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

LAURI, NATALIA; LEAL DENIS, MARÍA F.; LEFEVRE, S.D.; ALVAREZ, CORA L.; ENRIQUE, NICOLÁS; RINALDI, D.E.; VECCHIO, L.E.; STRINGA, P.; MUÑOZ-GARAY, C.; MILESI, VERÓNICA; OSTUNI, MARIANO A.; HERLAX, VANESA; SCHWARZBAUM PABLO JUIO

La Plata

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

α-hemolysin (HlyA) of E. coli irreversibly binds to human erythrocytes (RBCs) and trigger changes in extracellular ATP and cell volume, ultimately leading to hemolysis.Here we analyzed the capacity of HlyA treated RBCs to release ATP, change shape, adhere, deform and/or aggregate. Cells were treated with HlyA (HlyA-RBCs) or with the un-acylated protoxin ProHlyA (Pro-RBCs), and compared to control conditions (c-RBCs).HlyA-RBCs showed an acute 1.3-2.2 fold increase of Ca2+i, increased crenation followed by swelling, and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed high adhesion to activated HMEC cells at low flow (0.2 dyn/cm2), although higher flows induced rapid detachment. Except for crenation, none of these changes occur in Pro-RBCs.Deformability changes were analyzed by ektacytometry as a function of osmolarity or shear stress. In osmoscan curves, c- and Pro-RBCs deformed optimally in isosmolar conditions, while with HlyA-RBCs the toxin time-dependently shifted the curve to the right, suggesting increased sphericity. HlyA-RBCs displayed highly reduced elongation under shear stress, and very low aggregation in syllectograms.RBCs were then perfused through a rat mesentery, where intravascular levels of ATP and free hemoglobin (to estimate lytic ATP release) were measured in perfusate samples. Results showed that HlyA-RBCs, unlike c- and Pro-RBCs, increased intravascular ATP 83-fold, and hemolysis -56-fold, with parallel increments in arterial bed pressure. Non-lytic ATP release of HlyA-RBCs was the major component explaining intravascular ATP.Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.