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. What do we know? Cryopreserved platelets are an alternative to overcome issues with the short shelf-life of room-temperature stored platelets After thawing, cryopreserved platelets exhibit changes in cell structure and receptor abundance Activated platelets can attach to leukocytes, forming platelet-leukocyte aggregates and altering their immune function Platelet-leukocyte aggregates can increase inflammation, which is associated with adverse events after transfusion, which can negatively affect patient outcomes What did we discover? Cryopreservation results in a heterogenous mix of platelet subpopulations Cryopreserved 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 platelets The increase in cryopreserved platelet-THP-1 cell aggregates was largely due to an increase in procoagulant platelets What is the impact? Our data demonstrate that cryopreservation increases platelet interaction with a monocyte-like cell line This may mediate immune responses and/or circulation time of transfused platelets