alpha‐hemolysin from E.coli induces P2 receptor activation, shrinkage and phosphatidyl serine exposure in human erythrocytes

Recently, we demonstrated that pore‐forming α‐hemolysin (HlyA) induces haemolysis via activation of P2 receptors on the erythrocytes (1). In addition, we observed that the erythrocyte swelling and lysis was preceded by a considerable volume reduction (1, 2). The present study documents the underlying mechanism for this shrinkage. By a combination of flow cytometry and live cell microscopy, we could demonstrate an early Ca 2+ ‐influx, which preceded the volume reduction. Reduced activity in either the K + channel K Ca 3.1 or the Cl − channel TMEM16A reduce the shrinkage, and potentiate the HlyA‐induced haemolysis. We also found that HlyA triggers phosphatidyl serine (PS) exposure in the outer leaflet of the plasma membrane. This PS exposure is significantly reduced by K Ca 3.1 channel blockers and P2 receptor antagonists. The latter implies that extracellular ATP is involved in the breakdown of the membrane asymmetry. By luciferin/luciferase method we were able to show that HlyA provokes ATP release within minutes of exposure, before any detectable hemolysis. In conclusion, this study shows that HlyA triggers ATP release important for shrinkage via K Ca 3.1 and TMEM16A and PS‐exposure. This mechanism protects the cells against early lysis, and potentially allows removal of damage erythrocytes from the bloodstream and, thereby, reduces the risk of intravascular hemolysis.