Debora Bertaggia Calderara

Background: Production of platelet concentrates (PCs) involves several steps that significantly affect platelet behavior. To gain a deeper understanding of how storage conditions impact donor platelet recirculation and functionality post‐transfusion, ex vivo platelet labeling is a feasible approach. However, before pursuing clinical investigations of platelet recirculation and function in humans, we aimed to determine the effects of pathogen inactivation technology (PIT) and storage conditions (4°C vs. room temperature [RT]) on phenotype and function of biotinylated platelets compared to conventional PIT PCs for transfusion.Methods: Nine PCs were prepared in 61% additive solution from 45 buffy coats (five buffy coats each). A pool‐and‐split of three units was used to prepare three equivalent PCs: two labeled with biotin and stored at RT or 4°C, and one without labeling and stored at RT. All PCs were then treated by PIT (amotosalen/UVA) and stored for 14 days. Labeling efficiency, platelet concentration, metabolic parameters, aggregation response (ADP, collagen, co‐aggregation with epinephrine), and platelet phenotype (CD42b, CD62‐P, phosphatidylserine) at the basal stage and upon stimulation (ADP or TRAP‐6) were performed.Results: Labeling efficiency of PIT and 4°C PCs was stable over 14 days of storage. Differences in platelet function and phenotype were mainly due to the storage temperature and not the biotinylation process. Phenotypes at baseline or after stimulation were equivalent in biotin‐positive and biotin‐negative platelets.Conclusion: Biotin‐labeled platelets can effectively enable investigation of the effects of PIT and storage temperature for clinical studies. This method shows great potential for improving platelet transfusion knowledge.

Introduction ADP-stimulated elevation of cytosolic Ca²⁺ is an important effector mechanism for platelet activation. The rapidly elevating cytosolic Ca²⁺ is also transported to mitochondrial matrix via Mitochondrial Ca²⁺ Uniporter (MCU) and extruded via Na⁺/Ca²⁺/Li⁺ Exchanger (NCLX). However, the exact contribution of MCU and NCLX in ADP-mediated platelet responses remains incompletely understood. Methods and results The present study aimed to elucidate the role of mitochondrial Ca²⁺ transport in ADP-stimulated platelet responses by inhibition of MCU and NCLX with mitoxantrone (MTX) and CGP37157 (CGP), respectively. As these inhibitory strategies are reported to cause distinct effects on matrix Ca²⁺ concentration, we hypothesized to observe opposite impact of MTX and CGP on ADP-induced platelet responses. Platelet aggregation profiling was performed by microplate-based spectrophotometery while p-selectin externalization and integrin αIIbβ3 activation were analyzed by fluorescent immunolabeling using flow cytometery. Our results confirmed the expression of both MCU and NCLX mRNAs with relatively low abundance of NCLX in human platelets. In line with our hypothesis, MTX caused a dose-dependent inhibition of ADP-induced platelet aggregation without displaying any cytotoxicity. Likewise, ADP-induced p-selectin externalization and integrin αIIbβ3 activation was also significantly attenuated in MTX-treated platelets. Concordantly, inhibition of NCLX with CGP yielded an accelerated ADP-stimulated platelet aggregation which was associated with an elevation of p-selectin surface expression and αIIbβ3 activation. Conclusion Together, these findings uncover a vital and hitherto poorly characterized role of mitochondrial Ca²⁺ transporters in ADP-induced platelet activation. Graphical Abstract

