Autor:innen:
I. Marini (Tübingen, DE)
Y. Tamamushi (Tübingen, DE)
L. Pelzl (Tübingen, DE)
S. Nowak-Harnau (Tübingen, DE)
T. Bakchoul (Tübingen, DE)
Introduction:
Apheresis-derived platelet concentrates (APCs) is an essential medical therapy use to treat bleeding. Nevertheless, the storage at room temperature increases the risk of bacterial-infection post transfusion. Recently, we reported that cold-stored APCs show better platelet functionality but decreased platelet half-life caused by apoptosis activation (Marini et al., Haematologica 2019). In this study, we investigated the impact of an apoptosis inhibitor on platelet functionality and half-life during cold storage.
Methods: APCs were collected from healthy donors and stored for 7 and 10 days at 4°C in the presence or in the absence of an apoptosis inhibitor (G04). Platelet apoptosis was assessed measuring the phosphatidylserine (PS) exposure and the mitochondrial membrane potential (MMP) by flow cytometry. Platelet functionality was investigated analyzing the expression of CD62, CD63 and PAC1 upon TRAP stimulation, by flow cytometry. Platelet aggregation ability, after TRAP and ristocetin incubation, was tested using an aggregometer. Next, thrombin generation was measured using a thrombogram. While, the adhesion ability was determined performing an in vitro assay. Platelet survival was analyzed using a NSG mouse model.
Results:
Upon incubation with the apoptosis inhibitor a significant reduction of PS externalization was detected after 10 days of storage (p=0.002). Accordingly, significant higher levels of MMP were observed after G04 incubation (p=0.034). Interestingly, the inhibitor did not affect the platelet functionality. In fact, CD62 expression was comparable to untreated cells (p=0.086). While, the responsiveness of CD63 and PAC1 was better conserved in platelets stored with the inhibitor (day 10, CD63 p=0.035 and PAC1, p=0.005). Furthermore, on day 10 no differences in the platelet aggregation ability were detected with or without inhibitor (TRAP, p=0.591 and Ristocetin p=0.998) as well as comparable thrombin formation (p=0.602). Interestingly, a significant increase of the number of adherent cells was detected after platelets were treated with G04 (day 7, p= 0.0465). More importantly, a higher percentage of circulating human cold-stored platelets was detected in the mouse bloodstream upon incubation with G04 after 7 days of cold storage compared to untreated cells (5h post injection, p=0.046).
Conclusion: Our results show that the cold-induced platelet apoptosis, responsible for the faster destruction of cold-stored platelets in vivo, can be efficiently prevented using an apoptosis inhibitor. Furthermore, the inhibitor did not impair platelet functions like activation, aggregation, thrombin formation and adhesion. More importantly, the inhibitor better maintains cell survival in vivo. Therefore, incubation of APCs with apoptosis inhibitor(s) during cold storage, might be a promising strategy to prolong the storage time without impairing platelet’s functionality and improving cell half-life.