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

To the editor:

In their recent report published in Blood,¹  Sikora and colleagues correlate cell-free hemoglobin (Hb) and extracellular adenosine triphosphate (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 sulfoxide [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.^{2-4,6-8,10,11,13}  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 signaling. 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 the hypothesis that hemolysis is the primary cause for [ATP]_ec elevations in response to selected stimuli. However, multiple experimental design flaws exist, limiting the conclusions.

First, the lysis-induced ATP liberation was not discriminated from genuine ATP release. Lysis can mask regulated ATP release because the expulsion of intracellular nucleotidases leads to rapid ATP hydrolysis (as in Figure 2 of their article), so that the measure of [ATP]_ec would underestimate actual ATP release, and because intracellular [ATP] far exceeds exported [ATP].^2,3,11  Masking may also arise from: centrifugation (mechanical stress) used to separate the ATP_ec and free Hb from RBCs, the vulnerability of the banked RBCs studied (especially given their deficient ATP-release capacity^8,10 ), DMSO solvent (other vehicles can dissolve cyclic AMP [cAMP] analogs^3,11 ), RBC temperature shift (promotes vulnerability), and poly-l-lysine (higher concentrations perturb distribution of band 3,³  a determinant of controlled ATP export^7,9 ). In the case of the ATP-release stimulus hypoxia, special caution and controls using normoxic gas flow are lacking in Sikora’s study but necessary because the force of gas flow itself can lyse cells. Therefore, the signal from genuine ATP release was likely overwhelmed here by the noise of hemolysis.

Second, Sikora and colleagues observed no ATP release in response to cAMP-stimulating reagents (forskolin + papaverine + isoproterenol, hereafter referred to as “3V”). In contrast, adding 100% DMSO lysed RBCs, but 10% DMSO was innocuous. Consequently, they propose that hemolysis rather than cAMP could have caused ATP release in Montalbetti et al.¹¹  This argument is unsustainable because in Montalbetti¹¹ : (1) identical volumes of 3V (as 10 times stock) and vehicle (phosphate-buffered saline [PBS]) were added to cells in 1% DMSO; (2) cAMP-elevating isoproterenol (in PBS) markedly potentiated forskolin-evoked ATP release; (3) dibutyryl-cAMP (aqueous solution) promoted ATP release; and (4) inhibitors of Px1 abolished ATP release in the absence of measurable hemolysis. Lastly, Sikora and colleagues ignored the observation that 3V promoted ATP release in RBCs from wild-type but not Px1^−/− mice.³  The fact that the authors detected no cAMP-evoked ATP release in 0- to 14-day-old RBCs from hemochromatosis patients cannot invalidate studies using fresh, normal human and mouse RBCs.^3,11  Notably, ATP export has been shown to decline with RBC storage time,¹⁰  and the report by Sikora and colleagues might have been strengthened by showing the data in support of their assertion that hemolysis and ATP_ec did not differ as a function of RBC storage for 0 to 2 days vs up to 14 days.

Finally, the authors leave unresolved the intriguing but contradictory observation that in their experiments, the increases in ATP_ec precede the hemolysis.

(4) In vivo evidence on ATP and hemolysis

One can definitively determine RBC hemolysis in vitro, but direct study of intravascular hemolysis in vivo is more challenging. Nevertheless, plasma ATP rises during physiological stress in vivo and cannot be attributed to hemolysis. These data are consistent with regulated ATP export.² 

We agree that RBC hemolysis elevates [ATP]_ec as demonstrated by Sikora and colleagues. However, in our opinion, their findings are correlational and not mechanism-probing, and contrast with well-controlled experiments showing RBC ATP export independent of hemolysis and inhibited by agents that antagonize authentic ATP release. The evidence does not support hemolysis as a primary ATP-release mechanism in human RBCs.