Molecular Mechanism Underlying Excessive Phosphatidylserine Exposure in Sickle Red Blood Cells and Potential Therapeutics

Background:

Phosphatidylserine (PS) exposure on red blood cells (RBCs) promotes blood coagulation, enhances RBC adhesion to endothelial cells, and facilitates phagocytic clearance of RBCs (Weisel et al, JTH, 2019; Byrnes et al, Blood, 2017; Closse et at, BJH,1999; Boas et al, PNAS,1999). In sickle cell disease (SCD), excessive exposure of PS on sickle RBCs (SS RBCs) has long been associated with complications such as hypercoagulability, vaso-occlusion, and anemia (Wood et al, Blood,1996; Setty et al, Blood, 2002). Preventing PS exposure in SS RBCs is, therefore, a promising and paradigm-shifting therapeutic strategy to mitigate SCD and improve patients’ quality of life. However, this effort has been hindered by the elusive molecular mechanism underlying excessive PS exposure in SS RBCs.

We recently reported that TMEM16F is the sole calcium-activated phospholipid scramblase (CaPLSase) in RBCs, responsible for rapid PS exposure upon intracellular calcium increase (Liang et al, Blood, 2024). We found that calcium influx through the mechanosensitive channel PIEZO1 can activate TMEM16F CaPLSase in RBCs, and this functional coupling is dramatically enhanced in RBCs from individuals with hereditary xerocytosis (HX) harboring gain-of-function mutations in PIEZO1. More importantly, we discovered that benzbromarone, an anti-gout medicine, is a novel PIEZO1 inhibitor that can effectively decouple PIEZO1-TMEM16F functional interactions and prevent PS exposure (Liang et al, Blood, 2024). Since PIEZO1 was recently implicated in calcium increase in SS RBCs (Nader et al, Br J Haematol, 2023), we hypothesized that the functional coupling between PIEZO1 and TMEM16F is enhanced in SS RBCs and that decoupling PIEZO1-TMEM16F interaction could be a novel strategy to prevent SCD complications.

Results:

Using the combination of fluorescence imaging, flow cytometry and electrophysiology, here we report that PIEZO1-TMEM16F coupling plays a pivotal role in mediating the excessive PS exposure in SS RBCs and potential PS exposure-induced complications such as thrombosis and vaso-occlusion. We found that deoxygenation or sickling, which triggers hemoglobin S (HbS) polymerization and increases cell rigidity, promotes calcium increase and PS exposure from 1 ± 0.07 % to 2.5 ± 0.4%. This basal increase of calcium and PS exposure induced by deoxygenation can be prevented by PIEZO1 inhibitor GsMTx4, further supporting that PIEZO1 is involved in the sickling process.

To further dissect PIEZO1’s role in SCD pathophysiology, we stimulated SS RBCs with 2 µM Yoda1, a PIEZO1-specific agonist. We found that Yoda1 stimulation dramatically increased PS-positive SS RBCs to 37.3 ± 5.3%, which was further augmented to 69.2 ± 5.5% after deoxygenation. Next, we used electrophysiology to elucidate the underlying mechanism for the PIEZO1-mediated excessive PS exposure in SS RBCs. Our patch clamp recordings showed that sickling-induced PIEZO1 activation and dehydration-induced cell volume reduction promote intracellular calcium increase and TMEM16F activation, which subsequently leads to PS exposure. Consistent with excessive PS exposure in SS RBCs, we further demonstrated that deoxygenation and Yoda1 stimulation synergistically promote PS exposure-mediated changes, including increased release of PS+ microparticle, augmented thrombin generation, and enhanced RBC-endothelial adhesion. All these SCD-related phenomena can be effectively prevented in vitro by inhibiting PIEZO1 and subsequent PIEZO1-TMEM16F coupling using benzbromarone.

Conclusions:

Our study uncovers enhanced PIEZO1-TMEM16F functional coupling as a underlying mechanism that drives excessive PS exposure in SS RBCs, offering new insights into the pathophysiology of SCD. By demonstrating the effectiveness of pharmacological inhibition of PIEZO1 to suppress PS exposure using benzbromarone, we provide an alternative therapeutic strategy to alleviate SCD complications including thrombosis and vaso-occlusion.