Platelets From Sickle Cell Disease Individuals Induce Endothelial Activation, Demonstrating ICAM-1 and E-Selectin Adhesion Molecule Expression, Inflammatory Cytokine Production and Activation of NFκB Transcription Factor Gene Expression.
Authors: Proença-Ferreira Renata , Brugnerotto Ana Flavia , Garrido Vanessa Tonin , de Fatima Reis Ribeiro Marilene , Traina Fabiola , Saad Sara T Olalla , Costa Fernando Ferreira , Conran Nicola 
Published: October 2019

Abstract
Abstract 2114

Introduction:
Sickle cell disease (SCD) pathophysiology is associated with a hypercoagulable state that may contribute to the initiation and propagation of vaso-occlusion. Increased platelet activation has been described in SCD and SCD platelets may present augmented adhesion to the vascular endothelium, potentially contributing to vaso-occlusion.

Aim:
This study investigated whether platelets (PLTs) from SCD individuals are able to activate endothelial cells per se.

Methods:
Human umbilical vein endothelial cells (HUVEC) were cultured (1×106cells/well) on 6-well plates (37°C, 5% CO2). Subsequently, HUVEC were co-cultured in direct contact, or not, with washed PLTs (1×108PLTs/well) from healthy control individuals (CON, n=23) or steady-state SCD patients (SCD, n=47; 26 of which were on hydroxyurea therapy; 20mg/Kg/day) for 4h, 37°C, 5%CO2. After incubation, PLTs were removed; supernatants were reserved for cytokine quantification by ELISA, and HUVEC were analyzed by flow cytometry for CD62E (E-Selectin) and CD54 (ICAM-1) surface expression; gene expressions of ICAM1 and NFKBIA were analyzed by qPCR.

Results:
Basal ICAM-1 expression on the surface of HUVEC (39.6±3.2%, n=15) was significantly increased following their incubation in direct contact with SCD PLTs (46.1±3.1%, n=26, p<0.05, Wilcoxon test), but not CON PLTs (41.3±4.7%, n=12). E-selectin expression was also low level on the surface of HUVEC (0.9±0.2%, n=17), and was slightly but significantly increased following incubation of cells with SCD PLTs (6.1±2.2%, n=26, p<0.001), but not CON PLTs (3.6±1.5%, n=12, p>0.05). Repetition of these assays, but with the placement of transwell inserts in culture plates to separate the PLTs from HUVEC resulted in a 45% decrease in ICAM-1 expression (p<0.05), and 85% decrease in E-selectin (p<0.05) expression on the surface of HUVEC, following their incubation with SCD PLTs. HUVEC produce and release interleukin-8 (IL-8); basal IL-8 production by HUVEC (1×106cells/well) was 1.160±0.187ng/mL (n=21); this production was augmented in the presence of SCD PLTs (1.280±0.149ng/mL, n=42, p<0.01), but not by CON PLTs (1.127±0.157ng/mL, n=23). The influence of PLT IL-8 production on these values was negligible, as shown by data (not shown) demonstrating that PLT IL-8 production is low level and does not differ between CON and SCD PLTs. IL-1β is produced and released by PLTs (CON, 3.4±1.4ρg/mL, n=12; SCD, 6.5±1.5 ρg/mL, n=25, p>0.05), but this production was further increased when PLTs were co-cultured with HUVEC: SCD PLTs (10.3±4.2ρg/mL, n=47; p<0.01) and CON PLTs (5.8±2.4ρg/mL, n=25; p>0.05), compared to HUVEC alone (1.27±0.4ρg/mL, n=24). Gene expression of ICAM1 by HUVEC increased 6.3-fold in the presence of SCD PLTs (n=25, p<0.01), compared to basal expression (n=11), but was not altered in the presence of CON PLTs (n=11, p>0.05). The expression of the gene encoding the NFkB transcription factor, NKBIA, increased 3.4-fold in HUVEC following incubation with SCD PLTs (n=25, p<0.05), compared to basal NFKBIA expression (n=12); however NFKBIA expression in HUVEC was not significantly altered by CON PLTs (n=10, p>0.05).

Conclusions:
Results indicate that the contact of platelets, or products released from platelets, from patients with SCD may activate endothelial cells, in vitro, increasing adhesion molecule and IL-8 production, associated with an augmented expression of the gene encoding NFkB. Platelets produce IL-1β in greater quantities in the presence of endothelial cells, possibly contributing to endothelial cell activation; however the fact that transwell inserts significantly reduced SCD PLT-mediated endothelial activation indicates that the direct contact of PLTs (possibly via adhesion) is required for this activation. Data indicate that platelets adhered to vessel walls may play an important role in endothelial activation and, therefore, vaso-occlusive mechanisms in SCD.

Disclosures:
No relevant conflicts of interest to declare.