PB0772 - Platelet-Leukocyte Aggregates as Markers of (Pro)Thrombotic Inflammation

Background: Platelet-leukocyte aggregates are formed in the blood of patients with inflammatory diseases as a result of pathological activation of blood cells. They reflect relationships between hemostasis and immunity, but the mechanisms of the interactions between platelets and leukocytes and their pathophysiological significance are not fully understood.

Aims: The aim of this work was to study the conditions of formation and morphological characterization of platelet-leukocyte aggregates in the blood in vitro.

Methods: Flow cytometry was used to identify and quantify platelet-leukocyte aggregates in whole human blood. Gating of different cell types and cellular aggregates was performed using fluorescently labeled antibodies against specific surface antigens, namely CD45 (all leukocytes), CD16 (neutrophils), CD14 (monocytes) and CD41 (platelets). Phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), thrombin receptor-activating peptide (TRAP), and adenosine diphosphate (ADP) were used as cellular activators. For confocal fluorescent microscopy, cells were labeled with DiI and DAPI.

Results: After adding PMA to citrated blood, about 36% of neutrophils and 67% of monocytes were found within platelet-neutrophil and platelet-monocyte aggregates, respectively. ADP and TRAP had similar effects and caused incorporation of ~30% of neutrophils and 58-76% of monocytes into aggregates with platelets. Unlike other stimulants studied, LPS did not induce platelet co-aggregation with either neutrophils or monocytes, likely as a relatively weak platelet stimulant. Microscopy of the platelet-leukocyte aggregates showed that the prevailing and roughly equal platelet/neutrophil and platelet/monocyte ratios were 1:1 and 2:1 for both types of heterotypic aggregates.

Conclusion(s): Activation of blood cells with PMA, ADP, or TRAP promotes the formation of platelet-neutrophil and platelet-monocyte aggregates, whereas LPS does not cause platelet activation sufficient for expression of adhesive molecules needed for the heterotypic cellular aggregation. Therefore, platelet-leukocyte aggregates are induced by some, but not all, inflammatory mediators and metabolites and can be used as markers of (pro)thrombotic conditions associated with systemic inflammation.

Funding: the "Priority-2030" Program at KFU.