The Mechanism of Hemolysis in Sickle Cell Anemia.
Authors: Presley Tennille , Bain Lauren , Ballas Samir , Nichols James , Sabio Hernan , Gladwin Mark , Kato Gregory J. , Kim-Shapiro Daniel 
Published: April 2020

Abstract
Accumulating transgenic animal, large animal and human epidemiological evidence supports a role for hemolysis in the pathobiology of sickle cell disease. However, the mechanism of hemolysis or more specifically the relative contribution of sickling and oxidative damage has yet to be determined. Early studies have shown that repetitive sickling/unsickling via cycles of deoxygenation/reoxygenation lead to a decrease in sickle red cell deformability (even under oxygenated conditions), suggesting an important role for sickle hemoglobin polymerization probably associated with membrane loss and dehydration. However, all of these previous studies have used sickle (SS) cells which undergo cycles of sickling and unsickling in vivo and hence, have poor deformability even under aerobic conditions prior to in vitro experiments. In our study, we used sickle cell trait cells (AS) which do not sickle under physiological oxygen pressures, but can be sickled by exposing them to anoxia (zero percent oxygen). This novel approach allows us to study the effects of sickle hemoglobin polymerization on cells that have never contained polymers before, in order to gain information on the role of polymerization in intravascular hemolysis. We measured deformability in normal (AA), AS, and SS red cells using flow channel laser diffraction and obtained a deformability coefficient (the lower the coefficient the poorer the deformability). In addition, we measured mechanical fragility via shaking in the presence of glass beads followed by measurements of plasma hemoglobin using absorption spectroscopy. As expected, there was no difference in deformability measured for AA cells under aerobic or anaerobic conditions (2.1 ± 0.5 oxy vs. 1.9 ± 0.4 deoxy, n=3), while the deformability of deoxygenated SS or AS cells was substantially decreased, indicating that polymers formed for both SS and AS cells (1.6 ± 0.3 oxy SS vs. 1.34 ± 0.05 deoxy SS; 1.8 ± 0.4 oxy AS vs. 1.17 ± 0.04 deoxy AS, n=3). Likewise, whereas partial pressure of oxygen had no significant effect on the mechanical fragility of AA cells (2.1 ± 0.3 μM for oxy vs. 1.5 ± 0.9 μM for deoxy, n=3); deoxygenation greatly increased the mechanical fragility of both AS and SS cells (1.8 ± 0.2 μM oxy AS vs. 10.6 ± 3.2 μM deoxy AS; 0.8 ± 0.1 μM oxy SS vs. 2.7 ± 0.9 μM deoxy SS). Reoxygenation of SS cells following prolonged deoxygenation, tended to not regain the level of mechanical fragility of cells maintained in continuous aerobic conditions (1.9 ± 0.4 μM reoxy vs. 0.6 ± 0.1 μM oxy); consistent with previous findings that repeated sickling and unsickling leads to diminished red cell deformability. On the other hand, AS cells fully regained their lower mechanical fragility following reoxygenation after prolonged deoxygenation (1.0 ± 0.2 μM reoxy vs. 1.1 ± 0.4 μM oxy). Our data support two important conclusions:
The observed poor rheology of SS cells under aerobic conditions does not result from a single or prolonged sickling event, but rather is likely to include contributions from oxidative damage. This conclusion is based on the observation that rheological properties of deoxygenated AS cells return to normal following reoxygenation. A substantial amount of intravascular hemolysis occurs in vivo in cells that contain sickle cell hemoglobin polymers.
This is suggested by the dramatic increase in mechanical fragility upon deoxygenation of both AS and SS cells. AS cells at zero oxygen pressure are likely to contain similar amounts of polymers as SS cells under physiological conditions. Thus, it is likely that many cells that hemolyze in vivo do so upon the first sickling event.