Paraformaldehyde induces elevation of intracellular calcium and phosphatidylserine externalization in platelets

Abstract

Paraformaldehyde fixation of platelets stabilizes surface antigens while altering some that are associated with cellular activation. Present experiments show that the asymmetric distribution of phosphatidylserine in platelets was especially sensitive to paraformaldehyde treatment. It was found that this reagent induced a dose- and time-dependent translocation of phosphatidylserine to the membrane surface as measured by annexin V binding and flow cytometry. The percent phosphatidylserine-positive cells increased from about 5% to > 90%. Chelation of extracellular Ca 2+ with EGTA partially blocked this translocation. Spectrofluorimetric analysis of fluo-3 loaded platelets indicates that paraformaldehyde caused a concomitant elevation of intracellular Ca 2+ concentrations, [Ca 2+] i. ATP levels also declined in paraformaldehyde-treated cells, suggesting that the rise in [Ca 2+] i ensued in part from decreased activity of calcium pumps. Previous studies indicate that phosphatidylserine externalization arises from Ca 2+-activated randomization of membrane phospholipids and decreased transport of phosphatidylserine from the outer to the inner leaflet of the plasma membrane. In light of present results, paraformaldehyde fixation is best avoided particularly in studies involving platelet apoptosis or activation.