PL2-SN-08 - Platelet Cold Storage Alters Platelet Extracellular Vesicle Phenotype, Inducing Pro-Inflammatory Responses in Monocytes

Background/Case Studies: The Food and Drug Administration issued guidance allowing use of cold-stored platelets (CSP) for up to 14 days in bleeding patients when room temperature platelets are unavailable or their use is not practical. However, the impact of extended cold storage (up to 21 days) on extracellular vesicle (EV) composition and their immunomodulatory activity remains unclear. This study objective was to determine how extended CSP storage alters EV phenotype and EV cytokine content and whether this impacts monocyte inflammatory responses in vitro.

Study

Design/Methods: Apheresis platelets (Trima Accel collection, stored in 100% plasma, N=9 healthy donors) were stored at 22°C with agitation and collected on day 7, or at 4°C without agitation and collected on day 0 (baseline), 7, 14, and 21. EVs were isolated using differential ultracentrifugation. EV concentration and size were analyzed by nanoparticle tracking analysis. Phenotypic characterization was performed using a spectral flow cytometer, including 10 markers related to platelets and tetraspanin proteins. Nine cytokines were measured in EV pellets and platelet-poor plasma (PPP) using a Luminex immunoassay. Monocyte activation was assessed by incubating EVs with PBMCs from a healthy donor for 6 hours, followed by intracellular cytokine staining for TNF-α, IL-1β, IL-6, MCP-1, and analysis by spectral flow cytometry. Statistical analysis was performed using the Kruskal-Wallis test and the Friedman test with Dunn’s post hoc comparisons (p< 0.05).

Results/Findings: Total EV concentration significantly increased in CSPs by days 14 and 21 compared to baseline. Cold storage significantly increased levels of EVs expressing CD41, CD42b, CD62P, and CD63 over time. On day 7, platelets stored at 22°C showed a trend toward higher CD62P+ and CD63+ EVs compared to those stored at 4°C (Fig. A-A). Levels of IL-1β, TNF-α, IL-6, IL-8, IFN-γ, VEGF, sCD40, and sCD62P were statistically significantly increased in EVs and PPP from CSPs on days 14 and 21 compared to baseline (Fig. A-B). These markers were also elevated under 22°C compared to 4°C conditions on day 7 (Fig. A-B). Monocytes exposed to CSP-derived EVs showed significantly higher TNF-α and IL-6 on days 7, 14, and 21 vs. baseline and unstimulated cells. On day 7, EVs from 22°C-stored platelets induced more TNF-α and IL-6 than CSP-derived EVs. IL-1β was significantly elevated in response to day 21 CSP-EVs.

Conclusions: EVs derived from CSPs with extended storage exhibit phenotypic alterations and carry elevated levels of inflammatory mediators. Furthermore, EVs derived from CSPs possess immunomodulatory properties that enhance monocyte activation pro-inflammatory cytokine release. Further research is warranted to explore their effects on recipient immune responses.