Response: Hemolysis is a primary and physiologically relevant ATP release mechanism in human erythrocytes

We appreciate the willingness of Kirby and colleagues to discuss methodological concerns in red blood cell (RBC) adenosine triphosphate (ATP) release studies. In our investigations, before moving to mechanism-probing experiments, we spent most of the time evaluating the different methods that have been used in the field to assess RBC ATP release and the contribution of hemolysis to that release. Although we began with the hypothesis that regulated ATP release mechanisms do operate in RBCs, after extensively verifying each experimental approach, we came to the conclusion expressed in the title: ie, that “Hemolysis is a primary ATP release mechanism in human erythrocytes.”¹ In the course of the study, while attempting to reproduce previously published findings, we identified a number of methodological pitfalls (the major ones are discussed in the supplemental Methods available on Blood Web site), which likely precluded a proper evaluation of ATP release and also the contribution of hemolysis in the earlier studies^2-10 cited by Kirby et al.

We agree that “liberation of ATP secondary to hemolysis is not mutually exclusive of regulated export.”¹¹ Our point is that without the controls described in our paper, a confounding effect of hemolysis cannot be excluded.

We determined that the most reliable and direct evaluation of a hemolysis contribution to RBC ATP release requires the following. (1) The use of RBC supernatants for extracellular ATP (ATP_ec) evaluation rather than RBC suspensions is necessary. We verified that centrifugation (500g, 10 minutes, 4°C), even when repeated 5 times, does not elevate ATP_ec above the low basal level seen after the first or second centrifugation. (2) The measurement of free hemoglobin (Hb) in an aliquot of the same supernatant used for ATP_ec determination is necessary. To the best of our knowledge, such paired measurements have been performed only for plasma ATP-Hb determinations by Gorman et al¹² and Kirby et al² or toxin-induced ATP release.¹³ When ATP_ec-Hb values of all samples were plotted to evaluate the correlation between these variables, they showed a linear relationship and exactly matched those obtained with freshly prepared RBC lysates (see Figure 1D in Sikora et al¹). Any “upward” deflection in the linear ATP-Hb relationship would indicate a contribution of nonlytic ATP release.

With these restrictions, we found that ATP and Hb release were tightly correlated for all stimuli tested and exactly matched expectations based on the number of lysed cells and independently determined intracellular ATP concentration. Our conclusions were fully confirmed by directly visualizing single-cell ATP release and cell lysis using luminescence ATP imaging and simultaneous infrared cell imaging, respectively. We encourage other investigators to use these criteria and to examine the correlation between ATP and Hb release in individual experiments, so that conclusions can be made concerning regulated, hemolysis-independent mechanisms.

Specific responses

Gas flow was used in both normoxia- and hypoxia-treated samples, although indeed this was not stated clearly in the paper. Fresh and stored RBCs gave qualitatively similar results, despite somewhat elevated hemolysis after prolonged storage. The measured increase in ATP apparently precedes RBC hemolysis due to the faster diffusion of ATP compared with Hb, consistent with hemolytic release mechanism (see Figure 2B-C and Discussion in Sikora et al¹).

Authorship

Contribution: R.G. wrote the manuscript; and all authors reviewed and approved the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Ryszard Grygorczyk, Centre de Recherche, Centre Hospitalier de l'Université de Montréal, Tour Viger, 900, rue St-Denis, Montreal, QC, Canada H2X 0A9; e-mail: ryszard.grygorczyk@umontreal.ca.