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

• PIEZO1- and KCNN4-HX RBCs show distinct differences in functional and metabolic properties. • Ex vivo PK activation leads to increased energy levels, but it 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 12 patients with PIEZO1-HX, 3 patients with KCNN4-HX, and 10 controls, with particular focus on the key glycolytic enzyme pyruvate kinase (PK). In PIEZO1-HX, RBC PK activity is relatively decreased (PK-to-hexokinase ratio, 4.7 vs 8.4 in controls), along with a decrease in PK thermostability (43.6% vs 77.8%) and PK protein levels. KCNN4-HX RBCs show a less pronounced decrease in activity (PK-to-hexokinase ratio, 6.3) and thermostability (64.7%), with no decrease in protein levels. Untargeted metabolomics demonstrated distinct differences in various metabolites, including carnitines, in PIEZO1-HX vs KCNN4-HX or controls. Interestingly, hydration (O hyper ) of PIEZO1-HX RBCs correlated negatively with the ratio of adenosine triphosphate to 2,3-diphosphoglycerate ( 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 treatment increased PK activity (>40%) in both disorders, but it 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 the understanding of the differences between PIEZO1-HX and KCNN4-HX and provide evidence for a potential response to PK activator treatment.