Inhibitors of second messenger pathways and Ca²⁺-induced exposure of phosphatidylserine in red blood cells of patients with sickle cell disease

The present work investigates the contribution of various second messenger systems to Ca²⁺-induced phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients. The Ca²⁺ dependence of PS exposure was confirmed using the Ca²⁺ ionophore bromo-A23187 to clamp intracellular Ca²⁺ over 4 orders of magnitude in high or low potassium-containing (HK or LK) saline. The percentage of RBCs showing PS exposure was significantly increased in LK over HK saline. This effect was reduced by the Gardos channel inhibitors, clotrimazole and charybdotoxin. Nevertheless, although Ca²⁺ loading in the presence of an outwardly directed electrochemical gradient for K⁺ stimulated PS exposure, substantial exposure still occurred in HK saline. Under the conditions used inhibitors of other second messenger systems (ABT491, quinacrine, acetylsalicylic acid, 3,4-dichloroisocoumarin, GW4869 and zVAD-fmk) did not inhibit the relationship between [Ca²⁺] and PS exposure. Inhibitors of phospholipase A2, cyclooxygenase, platelet-activating factor, sphingomyelinase and caspases, therefore, were without effect on Ca ²⁺-induced PS exposure in RBCs, incubated in either HK or LK saline.