Apoptosis Inhibition: A Promising Approach for Cold Storage of Apheresis Platelet Concentrates

Introduction Transfusion of platelet concentrates is routinely used to treat or prevent bleeding. Bacterial infection caused by storage at room temperature (RT) still remains a major drawback of this established therapeutic approach. Recently, we showed that cold-stored APCs are associated with better PLT functionality, such as adhesion and aggregation ability, but also with shorter survival in vivo (Haematologica 2018, PMID: 30115655). Cold-induced apoptosis was identified as a potential mechanism of the accelerated PLT clearance. In this study, we investigated the protective effect of apoptotic inhibitors during cold storage of APCs.

Methods Apheresis-derived platelet concentrates (APCs) were collected and stored at RT and 4°C in the presence or absence of a caspase-3 inhibitor. PLT apoptosis was assessed measuring the phosphatidylserine exposure and the mitochondrial membrane potential (MMP) using flow cytometry. The protein expression was quantified by Western blot.

Results Higher expression of the apoptotic marker phosphatidylserine was detected in cold-stored APCs (C-APCs) compared to RT ones (RT-APCs) (% apoptotic events mean ± SEM: 13 ± 1% vs. 5 ± 1%, C-APCs vs. RT-APCs, respectively, p = 0.018). In order to investigate if the apoptosis specifically involved the intrinsic pathway, the MMP was analyzed as a marker of live cells. Interestingly, after cold storage, a decrease in the MMP was observed in comparison to RT indicating the activation of the intrinsic pathway (mean fluorescence intensity TMRE mean ± SEM: 6.13 ± 1.89 vs. 18.53 ± 3.64, 4°C vs. RT-APCs, respectively, p = 0.038). In accordance, a decrease of the procaspase-3 protein level after cold storage was detected by Western blot. However, when PLTs were stored in the presence of a caspase-3 inhibitor, a significant rescue of the cold-stored cells viability was observed (TMRE staining: % alive cells mean ± SEM: 74 ± 3% vs. 22 ± 3%, caspase 3 inhibitor vs. ionomycin, p = 0.005). This indicates that the cold-induced apoptosis can be prevented in the presence of caspase inhibitor.

Conclusion Our results show that the decrease of cold-stored PLT viability can be prevented by a specific caspase inhibitor. Consequently, cold storage, associated with a better PLT functionality, may become an efficient strategy for APC storage in combination with apoptotic inhibitors.

Publication History

Article published online:
13 November 2020