Hot-cold
hemolysis is the phenomenon whereby red blood cells, preincubated at 37 degrees C in the presence of certain agents, undergo rapid
hemolysis when transferred to 4 degrees C. The mechanism of this phenomenon is not understood. PlcHR 2, a
phospholipase C/
sphingomyelinase from Pseudomonas aeruginosa, that is the prototype of a new
phosphatase superfamily, induces hot-cold
hemolysis. We found that the
sphingomyelinase, but not the
phospholipase C activity, is essential for hot-cold
hemolysis because the phenomenon occurs not only in human erythrocytes that contain both
phosphatidylcholine (PC) and
sphingomyelin (SM) but also in goat erythrocytes, which lack PC. However, in horse erythrocytes, with a large proportion of PC and almost no SM, hot-cold
hemolysis induced by PlcHR 2 is not observed. Fluorescence microscopy observations confirm the formation of
ceramide-enriched domains as a result of PlcHR 2 activity. After cooling down to 4 degrees C, the erythrocyte ghost membranes arising from
hemolysis contain large,
ceramide-rich domains. We suggest that formation of these rigid domains in the originally flexible cell makes it fragile, thus highly susceptible to
hemolysis. We also interpret the slow
hemolysis observed at 37 degrees C as a phenomenon of gradual release of aqueous contents, induced by the
sphingomyelinase activity, as described by Ruiz-Arguello et al. [(1996) J. Biol. Chem. 271, 26616]. These hypotheses are supported by the fact that
ceramidase, which is known to facilitate slow
hemolysis at 37 degrees C, actually hinders hot-cold
hemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHR 2 demonstrates the presence of
ceramide-rich domains that are rigid at 4 degrees C but fluid at 37 degrees C.
Ceramidase treatment causes the disapperance of the calorimetric signal assigned to
ceramide-rich domains. Finally, in
liposomes composed of SM, PC, and
cholesterol, which exhibit slow release of aqueous contents at 37 degrees C, addition of 10 mol %
ceramide and transfer to 4 degrees C cause a large increase in the rate of solute efflux.