Interactions between metal ions and poly(ethylene glycol) in the fusion of human erythrocytes.

Diffusion of the fluorescent membrane probe, Dil-C16 (3), from labelled to unlabelled human erythrocytes has been employed to monitor hemi-fusion (membrane fusion) in monolayers of cells exposed to poly(ethylene glycol) (PEG). Diffusion of the cytoplasmic probe, 6-carboxyfluorescein, was used similarly to monitor cell fusion (cytoplasmic mixing). Hemi-fusion, which is normally seen when erythrocytes are exposed to dehydrating concentrations of commercial PEG 6000, did not occur when the PEG was pretreated with Chelex 100 resin to remove metal ions.

Divalent cations, phospholipid asymmetry and osmotic swelling in electrically-induced lysis, cell fusion and giant cell formation with human erythrocytes.

We have previously reported that acidic phospholipids are exposed at the surface of human erythrocytes when the cells are subjected to electrical breakdown. It has now been shown that the prothrombinase assay, which was used previously for the determination of acidic phospholipids, is specific for phosphatidylserine under the conditions of our experiments. In the light of this finding, we have investigated and characterised factors that govern cell lysis, cell fusion, and the formation of giant cells induced by electrical breakdown with human erythrocytes in media of low ionic strength.

Relationships between the surface exposure of acidic phospholipids and cell fusion in erythrocytes subjected to electrical breakdown.

The procoagulant activity of human erythrocytes, which provides a measure of the translocation of acidic phospholipids from the inner to the outer monolayer of the plasma membrane, has been compared with the percentage cell fusion in experiments on the effects of electrical breakdown pulses under differing experimental conditions. After treatment with breakdown pulses of 20 microseconds or longer (5 kV cm-1), the plasma membranes of erythrocytes in 250 mM sucrose exhibited an almost complete loss of asymmetry with respect to acidic phospholipids. As the breakdown voltage was increased from 2 to 5 kV cm-1 (with breakdown pulses of 99 microseconds), the surface exposure of acidic phospholipids and cell fusion increased approximately in parallel.

Surface exposure of phosphatidylserine is associated with the swelling and osmotically-induced fusion of human erythrocytes in the presence of Ca2+.

An assay for procoagulant activity has been used to investigate the Ca2(+)-dependent exposure of phosphatidylserine at the surface of human erythrocytes that were induced to swell and to fuse osmotically. Since the phosphatidylserine of human erythrocytes is located in the inner leaflet of the plasma membrane, it is inaccessible in intact cells which therefore had no procoagulant activity in an isotonic solution of sucrose. The procoagulant activity of erythrocytes incubated in increasingly hypotonic, sucrose solutions containing Mg2+ paralleled the percentage haemolysis, reflecting the accessibility of phosphatidylserine in an increasing number of lysed cells.