Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients.
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.
Affiliation: Cell Physiology Research Group, Department of Physiology, University of Extremadura, Cáceres, Spain.Show MeSH
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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.
Affiliation: Cell Physiology Research Group, Department of Physiology, University of Extremadura, Cáceres, Spain.