AB0080\u2005ALTERED CONCENTRATIONS OF DIFFERENT SMALL EXTRACELLULAR VESICLE POPULATIONS IN PLASMA OF PATIENTS WITH ANTIPHOSPHOLIPID SYNDROME

Abstract

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by thrombosis, obstetric complications, and the presence of antiphospholipid antibodies (aPL) that cause endothelial injury and thrombophilia [1]. Extracellular vesicles (EVs) are involved in various thrombotic disorders [2], including APS [3, 4], and therefore may influence the prothrombotic status of APS patients. One of the hallmarks of activated endothelium is the expression of adhesion molecules, such as ICAM-1 (CD54) and E-selectin (CD62E), that play a key function in the interactions with leukocytes and platelets.To determine the level of total tetraspanin (CD81/CD63/CD9)-positive vesicles and specific EV populations (CD54- and CD62E-positive EVs) in plasma from APS patients.Whole blood was collected from 4 APS patients and 3 healthy blood donors (HBDs) and processed to obtain platelet-depleted plasma. The size and concentration of EVs were determined using ExoView platform (NanoView Biosciences, MA, USA). In brief, EVs were captured and immobilized on ExoView Tetraspanin chips using the capture antibodies CD81, CD63, and CD9. Size was determined using SP-IRIS technology. Concentration measurements were performed with fluorescently labeled detection antibodies, specifically tetraspanins (CD81/CD63/CD9) for total EVs and CD54 and CD62E for specific EV populations.Analysis of EVs size confirmed the presence of small EVs (sEVs, < 70\u2009nm) in both study groups. The size of EVs did not vary significantly between study groups (mean size ± SD in APS vs. HBD; CD81 (63.1 ± 1.5\u2009nm vs. 66.6 ± 10.6\u2009nm), CD63 (63.3 ± 1.64\u2009nm vs. 69.2 ± 13.2\u2009nm), and CD9 capture spot (61.8 ± 1.18\u2009nm vs. 64.1 ± 7.64\u2009nm)). The levels of total EVs (tetraspanin-positive) were increased 1.7-fold, 1.4-fold, and 2.2-fold for CD81, CD63, and CD9 capture spots, respectively, in APS patients compared to HBDs (Figure 1A). In addition, CD54- and CD62E-positive EVs represented a small population (< 2 %) of the total EVs (Figure 1B). The levels of CD54 were increased 2.9-fold, 3.0-fold, and 2.5-fold on CD81, CD63, and CD9 capture spots, respectively, and similarly, the levels of CD62E were increased 2.2-fold, 2.7-fold, and 2.0-fold on CD81, CD63, and CD9 capture spots, respectively, in APS patients compared to HBDs (Figure 1A).Figure 1.Concentration of tetraspanin- (CD81/CD63/CD9), CD54- and CD62- positive sEVs in APS patients and HBDs.Higher levels of sEVs and increased percentage of CD54- and CD62E-positive sEVs in plasma of APS patients could indicate an altered and activated endothelium in those patients.[1]Miyakis S. Journal of thrombosis and haemostasis. 2006.[2]Zara M. Int J Mol Sci. 2019.[3]Chaturvedi S. Semin Thromb Hemost. 2018.[4]Stok U. Cells. 2020.[5]Oggero S. Front Pharmacol. 2019.Ula Stok: None declared, Alex Shephard Employee of: NanoView Biosciences., Sasa Cucnik: None declared, Snežna Sodin-Šemrl: None declared, Polona Zigon: None declared