Altered membrane skeleton of hydroxyethylstarch-cryopreserved human erythrocytes

Attempts have been made to use hydroxyethylstarch (HES) as an alternative to glycerol for cryopreservation of erythrocytes. However, HES cryopreservation causes significant transient rheological alterations in erythrocytes. Membrane proteins play a critical role for erythrocyte rheology. This study was undertaken to analyze erythrocyte membrane proteins during HES cryopreservation. Erythrocyte membranes with submembrane skeleton (ghosts) and the submembrane skeleton alone (unstripped skeletons) were prepared before freezing (native), after thawing and following 3 h reconditioning in glucose-enriched Ringer's solution (Ringer plus glucose), or in autologous fresh frozen plasma (AFFP). After electrophoresis protein concentrations (percentage of total protein) were determined by densitometry. In ghosts, no significant changes were found, whereas in unstripped skeletons the following results could be seen: beta-Spectrin: 31.8 +/- 2.2% (native), 22.1 +/- 0.8% (postthawing, P < 0.05 vs native), 22.4 +/- 1.6% (Ringer plus glucose, P < 0.05 vs native), 31.0 +/- 2.8% (AFFP). Other proteins remained unchanged. Since a significant decrease in beta-spectrin concentration after HES cryopreservation and after subsequent reconditioning in Ringer's solution with glucose was only detected in unstripped skeletons, this cannot be interpreted as in vivo protein loss. More likely, HES cryopreservation may have created changes in protein-protein associations. The course of beta-spectrin concentration parallels certain rheological and biochemical changes and might explain the transient rheological changes seen after HES cryopreservation.