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Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients.

López E, Berna-Erro A, Bermejo N, Brull JM, Martinez R, Garcia Pino G, Alvarado R, Salido GM, Rosado JA, Cubero JJ, Redondo PC - J. Cell. Mol. Med. (2013)

Bottom Line: Blood samples were drawn from healthy volunteers and kidney transplant patients long-term medicated with rapamycin: sirolimus and everolimus.Our results indicate that rapamycin evokes a biphasic time-dependent alteration in calcium homeostasis and function in platelets from kidney transplant patients under rapamycin regime, as demonstrated by the reduction in granule secretion observed and subsequent impairment of platelet aggregation in these patients compared with healthy volunteers.Platelet count was also reduced in these patients, thus 41% of patients presented thrombocytopenia.

View Article: PubMed Central - PubMed

Affiliation: Cell Physiology Research Group, Department of Physiology, University of Extremadura, Cáceres, Spain.

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Granule secretion in patients medicated with sirolimus. Platelets isolated from kidney-transplanted patients treated with sirolimus were incubated in plasma rich platelets (PRP) for 30 min. at 37 °C with anti-CD41 and quinacrine (10 μM). Platelets were then left under resting condition or stimulated for 10 min. with Thr (0.1 U/ml), and simultaneously, anti-CD62 antibody (P-selectin; diluted 1:50) was added to medium. Fluorescence of CD41 was used for selecting platelet positive cells. Fluorescence was analysed using flow cytometry, from patients under sirolimus medication for different periods of time (<24: I), (24–36: II),(36–60: III) and (>60 months: IV). The fluorescence results of recording CD41 and CD62-positive platelets (A; α-granules) and simultaneously, CD41 and quinacrine (B; δ-granules) in the same platelets sample. Histograms show either the increase in P-selectin membrane exposition or quinacrine fluorescence remaining in the platelets in fold increase. *, ** and ***, represent P < 0.05, <0.01 and <0.01 compared with values found in resting platelets from healthy individuals (n = 6). While, $, $$, $$$, represent P < 0.05, <0.01 and <0.01 compared with Thr-stimulated values found in healthy individuals.
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fig02: Granule secretion in patients medicated with sirolimus. Platelets isolated from kidney-transplanted patients treated with sirolimus were incubated in plasma rich platelets (PRP) for 30 min. at 37 °C with anti-CD41 and quinacrine (10 μM). Platelets were then left under resting condition or stimulated for 10 min. with Thr (0.1 U/ml), and simultaneously, anti-CD62 antibody (P-selectin; diluted 1:50) was added to medium. Fluorescence of CD41 was used for selecting platelet positive cells. Fluorescence was analysed using flow cytometry, from patients under sirolimus medication for different periods of time (<24: I), (24–36: II),(36–60: III) and (>60 months: IV). The fluorescence results of recording CD41 and CD62-positive platelets (A; α-granules) and simultaneously, CD41 and quinacrine (B; δ-granules) in the same platelets sample. Histograms show either the increase in P-selectin membrane exposition or quinacrine fluorescence remaining in the platelets in fold increase. *, ** and ***, represent P < 0.05, <0.01 and <0.01 compared with values found in resting platelets from healthy individuals (n = 6). While, $, $$, $$$, represent P < 0.05, <0.01 and <0.01 compared with Thr-stimulated values found in healthy individuals.

Mentions: Ca2+ homeostasis regulates several intracellular mechanisms in human platelets like actin cytoskeleton reorganization, shape change or granule secretion. Hence, using flow cytometry, we gated CD41+ cells (platelet positive staining), and fluorescence of anti-P-selectin (CD62P) antibody and quinacrine was monitored. Fluorescence protocols have been widely used to evaluate alpha (α-) and dense (δ-) granule secretion 39. As shown in Figure 2A, platelets present low levels of surface-exposed P-selectin under resting conditions (Fig. 2A; C: white bars representing resting platelets from healthy individuals), which is drastically enhanced upon α-granule secretion stimulated by Thr. Furthermore, we found that sirolimus-treated patients presented enhanced P-selecting membrane exposure under resting conditions, and subsequently, Thr-evoked P-selectin exposure was significantly lower (P < 0.001; n = 6); thus, the reduction in the fold increase observed between platelets from the group II of patients treated with sirolimus compared with control was of 0.18 ± 0.06 (Fig. 2A, right-hand side histogram; P < 0.05; n = 6). P-selecting exposition reached a 4.6 ± 0.1 fold increase (P < 0.001; n = 6) in Thr-stimulated platelets from healthy individuals. Hence, α-granule secretion was altered by sirolimus in a time-dependent manner (Fig. 2A, right-hand side histogram). Regarding everolimus patients, the most samples in resting conditions presented a very high elevated P-selectin exposure under resting condition, which makes subsequent evaluation of granule secretion difficult.


Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients.

