Modulation of red cell vesiculation by protease inhibitors

Release of vesicles from human red cell membranes was induced either by ATP-depletion or by incubation of the cells in presence of sonicated dimyristoylphosphatidylcholine (DMPC) vesicles. Vesicles released from ATP-depleted red cells but not the DMPC-induced vesicles contained degradation products of band 3 protein. Furthermore, in ATP-depleted erythrocytes proteolytic breakdown products could be demonstrated that were not detected in cells incubated with DMPC. Proteolysis was neither significantly affected by the protease inhibitor N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) nor by other protease inhibitors tested in this study (diisopropylfluorophosphate, N-ethylmaleimide and phenylmethylsulfonyl fluoride). Both vesiculation processes were inhibited in a concentration dependent way by TLCK while other protease inhibitors did not significantly influence membrane vesiculation. Phase contrast microscopy showed that TLCK diminished the DMPC-induced formation of echinocytes which is known to precede vesicle release. These results suggest that the influence of TLCK on membrane vesiculation is not primarily due to inhibition of proteolysis but to a direct interaction of the inhibitor with the intrinsic domain of the erythrocyte membrane.