Complement‐induced hemolysis and purinergic signaling

Activation of the complement system is important in the pathogenesis of certain autoimmune hemolytic anemias, via formation of the lytic membrane attack complex. Previously, we have shown that another pore‐former α‐hemolysin (HlyA) form E. coli requires P2X‐receptor activation to lyse erythrocytes (1). Here we investigate if this mechanism also implies for complement‐induced hemolysis. Ovine and human erythrocytes were incubated with anti‐sheep erythrocyte antibodies or anti‐Rh (D) antibodies respectively, with either human plasma or guinea pig serum as complement donors. Non‐selective P2‐antagonists (PPADS and suramin) concentration‐dependently inhibit complement‐induced hemolysis. More specific P2‐receptor antagonists imply that P2X 7 and P2X 1 are the main receptors involved. Similar to HlyA, complement‐activation produces a sustained increase in the intracellular Ca 2+ , which triggers significant erythrocyte shrinkage that precedes swelling and lysis. This early volume reduction is likely to result from activation of the K + channel Kca3.1 as TRAM34 and clotrimazole augment the complement induced hemolysis. These results indicate that complement, similar to HlyA , requires purinergic signaling for full hemolysis, and that activation of the erythrocyte volume regulation protracts the lysis. This finding points several new pathways to interfere with hemolytic diseases. The project was founded by the Danish Council for Independent Research, Medical Sciences.