aortic valve disease (CAVD) resulting in life-threatening aortic stenosis, heart failure and death, develops earlier and progresses more rapidly in patients with bicuspid aortic valves (BAV) than tricuspid aortic valves (TAV), yet the circulating molecular drivers remain poorly understood. Extracellular vesicles (EVs) capture disease-relevant cellular activation and offer a powerful window into a BAV-specific pathophysiology. Methods: Circulating large extracellular vesicle (LEV) abundance and morphology were characterized by complementary single-vesicle nano-flow cytometry and super-resolution microscopy. Proteomic profiling further delineated LEV molecular cargoes and associated biological pathways between patients with BAV and TAV. Results: We demonstrate that patients with BAVs exhibited fundamentally altered circulating LEV profiles compared to TAVs. Super-resolution imaging revealed BAV-specific organization of platelet- and leukocyte-associated adhesion molecules into discrete EV membrane microdomains. Proteomic profiling identified distinct LEV cargoes between BAV and TAV, including a BAV-specific, previously unrecognized neutrophil-associated signature enriched for proteins involved in neutrophil degranulation and innate immunity. Consistent with this phenotype, BAV displayed alterations in platelet- and neutrophil-associated LEV populations, suggesting enhanced (thrombo-)inflammatory activity. Lastly, distinct associations between LEV populations and clinical parameters in BAV and TAV suggest that LEV profiles reflect morphology-specific aspects of disease severity. Conclusions: Comprehensive single-vesicle analysis and bulk proteomic profiling identified a distinct circulating LEV signature in BAV-associated CAVD, characterized by endothelial activation and reciprocal platelet-neutrophil interactions. These findings reveal previously unrecognized differences in circulating LEVs between BAV and TAV and highlight their potential as biomarkers of valve morphology-specific pathophysiology.