Intravascular hemolysis: the sacrifice of few…

In this issue of Blood, Sikora et al demonstrate unequivocally that when erythrocytes are submitted to shear stress and hypoxia, only hemolysis contributes to the release of adenosine triphosphate (ATP), suggesting erythrocyte sacrifice as a primary mechanism for in vivo local purinergic signaling and blood flow regulation.¹ 

Long ago, August Krogh arrived to the concept that “in the normal resting muscle a comparatively small number of capillaries…should be open, so as to allow the passage of blood while muscular work should cause the opening up of a larger number.”²  A century later, regulation of blood flow through capillaries and the role played by erythrocytes in this process are still in debate. The elegant approach of experimental physiology presented by Sikora et al constitutes a decisive breakthrough in the field.

It has been well documented that mechanical deformation, decreased oxygen partial pressure, and reduced pH, such as occur in microvessels of exercising muscle, induce ATP release and that this release activates purinergic receptors of vascular endothelial cells, resulting in synthesis of nitric oxide and metabolites of arachidonic acid known as powerful relaxation factors of smooth muscle vasculature contributing to match oxygen delivery with local need.³ 

However, the actual mechanism underlying ATP release remained in debate and was frequently attributed to complex signaling pathways opening up cystic fibrosis transmembrane conductance regulator (CFTR) channels.³  Unfortunately, no evidence of CFTR protein was found,⁴  and no CFTR single channel electrical activity was recorded in the erythrocyte membrane. The major contribution of Sikora et al is to provide an alternative and more simple mechanism not involving channels of intact cells: ATP release results from intravascular hemolysis! This result is not so surprising if we consider that blood is not a homogenous population of cells but rather is composed of a wide range of cells displaying very different homeostatic states, among them the senescent cells that are in a prolytic^5,6  state before removal from circulation and constitute an easily accessible pool of ATP for local release.

The role played by hemolysis is not novel because hemolysis has always been considered a potential factor contributing to stimulated ATP release in most previous investigations, but because of inappropriate evaluation of its actual involvement, due to a lack of paired measurement of free hemoglobin and ATP, its contribution to ATP release has been underestimated or simply overlooked. The novelty is that for the first time, Sikora et al were able to show that all regions displaying ATP release perfectly matched sites where cells had lysed. The clever use of ATP luminometry and luminescence ATP imaging demonstrated very nicely that brief spikes of luminescence due to ATP release from lysing cells was the sole mechanism of ATP release in these experiments.

The authors show that in response to stimuli that elevate cyclic adenosine monophosphate (cAMP), the ATP releases were indistinguishable from those evoked by an equivalent volume of the solvent dimethyl sulfoxide (DMSO) alone, which rules out a possible contribution of cAMP agonists in ATP release but interestingly shows an effect of DMSO on ATP release, which illustrates the deleterious effects of DMSO on cell homeostasis.

What does all this mean? Obviously, as clearly stated by Sikora et al, because hemolysis also occurs in vivo in physiological situations, such ATP release mechanisms are powerful physiological tools of blood flow regulation. It means that every factor influencing erythrocyte membrane fragility will also contribute to modulation of ATP release via hemolysis, but it also means that in absence of regulation these factors could be the origin of pathologies. Altered erythrocyte fragility and hemolysis are observed in many situations such as in vitro experimental conditions, blood storage, senescence, and pathologies.

In light of the present work, it appears that there is an urgent need to better understand the mechanisms underlying hemolysis, but first, more caution should be taken when processing with blood samples. As the authors state, from the moment of blood collection to final assessment, erythrocytes are exposed to various physical perturbations that unavoidably cause rupture and hemolysis of many cells. For instance, the use of filters for removing white cells before experiments causes massive hemolysis and deprives the investigator of invaluable information on prolytic red cells and on stimuli intended to trigger regulated ATP release.

It is a matter of fact that we know very little about conditions prevailing in cells toward the final days in the circulation. Lew et al⁷  have shown that the progressively increased intracellular calcium together with erythrocyte dehydration would activate a nonselective cationic channel,^5,6  eventually leading to an unexpected light-density terminal state. Renewed attention should be paid to this phenomenon, which was also observed in sickle cells,⁸  to evaluate the role played by cationic channels as a possible triggering factor of hemolysis.

The findings of Sikora et al also shed new light on the crucial problems of blood storage and transfusion. Today, the blood preservation standards for transfusion require that ≥75% of the cells are still in the circulation 24 hours after transfusion and that hemolysis remains <1% at the end of the storage period, but the key mechanisms responsible for hemolysis and cell disappearance remain unknown, and we can suspect the subsequent massive release of ATP to play a major role in posttransfusion harmful clinical outcomes.

The work of Sikora et al raises many fascinating basic questions such as the role played by calcium in modulation of osmotic fragility and the link between altered membrane function in erythrocyte disorders⁹  (such as hereditary spherocytosis, elliptocytosis, ovalocytosis, stomatocytosis, sickle cell disease, and thalassemias) and the constant observation of cation leaks in these diseases. The possible collateral effects of the other molecules released together with ATP (ie, carbonic anhydrase) and the possible role played by microvesicles¹⁰  in this hemolytic process also deserve further investigation.