Introduction: Atrial fibrillation (AF) is the most common arrythmia in humans. The most important sequel of AF is intracardiac thrombus formation and peripheral embolization leading to serious ischemic organ damage. Thrombus formation typically takes place in the left atrial appendage and occurs at different rates in different types of atrial fibrillation (paroxysmal, persistent, permanent). Recently, it has been shown that different types of AF involve structural remodeling of the atrium to a varying extent. However, it remains unclear how this translates into different rates of thrombus formation. Over the past decade, circulating extracellular vesicles (EVs) have been shown to be potent regulators of platelet activation and coagulation. Large EVs (Microvesicles) share surface receptors with their mother cells and can therefore be characterized via flowcytometry. In this study, we aimed to investigate the impact of different types of atrial fibrillation on the abundance of platelet- and endothelial cell-derived EV subspecies as potential mediators of thrombus formation.

Methods and Results: In a cohort of the 59 patients undergoing cardiac intervention by catheter, we collected blood samples from the right atrium, the left atrium and the left atrial appendage. Of the 59 patients 10 had no history of AF, 20 had a history of non-permanent AF and 29 had a permanent AF. The diagnosis was confirmed via an electrocardiogram upon inclusion in the study. Large EV were isolated from 150 µL citrate plasma by a four-stage differential centrifugation protocol (1500g x 15min, 13000rpm x 2min, 20000g x 40min, 20000g x 40min). The EV were characterized by immunoblotting and nanoparticle tracking analysis. The size of the EV ranged between 50 and 600 nm and the EV were shown to carry typical markers such as Annexin A1 and Flotilin-1, while Albumin was only present in traces. For flow cytometric analysis Calcein AM, CD31-PE, CD41-APC, CD235a-PE-Cy7 were used for staining. Specificity of the staining was confirmed in a degradation assay (Triton X-100) and through fluorescence minus one controls. EV numbers were assessed in all cardiac localizations and relative proportions of different EV subtypes were calculated. Regarding the type of atrial fibrillation, patients with permanent AF showed significantly higher numbers of platelet-derived EVs in the RA and LAA compared to patients with non-permanent AF (RA, non-permanent AF: 25.00 ± 8.11 % vs. permanent AF 29.99 ± 5.03 %, p = 0.049; LAA non-permanent 25.87 ± 6.09 % vs. permanent AF 31.21 ± 5.04 %, p = 0.028, 2-way ANOVA + Tukey’s multiple comparison test) No differences between the AF subtypes and localizations were detected for total EV numbers and red blood cell and endothelial cell-derived EVs.

Conclusion: In the present study, we found that the type of atrial fibrillation (permanent vs non-permanent) influences the presence of different EV subsets in the left atrial appendage. As EVs are known to influence coagulation and thrombus formation locally, these results contribute to our knowledge how different types of atrial fibrillation influence thrombus formation in the left atrial appendage.