S299: ROLE OF RED BLOOD CELL MEMBRANE-DERIVED PARTICLES ON ENDOTHELIAL DAMAGES DURING ONSET STAGE OF DELAYED HEMOLYTIC TRANSFUSION REACTION

Abstract

Background: Delayed hemolytic transfusion reaction (DHTR) is a life-threatening complication of red blood cell (RBC) transfusion in sickle cell disease (SCD) patients, leading to the increase of plasma hemoglobin (Hb) and heme which are responsible for endothelial damage and organs failure. By the development of an in vitro model reproducing endothelial damages at the early phase of DHTR (Nguyen et al, ASH 2018), our previous study suggested that RBC membrane-derived particles released during the onset stage of DHTR could be also involved in DHTR physiopathology. Aims: In this study, we aim to determine the mechanism of endothelial damages induced by these RBC particles. Methods: Human Umbilical Vein Endothelial Cells (HUVECs) cultured in flow condition for 24 hours were perfused by either serum only (decomplemented or not) or serum containing either whole hemolysate (sonicated RBCs) or different hemolysate components or RBCs membranes (ghosts) under shear stress 1 dyne/cm2. Annexin V and Eculizumab were used for complement studies. Endothelial activation and damages were assessed by membrane CD54 (PECAM-1, Platelet endothelial cell adhesion molecule 1), CD106 (VCAM-1, Vascular Cell Adhesion Molecule-1) and actin network staining. The whole blood adhesion assays and platelets aggregation study on hemolysate-preconditioned HUVECs were performed in continuous flow. Confocal microscopy was used to study interaction between HUVECs and hemoglobin (Hb) and RBC membrane-derived particles. Results: Our fluidic model reproduced a hemolysate-induced pro-inflammatory phenotype and damages of HUVECs via NFkB signaling pathway in a TLR-4 independent manner. The most deleterious effects were observed in whole hemolysate conditions in which, an important quantity of RBC large-size particles (> 2 μm) mostly positive for C3 was detected. These effects on HUVECs were significantly reduced after their elimination by 14000 g centrifugation (Figure 1). Noteworthy, the suspension of ghosts induced a similar activation on HUVECs compared to whole hemolysate suggesting the major role of RBC membrane-derived particles in early hemolysis-induced endothelial damages. Confocal microscopy for intracellular compartment of hemolysate-treated HUVECs demonstrated that while the CD235a+ Hb+ particles were only found in whole hemolysate conditions, the Hb was detected similarly in all conditions tested. Selective blocking of RBCs particles and Hb endocytosis did not affect hemolysate-induced HUVECs activation. These observations demonstrate that in the very early stage (≤4H) of intravascular hemolysis, erythrocytes large-size particles can be internalized in HUVECs, but their internalization is not necessary for endothelial activation effect. Complement inactivation of serum or treatment of RBC particles by either Annexin V or Eculizumab could reduced their effects on endothelial activation suggesting a complement-dependent mechanism Image:Summary/Conclusion: This study suggested for the first time the major role of RBCs large-size particles on endothelial damages at the onset of DHTR in a complement-dependent manner justifying the complement blocking treatment in certain DHTR patients. Furthermore, we have also demonstrated the endocytosis of free Hb by HUVECs, but its pathological impact is still unclear. This endocytosis would facilitate the Hb clearance during a massive hemolysis? These new understandings will allow a better and earlier management to preserve endothelial functions.