Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis

LEAL DENIS FLORENCIA; LEFEVRE, SOPHIE; ALVAREZ CORA; LAURI NATALIA; NICOLAS ENRIQUE; RINALDI DEBORA; GONZALEZ LEBRERO RODOLFO ; VECCHIO LEANDRO; ESPELT VICTORIA ; STRINGA PABLO ; MUÑOZ GARAY CARLOS; MILESI VERONICA; OSTUNI MARIANO; HERLAX VANESA; SCHWARZBAUM PABLO JULIO

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2019

0167-4889

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds tohuman erythrocytes (RBCs) and triggers activation of ATP release and metabolicchanges ultimately leading to hemolysis.We studied the regulation ofextracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used toassess ATP release and ATPe hydrolysis, whereas changes in cell volume andmorphology were determined by electrical impedance, ektacytometry andaggregometry.Exposure of RBCs to HlyA induced a strong increase of[ATPe] (3-36-fold) and hemolysis (1-44-fold), partially compensated 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 analogHlyA∆914-936 were unable to induce ATP release or hemolysis.For HlyA treated RBCs, a data driven mathematicalmodel showed that simultaneous lytic and non-lytic release mainly governed ATPekinetics, while ATPe hydrolysis became important after prolonged toxinexposure.HlyA induced a 1.5-fold swelling, while blocking thisswelling reduced ATP release by 77 %. Blocking ATPe activation of purinergicP2X receptors reduced swelling by 60-80 %. HlyA-RBCs showed an acute 1.3-2.2 foldincrease of Ca2+i, increased crenation and externalization ofphosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strongadhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCsexhibited increased sphericity under osmotic stress, reduced elongation undershear stress, and very low aggregation in viscous media.Overall results showed that HlyA-RBCs displayedactivated ATP release, high but weak adhesivity, low deformability andaggregability and high sphericity.