AbstractBackgroundAtrial fibrillation (AF) is associated with an increased risk of thrombosis of the left atrial appendage (LAA). However, the molecular mechanisms underlying this site‐specificity remain poorly understood. Here, we present a comparative single‐cell transcriptional profile of paired atrial appendages from patients with AF and illustrate the chamber‐specific properties of the main cell types.MethodsSingle‐cell RNA sequencing analysis of matched atrial appendage samples from three patients with persistent AF was evaluated by 10× genomics. The AF mice model was created usingTbx5knockout mice. Validation experiments were performed by glutathione S‐transferase pull‐down assays, coimmunoprecipitation (Co‐IP), cleavage assays and shear stress experiments in vitro.ResultsIn LAA, phenotype switching from endothelial cells to fibroblasts and inflammation associated with proinflammatory macrophage infiltration were observed. Importantly, the coagulation cascade is highly enriched in LAA endocardial endothelial cells (EECs), accompanying the up‐regulation of a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) and the down‐regulation of the tissue factor pathway inhibitor (TFPI) and TFPI2. Similar alterations were verified in an AF mouse model (Tbx5+/−) and EECs treated with simulated AF shear stress in vitro. Furthermore, we revealed that the cleavage of both TFPI and TFPI2 based on their interaction with ADAMTS1 would lead to loss of anticoagulant activities of EECs.ConclusionsThis study highlights the decrease in the anticoagulant status of EECs in LAA as a potential mechanism underlying the propensity for thrombosis, which may aid the development of anticoagulation therapeutic approaches targeting functionally distinct cell subsets or molecules during AF.