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

LEAL DENIS MARÍA FLORENCIA; ENRIQUE NICOLÁS; STRINGA PABLO; OSTUNI MARIANO ANIBAL; LEFEVRE SOPHIE DENISE; RINALDI DÉBORA EUGENIA; MUÑOZ-GARAY CARLOS; HERLAX, VANESA; LAURI NATALIA; ALVAREZ CORA LILIA; LEANDRO VECCHIO; MILESI, VERÓNICA; SCHWARZBAUM PABLO JULIO

La Plata

Congreso; Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofísica

α-hemolysin (HlyA) of E. coli irreversibly binds to human erythrocytes (RBCs) andtrigger changes in extracellular ATP and cell volume, ultimately leading tohemolysis.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 withthe 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 crenationfollowed by swelling, and externalization of phosphatidylserine. Perfusion of HlyARBCsthrough adhesion platforms showed high adhesion to activated HMEC cells atlow flow (0.2 dyn/cm2), although higher flows induced rapid detachment. Exceptfor crenation, none of these changes occur in Pro-RBCs.Deformability changes were analyzed by ektacytometry as a function of osmolarityor shear stress. In osmoscan curves, c- and Pro-RBCs deformed optimally inisosmolar conditions, while with HlyA-RBCs the toxin time-dependently shifted thecurve to the right, suggesting increased sphericity. HlyA-RBCs displayed highlyreduced elongation under shear stress, and very low aggregation in syllectograms.RBCs were then perfused through a rat mesentery, where intravascular levels ofATP and free hemoglobin (to estimate lytic ATP release) were measured inperfusate samples. Results showed that HlyA-RBCs, unlike c- and Pro-RBCs,increased intravascular ATP 83-fold, and hemolysis -56-fold, with parallelincrements in arterial bed pressure. Non-lytic ATP release of HlyA-RBCs was themajor component explaining intravascular ATP.Overall results showed that HlyA-RBCs displayed activated ATP release, high butweak adhesivity, low deformability and aggregability and high sphericity.