PIEZO1 activation alters membrane remodeling and aggregability of stored red blood cells

BACKGROUND: Transfused red blood cells (RBCs) actively contribute to post-injury hemostasis due to surface phosphatidylserine (PS) exposure. We hypothesized that storage sensitizes erythrocytes to the PIEZO1-induced, calcium‑dependent, PS externalization through calcium and protein kinase C (PKC) dependent activation of the phospholipid scramblase TMEM16F. METHODS: Five units of type O whole blood were obtained for analysis at days 21 and 42 of storage. RBC PS was quantified by annexin V staining on flow cytometry at increasing extracellular CaCl₂ concentrations (0, 0.5, 1.0, 2.0, and 5.0 mmol/L), with and without the PIEZO1 agonist Yoda1 (0.2 μM), PKC agonist Phorbol 12-myristate 13-acetate (PMA; 6 μM) and antagonist chelerythrine chloride (CC; 10 μM), and the TMEM16 family inhibitor niclosamide (2 μM). RBC aggregometry was assessed by multiplate. Data were analyzed by analysis of variance and mixed-effects modeling. RESULTS: PS exposure increased in a Ca²⁺‑dependent manner in both day 21 and 42 RBCs ( p <0.001). PIEZO1 stimulation enhanced this response to a greater extent in day 42 RBCs, with greater change in PS at 1 mmol/L (2.21% vs. −0.44%, p =0.021), 2 mmol/L (7.36% vs. 0.53%, p <0.001), and 5 mmol/L (12.09% vs. 6.40%, p <0.001) CaCl 2 . Compared with calcium alone, Yoda1 and PMA (+61.3%, p <0.001 for both) produced similar increases in PS externalization, whereas PKC inhibition (−45.6%, p =0.009) and TMEM16F inhibition (−4.6%, p =0.99) negated the PIEZO1 response. Yoda1 agonism preserved RBC aggregability after 42 days of storage (51.2±39.0 vs. 2.0±3.5 area under the curve, p <0.001). CONCLUSION: Storage sensitizes RBCs to PIEZO1-mediated, calcium-dependent, PS externalization through PKC- and TMEM16F-dependent signaling. Given its endogenous mechanosensitive properties, pharmacologic PIEZO1 activation highlights a potential mechanism by which mechanical stress encountered during trauma and transfusion through the injured microcirculation may promote erythrocyte phospholipid membrane remodeling and aggregability, contributing to the procoagulant activity of transfused RBCs.