Extracellular Vesicles from Sickle Cell Disease Patients with Acute Chest Syndrome Suggest Activation of Endothelial Cells and Coagulation
Authors: Souza Cristiane Maria , Pereira dos Santos Irene , Lanaro Carolina , Sesti-Costa Renata , Saad Sara Teresinha Olalla , Benites Bruno D , Costa Fernando Ferreira 
Published: December 2024

Introduction: Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by a complex physiopathology. This pathophysiology, which includes hemolysis, inflammation and vaso-occlusion, contributes to a number of different clinical abnormalities. The endothelial damage and activation that is promoted by a cascade of events following red cell sickling is probably directly involved in many of the complications observed in SCD patients, such as acute chest syndrome (ACS), a complication that appears also to be influenced by the hypercoagulability state. Extracellular vesicles (EVs) are small structures released by cells that express and carry several molecules and proteins that are actively involved in cellular communication and have an important role in various diseases. Although several studies suggest the participation of EVs in SCD events, their role in SCD is largely unclear. The possible role of EVs in the pathogenesis of ACS is unknown and we aim, here, to further understand whether EVs could reflect the endothelial damage and coagulation activation in patients who have developed ACS.

Aim: Evaluate the profile of endothelial derived EVs and EVs expressing tissue factor (TF) in patients with SCD who have not experienced episodes of ACS during the previous 12 months and those who presented at least one episode of ACS in the 12 months prior to participating in the study.

Methods: Patients with at least one episode of ACS in the 12 months before sample collection and patients without ACS were included (n=08 and 70, respectively).Samples were collected and processed as previously described (Olatunya et al., 2019). To quantify and characterize endothelial EVs, we used CD146pos and CD45neg identification. To identify the expression of TF we used the CD142 positivity. The Cytoflex (Beckman Coulter) was used to quantify EVs by flow cytometry and analysis was performed using CytoExpert software. Statistical tests were performed according to the non-parametric distribution for comparative analyzes (Kruskal wallis, Mann Whitney). Values of p&lt;0.05 were considered statistically significant.

Results and Discussion: We found higher numbers of EVs expressing the CD142 marker (p=0.019) and of endothelial cell-derived EVs expressing the CD142 marker (p=0.020) in patients with previous ACS, compared to patients that had not experienced ACS during the last 12 months before sample collection. Some functional and observational studies have shown the possible participation of EVs in ACS. A previous study (Jutant et al., 2021) showed that patients present higher plasma levels of EVs from red blood cells, platelets, and pro-coagulant EVs during an ACS episode. In this study, although the sample collection was not carried out at the moment of the event, the EV profile may reflect endothelial activation and the hypercoagulability state in these patients, as augmented levels of endothelial derived-EVs expressing TF were observed in these patients. No difference was observed for endothelial cell-derived EVs with no CD142 marker (p&gt;0.05). As TF is an activator of the coagulation process, we speculate that this could reflect a hypercoagulability state in patients who have had ACS. Moreover, we previously demonstrated (Souza CM, ASH 2023) that EVs from patients with sickle cell anemia in steady state carried proteins that stimulate coagulation. Further analysis should be conducted with samples collected at the time of the complication to better understand the participation of EVs in ACS and how they can be used as a biomarker for ACS.

In summary, our data suggest that EVs may provide important insights into the clinical condition of patients with SCD, as they may reflect patterns related to the key events involved in ACS.