Cooling and Freezing Damage Platelet Membrane Integrity

Abstract

Cytoskeletal rearrangements and a membrane lipid phase transition (liquid crystalline to gel) occur in platelets on cooling from 23 to 4°C. A consequence of these structural alterations is irreversible cellular damage. We investigated whether platelet membrane integrity could be preserved by (a) previously studied combinations of a calcium chelator (EGTA) and microfilament stabilizer (cytochalasin B) with apparent benefit in protecting platelets from cooling injury or (b) agents of known benefit in protecting membranes and proteins from freezing injury. Platelet function and activation before and after freezing or cooling were measured by agglutination with ristocetin, aggregation with thrombin or ADP, platelet-induced clot retraction (PICR), and expression of P-selectin. Platelets were loaded with 10 nM fluorescein diacetate. After freezing or cooling, the preparations were centrifuged and the supernatant was measured for fluorescein. For cooling experiments, fresh platelets were chilled at 4°C for 1 to 21 days with or without the combination of 80 μM EGTA/AM and 2 μM cytochalasin B (EGTA/AM-CytoB) and then warmed rapidly at 37°C. For freezing experiments, 5% dimethyl sulfoxide (Me2SO) or 5 mM glycerol were added to fresh platelets. The preparations were then frozen at −1°C/min to −70°C and then thawed rapidly at 37°C. Platelet membrane integrity, as measured by supernatant levels of fluorescein, correlated inversely with platelet function. Chilling platelets at 4°C with EGTA/AM-CytoB showed a gradual loss of membrane integrity, with maximum loss reached on day 7. The loss of membrane integrity preceded complete loss of function as demonstrated by PICR. In contrast, platelets chilled without these agents had complete loss of membrane integrity and function after 1 day of storage. Freezing platelets in Me2SO resulted in far less release of fluorescein than did freezing with or without other cryoprotectants (P< 0.001). This result correlated with enhanced function as demonstrated by PICR and supports earlier observations that Me2SO protects platelet membranes from freezing injury. Release of fluorescein into the surrounding medium reflected loss of membrane integrity and function in both cooled and frozen platelets. Membrane cytoskeletal rearrangements are linked to membrane changes during storage. These results may be generally applicable to the study of platelet storage.