Platelet membrane integrity during storage and activation.
BACKGROUND
The platelet cell membrane appears to undergo a lipid-phase transition on cooling from 23 degrees C to 4 degrees C. Consequences of this phase transition are leakage of cellular material and irreversible cellular damage. Whether agents, of known benefit in protecting membranes and proteins from cooling and drying injury, could also protect platelets was investigated. Leakage of cytosolic components was assessed by measuring the release of fluorescein into the surrounding medium.


METHODS
Fresh platelets were suspended in 5 percent dimethyl sulfoxide (DMSO) or in 5 mM of one the following agents: glucose, trehalose, sucrose, glycerol, ethylene glycol, 1,2-propanediol, or L-proline. Platelets were loaded with 10 nMfluorescein diacetate (FD), chilled at 4 degrees C for 24 hours or frozen at -1 degree C per minute to -70 degrees C, warmed rapidly at 37 degrees C, and centrifuged, and the supernatant was measured for the presence of fluorescein. The effect of FD on platelets was assessed by agglutination with ristocetin, aggregation with thrombin and ADP, platelet-induced clot retraction, and expression of p-selectin. Platelet function and activation before and after freezing or cooling were measured by the same methods.


RESULTS
By flow cytometry, 98 percent of the platelets incorporated FD. The trapped fluorescein resulted in neither platelet activation (p = 0.9) nor reduction of platelet function (p = 0.12-0.94) from that in control platelets. Freezing of platelets in DMSO caused far less release of fluorescein than did freezing with other agents (p<0.001) or chilling of platelets at 4 degrees C for 24 hours (p<0.0001). Supernatant levels of fluorescein correlated inversely with platelet function. Fluorescein was also shown to be released during aggregation with thrombin or ADP but not during agglutination with ristocetin.


CONCLUSIONS
Release of fluorescein into the surrounding medium indicated a loss of platelet membrane integrity and function. Cellular loading with FD is a simple method of studying membrane integrity of platelets and other cells.
Reid
T J
TJ
Department of Hematology and Vascular Biology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA. reidt@wrsmtp-ccmail.army.mil
Esteban
G
G
Clear
M
M
Gorogias
M
M
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology; D015925:Cryopreservation; D005452:Fluoresceins / Q000378:metabolism / Q000494:pharmacology; D006384:Hemagglutination / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D019007:P-Selectin / Q000097:blood; D015539:Platelet Activation / Q000502:physiology; D010974:Platelet Aggregation / Q000187:drug effects; D012310:Ristocetin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D005452:Fluoresceins; D019007:P-Selectin; D012310:Ristocetin; D000244:Adenosine Diphosphate; D013917:Thrombin; C018506:diacetylfluorescein