Red blood cell properties in hereditary xerocytosis and their response to ex vivo pyruvate kinase activation: a preliminary study. Open Access

• PIEZO1- and KCNN4-hereditary xerocytosis red blood cells show distinct differences in functional and metabolic properties.

• Ex vivo pyruvate kinase activation leads to increased energy levels, yet does not evidently improve hydration in PIEZO1-HX.

Hereditary xerocytosis (HX, also known as dehydrated stomatocytosis) is a rare red blood cell (RBC) disorder associated with hemolysis and iron overload. Gain-of-function mutations in either PIEZO1 or KCNN4 lead to disturbed RBC ion homeostasis. PIEZO1-HX and KCNN4-HX are mechanistically different and have distinct phenotypes, yet these differences are poorly understood. Here, we study RBC hydration and metabolism in twelve PIEZO1-HX patients, three KCNN4-HX patients and ten controls, with particular focus on the key glycolytic enzyme pyruvate kinase (PK). In PIEZO1-HX, RBC PK activity is relatively decreased (PK/hexokinase ratio 4.7 versus 8.4 in controls), alongside a decrease in PK thermostability (43.6% versus 77.8%) and PK protein levels. KCNN4-HX RBCs show a less pronounced decrease in activity (PK/hexokinase ratio 6.3) and thermostability (64.7%), and no decrease in protein levels. Untargeted metabolomics demonstrated distinct differences in various metabolites, including carnitines, in PIEZO1-HX versus KCNN4-HX or controls. Interestingly, hydration (O_hyper) of PIEZO1-HX RBCs correlated negatively with the ATP/2,3-DPG ratio (r=-0.839). To evaluate whether enhancing glycolysis by PK activation could improve hydration, we treated RBCs ex vivo with the PK activator tebapivat. This increased PK activity (>40%) in both disorders yet did not negate dehydration in most PIEZO1-HX RBCs. In contrast, O_hyper did improve in KCNN4-HX (>10 mOsm/kg increase). Our results indicate significant metabolic differences between PIEZO1-HX and KCNN4-HX, and different responses to ex vivo PK activation. Our findings increase our understanding of the differences between PIEZO1-HX and KCNN4-HX and provide evidence for a potential response to PK activator treatment.