Biomedical subjects

M Gorogias

Publications and source records attributed to M Gorogias.

3 recordsLinked to original sources

PubMed2000

Correlation of in vivo and in vitro functions of fresh and stored human platelets.

BACKGROUND: Long-term storage of human platelets has been hindered by the loss of function of the platelets stored under current protocols. Novel preservation methods have encouraged examination of platelet function of cells preserved by cooling and freezing. The function of the platelets was assessed by using both in vitro assays and an in vivo rabbit bleeding model. STUDY DESIGN AND METHODS: Human platelets were stored in the presence or absence of 2 microM: cytochalasin B and 80 microM: EGTA/AM at 4 degrees C for 14 days or by freezing in the presence or absence of 5 percent DMSO. After the storage period, the platelets were resuspended in normal saline and infused into rabbits. Platelet function was assessed in vivo in a kidney bleeding model and in vitro by platelet-induced clot retraction and by platelet aggregation. RESULTS: Platelets stored at either 4 degrees C or -145 degrees C exhibited shorter survival times in the rabbit circulation than did fresh platelets. Platelets cooled to 4 degrees C, in both the presence or absence of cytochalasin B and EGTA/AM treatment, or frozen in the absence of DMSO were not effective in halting bleeding. However, frozen DMSO-treated platelets were as effective as fresh platelets in stopping bleeding. In vitro assays showed that cooled platelets treated with cytochalasin B and egtazic acid/AM and frozen DMSO-treated platelets retained 30 to 40 percent platelet function, while the cooled and frozen control samples exhibited no platelet-induced clot retraction. With thrombin as the agonist, only frozen DMSO-treated platelets exhibited a tendency to aggregate, although at only 22 percent of the aggregation function of fresh platelets. CONCLUSION: It is possible to freeze platelets and retain in vivo efficacy if the cryopreservative DMSO is included in the preparation. In vitro responses were greatly reduced by all of the storage protocols, but it may not be necessary to retain 100 percent in vitro function to have a platelet substitute or storage product that functions satisfactorily in vivo.

Animals↗

PubMed1999

Cooling and freezing damage platelet membrane integrity.

Cytoskeletal rearrangements and a membrane lipid phase transition (liquid crystalline to gel) occur in platelets on cooling from 23 to 4 degrees 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 degrees C for 1 to 21 days with or without the combination of 80 microM EGTA/AM and 2 microM cytochalasin B (EGTA/AM-CytoB) and then warmed rapidly at 37 degrees C. For freezing experiments, 5% dimethyl sulfoxide (Me2SO) or 5 mM glycerol were added to fresh platelets. The preparations were then frozen at -1 degrees C/min to -70 degrees C and then thawed rapidly at 37 degrees C. Platelet membrane integrity, as measured by supernatant levels of fluorescein, correlated inversely with platelet function. Chilling platelets at 4 degrees 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.

Adenosine Diphosphate↗

PubMed1999

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. STUDY DESIGN AND 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.

Adenosine Diphosphate↗