Liberation of ATP secondary to hemolysis is not mutually exclusive of regulated export.

Liberation of ATP secondary to hemolysis is not mutually exclusive of regulated export In their recent report published in Blood, 1 Sikora and colleagues correlate cell-free hemoglobin (Hb) and extracellular adenosine tri-phosphate (ATP ec), and conclude that cell rupture from hypotonic shock accounts for ATP ec increases in isolated red blood cell (RBC) suspensions. Although this conclusion may apply to the authors' experimental conditions (.25% reduction in tonicity with RBCs from hemochromatosis patients, stored 0-14 days), it cannot be generalized to studies in which (patho)physiologically relevant conditions (eg, hypoxia, acidosis, adrenergic stimulation, mechanical stress) were used to promote ATP release from RBCs freshly isolated from normal humans independently of hemolysis. 1-11 We raise the following concerns. (1) The selected stimuli directly and independently evoke hemolysis Cell lysis can result directly from stimuli such as hypotonic shock (a nonphysiological stimulus), toxins (eg, dimethyl sulfox-ide [DMSO]), and mechanical or hypoxic stress. RBCs, particularly , are readily ruptured, with expulsion of intracellular contents, including the classical hemolysis indicators, cell-free Hb, potassium , and lactate dehydrogenase. But liberation of ATP secondary to hemolysis is not mutually exclusive of regulated export. In some cases, the authors used banked RBCs, which are particularly vulnerable to osmotic lysis, in contrast to fresh human RBCs, which do not lyse in response to a 25% reduction in tonicity. 12 Moreover , Sikora and colleagues did not perform osmotic fragility curves for the hemochromatosis RBCs studied, or compare their susceptibility to lysis to that of freshly isolated normal human RBCs. 1 (2) Prior reports of extracellular ATP Contrary to the authors' statements, reports (some uncited) investigating RBC ATP release do address and preempt concerns over increased [ATP] ec via hemolysis, with rigorous intrasample comparison of cell-free [Hb] and [ATP] used to distinguish genuine ATP export from breakage of RBCs. Even the earliest investigations, from Bergfeld and Forrester, 9 parsed authentic ATP release from that due to cell damage, and numerous publications document regulated ATP export from RBCs while controlling for hemolysis. If increases in [ATP] ec are solely from hemolysis, then [ATP] ec should be unaffected by antagonizing ATP-export signal-ing. In fact, ATP export from mouse RBCs lacking pannexin 1 (Px1; an ATP conduit) or pretreated with Px1 inhibitors is low, or even absent, without altered hemolysis 3-5 ; Sikora and colleagues did not address this important point. (3) Experiment-specific results may preclude definitive conclusions The authors intended to test …