Abstract Body (Do not enter title and authors here): Objective Patients with congenital heart disease complicated by right ventricular outflow tract obstruction (RVOTO) often develop secondary pulmonary regurgitation (PR) after corrective surgery, leading to progressive right ventricular (RV) dysfunction that necessitates pulmonary valve replacement (PVR). This study employed proximity barcoding assay (PBA) to dynamically monitor changes in plasma exosome protein profiles and subpopulation characteristics during the perioperative period of transcatheter pulmonary valve replacement (TPVR).

Methods This study collected 120 human plasma samples from 10 patients undergoing TPVR at three time points, preoperative (T1 group), 1 day postoperative (T2 group), and 1 week postoperative (T3 group), as well as 10 healthy individuals (N group). Single-exosome analysis was performed using PBA. We performed combinatorial analysis and functional analysis of membrane proteins on single exosomes across different groups and evaluated their predictive performance as biomarkers using Receiver Operating Characteristic (ROC) curves. Finally, cluster analysis was performed on exosome subpopulations to identify subpopulation markers.

Results Compared to the control group, the disease group exhibited significantly reduced expression levels of the protein combination DSCAML1 + ALCAM + ITGA1 + CR1 (P<0.05; AUC=1), while showing significantly elevated expression levels of BCAM + CD36 + DSCAML1 + ITGB1 (P<0.05; AUC>0.82). Gene Ontology (GO) analysis indicated that the proteins are associated with pathways related to response to stress, membrane side, and protein binding, while Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed enrichment in the Hematopoietic cell lineage pathway. Subpopulation analysis revealed increased Cluster 1 and decreased Cluster 2 proportions in T1-T3 groups compared to the N group. Subpopulation markers showed CD8A dominance in Cluster 1, while DSCAML1 predominated in Cluster 2.

Conclusion This study was the first to implement PBA for the dynamic assessment of plasma exosome alterations in patients with TPVR. We identified downregulated protein combinations such as DSCAML1 + ALCAM + ITGA1 + CR1 and upregulated combinations such as BCAM + CD36 + DSCAML1 + ITGB1. Analysis of exosome subpopulations further demonstrated a predominance of CD8A protein expression in the disease group, whereas, in the control group, DSCAML1 expression was more prevalent.