Background Emicizumab is a bispecific antibody mimicking coagulation factor VIII (FVIII) employed to treat patients with hemophilia A (PwHA) regardless of FVIII inhibitor status. The identification of biological markers reflecting the hemostatic competence of patients under emicizumab therapy would have a great clinical value. Unfortunately, emicizumab over-corrects standard coagulation assays, precluding their use for evaluating the hemostatic correction achieved in vivo. Here, we investigated whether global coagulation assays (GCA) would allow monitoring the biological response to non-factor replacement therapy with emicizumab. Material and Methods Six adults PwHA received a weekly dose of emicizumab of 3 mg/kg during weeks (W) 1 4 and 1.5 mg/kg from W5 onwards. Response to treatment was monitored weekly by emicizumab plasma concentration, thrombin generation (TG), and fibrin clot formation (FCF) and structure. TG and FCF results were compared to patient baseline, FVIII replacement, and healthy donors. Results TG and FCF significantly increased in PwHA after the loading period, reaching a plateau that lasted until the end of monitoring. Similarly, fibrin clot network became denser with thinner fibrin fibers. However, TG contrary to FCF remained at the lower limits of reference values. Remarkably, despite having similar plateau concentrations of emicizumab some patients showed markedly different degrees of TG and FCF improvement. Conclusion Our study enriches the knowledge on the use of GCA to monitor non-factor replacement therapy, indicating that TG and FCF could act as direct markers of emicizumab biological activity. GCA allow to capture and visualize the individually variable response to emicizumab, leading a step forward to the personalization of patient treatment. D.B.C. and R.M.C. contributed to study design, performed research, collected laboratory data and analyzed results, wrote and critically revised the manuscript; M.G.Z. and A.A. critically revised the manuscript; A.P.B.M.S. coordinated sample and patient data collections and revised critically the manuscript; F.J.G. performed routine laboratory analyses including emicizumab measurements; A.C. contributed to study design, supervised fibrin clot structure and fibrinolytic assays, and critically revised the manuscript; L.A. designed the study, supervised patient treatment, laboratory assays, data analysis, and manuscript writing and revision. ^* D.B.C and R.M.C. share first authorship. ^** A.C. and L.A. share senior authorship. Received: 20 October 2022 Accepted: 01 May 2023 Article published online: 19 June 2023 © 2023. Thieme. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany

Background: Storage of platelet concentrates (PCs) has an impact on platelet quality and possibly affects their functions after transfusion. The influence of processing and storage conditions of PCs on their in vivo function upon transfusion is unknown. One option for investigating this question is to implement an ex vivo labeling of human platelets, to analyze them after transfusion into heathy volunteers and/or patients. In this study, we developed two labeling methods employing biotin.Methods: Two methods of biotinylation were compared to a control (standard PC). The “Bio‐Wash” process used washing steps to label all platelets within the PC; for the other method, “Bio‐Direct,” one fifth of the PC were directly labeled without washing steps. The control and the two biotinylated PCs were analyzed over 7 days of storage. Labeling efficiency, platelet counts, phenotypes, and functions, along with time and costs, were evaluated to select the best process.Results: Both methods achieved a stable labeling through the storage, with similar platelet counts and metabolism in comparison to control PCs. Bio‐Wash showed higher activation phenotype and lower aggregation response in comparison to the Bio‐Direct method. The Bio‐Direct was performed within 1.5 h versus 3 h for the Bio‐Wash. However, the Bio‐Direct required 12 mg of biotin instead of 8 mg for the other process.Conclusion: We set up two methods of biotinylation that can be easily implemented in a blood bank environment. The Bio‐Direct process was preferred to the Bio‐Wash because of its similarity, from a functional and phenotypic point of view, with standard PCs.

Procoagulant platelets are a subtype of activated platelets that sustains thrombin generation in order to consolidate the clot and stop bleeding. This aspect of platelet activation is gaining more and more recognition and interest. In fact, next to aggregating platelets, procoagulant platelets are key regulators of thrombus formation. Imbalance of both subpopulations can lead to undesired thrombotic or bleeding events. COAT platelets derive from a common pro-aggregatory phenotype in cells capable of accumulating enough cytosolic calcium to trigger specific pathways that mediate the loss of their aggregating properties and the development of new adhesive and procoagulant characteristics. Complex cascades of signaling events are involved and this may explain why an inter-individual variability exists in procoagulant potential. Nowadays, we know the key agonists and mediators underlying the generation of a procoagulant platelet response. However, we still lack insight into the actual mechanisms controlling this dichotomous pattern (i.e., procoagulant versus aggregating phenotype). In this review, we describe the phenotypic characteristics of procoagulant COAT platelets, we detail the current knowledge on the mechanisms of the procoagulant response, and discuss possible drivers of this dichotomous diversification, in particular addressing the impact of the platelet environment during in vivo thrombus formation.