Figure 7.
Figure 7. Rejuvenation of BAK/BAX-deficient platelets does not confer protection from the PSL at room temperature. Murine platelets were stored at RT (22°C) with gentle agitation in 5% plasma for up to 9 days. Mice were depleted of platelets (APS) 72 hours before platelet purification. (A) Caspase-3/7 activation (Caspase-Glo assay), (B) annexin V-positive platelets, (C) ATP activity (CellTiter-Glo assay), (D) TMRM-positive platelets, (E) GPIbα surface expression, and (F) P-selectin exposure were assessed at indicated points by flow cytometry or as otherwise indicated. Platelets counts were adjusted before storage. n = 11 to 12 biological replicates per genotype. Data are presented as mean ± SEM. Caspase activity is presented as ratio/start for each genotype. Statistical significance between 2 treatment groups was analyzed using an unpaired Student t test with 2-tailed P values. Data from Figure 1 are included in red and purple to allow statistical comparison with untreated samples (unpaired Student t test with 2-tailed P values). n.s., not significant. (G) Intrinsic apoptosis circumvents the functional decline of circulating platelets, but does not cause the platelet storage lesion. 1) Platelet storage. Mouse wild-type or BAK/BAX-deficient platelets were stored at the blood banking temperature (22°C) and at 37°C. In some experiments, the platelet age was synchronized to 3 days in both wild-type and BAK/BAX-deficient mice before ex vivo storage experiments (referred to as “Rejuvenation”). Loss of BAK and BAX did not prevent the development of the PSL at either temperature. 2) Platelet function. BAK/BAX-deficient and wild-type mice responded similarly in an experimental in vivo model of pulmonary embolism, which is highly dependent on thrombin generation. This indicates that intrinsic apoptosis does not contribute to procoagulant activity in vivo. BAK/BAX-deficient mice exhibited increased tail bleeding times and unstable thrombus formation in vivo, which was associated with reduced granule release from the aged BAK/BAX-deficient platelets in vitro. Rejuvenation (platelet age synchronization) rescued these defects, indicating that intrinsic apoptosis restricts platelet age to maintain platelet functionality.

Rejuvenation of BAK/BAX-deficient platelets does not confer protection from the PSL at room temperature. Murine platelets were stored at RT (22°C) with gentle agitation in 5% plasma for up to 9 days. Mice were depleted of platelets (APS) 72 hours before platelet purification. (A) Caspase-3/7 activation (Caspase-Glo assay), (B) annexin V-positive platelets, (C) ATP activity (CellTiter-Glo assay), (D) TMRM-positive platelets, (E) GPIbα surface expression, and (F) P-selectin exposure were assessed at indicated points by flow cytometry or as otherwise indicated. Platelets counts were adjusted before storage. n = 11 to 12 biological replicates per genotype. Data are presented as mean ± SEM. Caspase activity is presented as ratio/start for each genotype. Statistical significance between 2 treatment groups was analyzed using an unpaired Student t test with 2-tailed P values. Data from Figure 1 are included in red and purple to allow statistical comparison with untreated samples (unpaired Student t test with 2-tailed P values). n.s., not significant. (G) Intrinsic apoptosis circumvents the functional decline of circulating platelets, but does not cause the platelet storage lesion. 1) Platelet storage. Mouse wild-type or BAK/BAX-deficient platelets were stored at the blood banking temperature (22°C) and at 37°C. In some experiments, the platelet age was synchronized to 3 days in both wild-type and BAK/BAX-deficient mice before ex vivo storage experiments (referred to as “Rejuvenation”). Loss of BAK and BAX did not prevent the development of the PSL at either temperature. 2) Platelet function. BAK/BAX-deficient and wild-type mice responded similarly in an experimental in vivo model of pulmonary embolism, which is highly dependent on thrombin generation. This indicates that intrinsic apoptosis does not contribute to procoagulant activity in vivo. BAK/BAX-deficient mice exhibited increased tail bleeding times and unstable thrombus formation in vivo, which was associated with reduced granule release from the aged BAK/BAX-deficient platelets in vitro. Rejuvenation (platelet age synchronization) rescued these defects, indicating that intrinsic apoptosis restricts platelet age to maintain platelet functionality.

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