Abstract
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. 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. 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. 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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.