Effects of α-hemolysin on human erythrocytes. Part 2. Morphology, rheology, adhesion and signaling.

LAURI, N.; LEAL DENIS, M.F.; LEFEVRE, S.D.; ALVAREZ, C.L.; ENRIQUE, N.; RINALDI, D.E; VECCHIO, L.E.; STRINGA, P.; MUÑOZ-GARAY, C.; MILESI, V.; OSTUNI, M.A.; HERLAX, V.; SCHWARZBAUM, P.J.

La PLata

Congreso; XLVII Reunion Anual de la Sociedad Argentina de Biofisica; 2018

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes(RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis.We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used toassess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined byelectrical impedance, ektacytometry and aggregometry.Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3?36-fold) and hemolysis (1?44-fold), partiallycompensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells.Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43?67%).The un-acylated toxin ProHlyA and the deletion analog HlyAΔ914-936 were unable to induce ATP release orhemolysis.For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lyticrelease mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure.HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPeactivation of purinergic P2X receptors reduced swelling by 60?80%. HlyA-RBCs showed an acute 1.3?2.2-foldincrease of Ca2+i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCsthrough adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment.