Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Química y Fisico-Química Biológicas (IQUIFIB) "Prof. Alejandro C. Paladini", Facultad de Farmacia y Bioquímica, Junín 956 Buenos Aires, Argentina.; Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Analítica, Cátedra de Química Química Analítica y Fisicoquímica, Junín 956 Buenos Aires, Argentina.
UMR-S1134, Integrated Biology of Red Blood Cells, INSERM, Université Paris Diderot, Sorbonne Paris Cité, Université de la Réunion, Université des Antilles, F-75015 Paris, France.; Institut National de la Transfusion Sanguine, Laboratoire d'Excellence GR-Ex, F-75015 Paris, France.
Biochim Biophys Acta Mol Cell Res. 2019 May;1866(5):896-915. doi: 10.1016/j.bbamcr.2019.01.018. Epub 2019 Feb 3.
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 to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry. 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 analog HlyA∆914-936 were unable to induce ATP release or hemolysis. For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release 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 ATPe activation of purinergic P2X receptors reduced swelling by 60-80%. HlyA-RBCs showed an acute 1.3-2.2-fold increase of Cai, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media. Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.
α-溶血素（HlyA）可不可逆地结合到人红细胞（RBC）上，触发 ATP 释放和代谢变化的激活，最终导致溶血。我们研究了暴露于 HlyA 的 RBC 细胞外 ATP（ATPe）的调节。用发光法评估 ATP 释放和 ATPe 水解，而通过电阻抗、变形测定和聚集测定来确定细胞体积和形态的变化。暴露于 HlyA 的 RBC 诱导 [ATPe]（3-36 倍）和溶血（1-44 倍）的强烈增加，部分由外切 ATP 酶和由死亡细胞释放的细胞内 ATP 酶水解来补偿。泛半通道蛋白 1 抑制剂 carbenoxolone 部分抑制 ATP 释放（43-67%）。非酰化毒素 ProHlyA 和缺失类似物 HlyA∆914-936 不能诱导 ATP 释放或溶血。对于 HlyA 处理的 RBC，数据驱动的数学模型表明，同时的裂解和非裂解释放主要控制 ATPe 动力学，而在延长毒素暴露后，ATP 水解变得重要。HlyA 诱导 1.5 倍的肿胀，而阻断这种肿胀可使 ATP 释放减少 77%。阻断 ATPe 激活嘌呤能 P2X 受体可使肿胀减少 60-80%。HlyA-RBC 显示 Cai 增加 1.3-2.2 倍，带负电荷的磷脂酰丝氨酸外翻和卷曲增加。将 HlyA-RBC 通过粘附平台灌注到 HMEC 细胞中，显示出对激活的 HMEC 细胞强烈的粘附，然后迅速脱落。在渗透应激下，HlyA 暴露的 RBC 表现出较高的球形度，在剪切应力下伸长率降低，在粘性介质中聚集性非常低。总体结果表明，HlyA-RBC 显示出激活的 ATP 释放、高但弱的粘附性、低变形性和聚集性以及高球形度。
