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JOURNAL ARTICLE
RESEARCH SUPPORT, U.S. GOV'T, P.H.S.
Apoptotic activity in stored human platelets.
Transfusion 2003 April
BACKGROUND: Platelets possess some of the machinery required for apoptotic cell death. However, disruption of mitochondria function, implicated in several models of cell death, has not been extensively studied in platelets. Mitochondrial viability and several other measures of apoptotic death in stored and experimentally stressed platelets were evaluated.
MATERIALS AND METHODS: Platelet mitochondrial transmembrane potentials (Deltapsim) were studied by staining platelets with JC-1, a dye that fluoresces at different wavelengths based on the state of mitochondrial polarization. Annexin V binding, a measure of phosphatidylserine (PS) exposure, and CD62P expression, an indicator of platelet activation, were determined by flow cytometry. Caspase-3 activity was measured with an enzyme assay and by Western blotting. Experimental platelet stressors included storage for 7 days, azide exposure, calcium ionophore stimulation, and plasma deprivation.
RESULTS: As measured by flow cytometry, Deltapsim values were similar in freshly drawn platelets and in platelet concentrates stored for up to 7 days. However, compared to fresh platelets, stored platelet concentrates had significantly increased PS exposure (3.1 vs. 5.1%, p = 0.015), CD62P expression (6.5 vs. 13.5%, p = 0.0067), and caspase-3 activity. Azide exposure, which decreased ATP release 20 to 30 percent, did not affect the Deltapsim. Stressed platelets exhibited higher degrees of mitochondrial depolarization in response to calcium ionophore stimulation than platelets that were not stressed. Plasma deprivation also resulted in significant alterations in Deltapsim, PS exposure, and CD62P expression.
CONCLUSIONS: Platelet mitochondria maintain Deltapsim when stored for up to 7 days under standard blood bank storage conditions. Therefore, changes in platelet mitochondria Deltapsim do not correlate with downstream markers of apoptotic death such as caspase activation and PS exposure.
MATERIALS AND METHODS: Platelet mitochondrial transmembrane potentials (Deltapsim) were studied by staining platelets with JC-1, a dye that fluoresces at different wavelengths based on the state of mitochondrial polarization. Annexin V binding, a measure of phosphatidylserine (PS) exposure, and CD62P expression, an indicator of platelet activation, were determined by flow cytometry. Caspase-3 activity was measured with an enzyme assay and by Western blotting. Experimental platelet stressors included storage for 7 days, azide exposure, calcium ionophore stimulation, and plasma deprivation.
RESULTS: As measured by flow cytometry, Deltapsim values were similar in freshly drawn platelets and in platelet concentrates stored for up to 7 days. However, compared to fresh platelets, stored platelet concentrates had significantly increased PS exposure (3.1 vs. 5.1%, p = 0.015), CD62P expression (6.5 vs. 13.5%, p = 0.0067), and caspase-3 activity. Azide exposure, which decreased ATP release 20 to 30 percent, did not affect the Deltapsim. Stressed platelets exhibited higher degrees of mitochondrial depolarization in response to calcium ionophore stimulation than platelets that were not stressed. Plasma deprivation also resulted in significant alterations in Deltapsim, PS exposure, and CD62P expression.
CONCLUSIONS: Platelet mitochondria maintain Deltapsim when stored for up to 7 days under standard blood bank storage conditions. Therefore, changes in platelet mitochondria Deltapsim do not correlate with downstream markers of apoptotic death such as caspase activation and PS exposure.
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