López E, Berna-Erro A, Bermejo N, Brull JM, Martinez R, Garcia Pino G, Alvarado R, Salido GM, Rosado JA, Cubero JJ, Redondo PC - J. Cell. Mol. Med. (2013)

Granule secretion in patients medicated with sirolimus. Platelets isolated from kidney-transplanted patients treated with sirolimus were incubated in plasma rich platelets (PRP) for 30 min. at 37 °C with anti-CD41 and quinacrine (10 μM). Platelets were then left under resting condition or stimulated for 10 min. with Thr (0.1 U/ml), and simultaneously, anti-CD62 antibody (P-selectin; diluted 1:50) was added to medium. Fluorescence of CD41 was used for selecting platelet positive cells. Fluorescence was analysed using flow cytometry, from patients under sirolimus medication for different periods of time (<24: I), (24–36: II),(36–60: III) and (>60 months: IV). The fluorescence results of recording CD41 and CD62-positive platelets (A; α-granules) and simultaneously, CD41 and quinacrine (B; δ-granules) in the same platelets sample. Histograms show either the increase in P-selectin membrane exposition or quinacrine fluorescence remaining in the platelets in fold increase. *, ** and ***, represent P < 0.05, <0.01 and <0.01 compared with values found in resting platelets from healthy individuals (n = 6). While, $, $$, $$$, represent P < 0.05, <0.01 and <0.01 compared with Thr-stimulated values found in healthy individuals.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3822816&req=5

fig02: Granule secretion in patients medicated with sirolimus. Platelets isolated from kidney-transplanted patients treated with sirolimus were incubated in plasma rich platelets (PRP) for 30 min. at 37 °C with anti-CD41 and quinacrine (10 μM). Platelets were then left under resting condition or stimulated for 10 min. with Thr (0.1 U/ml), and simultaneously, anti-CD62 antibody (P-selectin; diluted 1:50) was added to medium. Fluorescence of CD41 was used for selecting platelet positive cells. Fluorescence was analysed using flow cytometry, from patients under sirolimus medication for different periods of time (<24: I), (24–36: II),(36–60: III) and (>60 months: IV). The fluorescence results of recording CD41 and CD62-positive platelets (A; α-granules) and simultaneously, CD41 and quinacrine (B; δ-granules) in the same platelets sample. Histograms show either the increase in P-selectin membrane exposition or quinacrine fluorescence remaining in the platelets in fold increase. *, ** and ***, represent P < 0.05, <0.01 and <0.01 compared with values found in resting platelets from healthy individuals (n = 6). While, $, $$, $$$, represent P < 0.05, <0.01 and <0.01 compared with Thr-stimulated values found in healthy individuals.
Mentions: Ca2+ homeostasis regulates several intracellular mechanisms in human platelets like actin cytoskeleton reorganization, shape change or granule secretion. Hence, using flow cytometry, we gated CD41+ cells (platelet positive staining), and fluorescence of anti-P-selectin (CD62P) antibody and quinacrine was monitored. Fluorescence protocols have been widely used to evaluate alpha (α-) and dense (δ-) granule secretion 39. As shown in Figure 2A, platelets present low levels of surface-exposed P-selectin under resting conditions (Fig. 2A; C: white bars representing resting platelets from healthy individuals), which is drastically enhanced upon α-granule secretion stimulated by Thr. Furthermore, we found that sirolimus-treated patients presented enhanced P-selecting membrane exposure under resting conditions, and subsequently, Thr-evoked P-selectin exposure was significantly lower (P < 0.001; n = 6); thus, the reduction in the fold increase observed between platelets from the group II of patients treated with sirolimus compared with control was of 0.18 ± 0.06 (Fig. 2A, right-hand side histogram; P < 0.05; n = 6). P-selecting exposition reached a 4.6 ± 0.1 fold increase (P < 0.001; n = 6) in Thr-stimulated platelets from healthy individuals. Hence, α-granule secretion was altered by sirolimus in a time-dependent manner (Fig. 2A, right-hand side histogram). Regarding everolimus patients, the most samples in resting conditions presented a very high elevated P-selectin exposure under resting condition, which makes subsequent evaluation of granule secretion difficult.

Bottom Line: Blood samples were drawn from healthy volunteers and kidney transplant patients long-term medicated with rapamycin: sirolimus and everolimus.Our results indicate that rapamycin evokes a biphasic time-dependent alteration in calcium homeostasis and function in platelets from kidney transplant patients under rapamycin regime, as demonstrated by the reduction in granule secretion observed and subsequent impairment of platelet aggregation in these patients compared with healthy volunteers.Platelet count was also reduced in these patients, thus 41% of patients presented thrombocytopenia.

View Article: PubMed Central - PubMed

Affiliation: Cell Physiology Research Group, Department of Physiology, University of Extremadura, Cáceres, Spain.

Show MeSH
Related in: MedlinePlus