We have shown previously that red blood cells (RBCs) can be induced to influx Ca(2+)
when treated with lipid mediators, such as lysophosphatidic acid and prostaglandin
E(2), that are released during clot formation. Since calcium loading of RBCs can lead
to both protein kinase C (PKC) activation and phosphatidylserine (PS) exposure, we
decided to investigate the possible linkage between PKC activation and membrane PS
scrambling using phorbol 12-myristate-13-acetate (PMA), a commonly used activator
of PKC. Treatment of RBCs with PMA in a calcium-containing buffer caused immediate
PS exposure in an RBC subpopulation. The size of the subpopulation did not change
upon further incubation, indicating that not all RBCs are equally susceptible to this
treatment. Using a fluorescent indicator, we found a subpopulation of RBCs with elevated
intracellular calcium levels. In the absence of extracellular calcium, no PS exposure
was found. However, we did find cells with high levels of calcium that did not expose
PS, and a variable percentage of PS-exposing cells that did not show elevated calcium
concentrations. Inhibition of PKC with either calphostin C, a blocker of the PMA binding
site, or chelerythrine chloride, an inhibitor of the active site, diminished the level
of formation of PS-exposing cells. However, the inhibitors had different effects on
calcium internalization, indicating that a high calcium concentration alone was not
responsible for inducing PS exposure in the absence of PKC activity. Moreover, PKC
inhibition could prevent PS exposure induced by calcium and ionophore treatment of
RBCs. We conclude that PKC is implicated in the mechanism of membrane phospholipid
scrambling.