Cryopreservation significantly alters the phenotype of platelets; generating distinct subpopulations, which may influence the formation of platelet leukocyte aggregates (PLA). PLAs are immunomodulatory and have been associated with transfusion-associated adverse events. As such, the aim of this study was to examine the effect of cryopreservation on the ability of platelets to form PLAs, using a monocyte-like cell line (THP-1). Platelets were tested pre-freeze, post-thaw and following stimulation with TRAP-6 or A23187, both alone and following co-culture with THP-1 cells for 1 and 24 hours (n = 6). Platelet subpopulations and platelet-THP-1 cell aggregates were analyzed using multi-color imaging flow cytometry using Apotracker Green (ApoT), CD42b, CD62P, CD61, and CD45. Cryopreservation resulted in the generation of activated (ApoT-/CD42b+/CD62P+), procoagulant (ApoT+/CD42b+/CD62P+) and a novel (ApoT+/CD42b+/CD62P-) platelet subpopulation. Co-incubation of cryopreserved platelets with THP-1 cells increased PLA formation compared to pre-freeze but not TRAP-6 or A23187 stimulated platelets. P-selectin on the surface membrane was correlated with increased PLA formation. Our findings demonstrate that cryopreservation increases the interaction between platelets and THP-1 cells, largely due to an increase in procoagulant platelets. Further investigation is required to determine the immunological consequences of this interaction.
Keywords: Cryopreservation; THP-1; immunology; leukocytes; platelets.
What do we know? Cryopreserved platelets are an alternative to overcome issues with the short shelf-life of room-temperature stored plateletsAfter thawing, cryopreserved platelets exhibit changes in cell structure and receptor abundanceActivated platelets can attach to leukocytes, forming platelet-leukocyte aggregates and altering their immune functionPlatelet-leukocyte aggregates can increase inflammation, which is associated with adverse events after transfusion, which can negatively affect patient outcomesWhat did we discover? Cryopreservation results in a heterogenous mix of platelet subpopulationsCryopreserved platelets display increased adherence to a monocyte-like cell line (THP-1 cells). Platelet-THP-1 aggregate formation was linked to expression of CD62P on the surface of the plateletsThe increase in cryopreserved platelet-THP-1 cell aggregates was largely due to an increase in procoagulant plateletsWhat is the impact? Our data demonstrate that cryopreservation increases platelet interaction with a monocyte-like cell lineThis may mediate immune responses and/or circulation time of transfused platelets.