OC 44.1 - The characteristics of platelet neutrophil aggregates formation and its potential role in neutrophil extracellular trap formation

Background: Neutrophil extracellular trap (NET) plays an important role in deep vein thrombosis. However, how NETs form in vivo was rarely known. In the previous study, we found elevated level of platelet-neutrophil aggregates (PNAs) in patients with DVT, which was consistent with the increased level of dsDNA, one of the important components of NET, compared with that in normal control group.

Aims: In the current study, we tried to investigate the characteristics of PNAs formation in DVT patients and its underlying correlation to NETs formation.

Methods: Platelets and neutrophils were separated from health volunteers. Arachidonic acid (AA) was added into platelets and neutrophils mix solution. The activation level of platelets, PNAs and Mac-1 expressed on the surface of neutrophils were detected by flow cytometry. mRNA level of Mac-1 was detected by real-time PCR. Immunofluorescence was applied to investigate the distribution of Mac-1 in neutrophils.

Results: The expression of PNAs increased with the concentration of AA and incubation time, which was consistent with the level of platelet activation. (Figure 1) Meanwhile, both the percentage of neutrophils expressing Mac-1 and the MFI increased, compared to that in the control group (Figure 2A). Furthermore, the addition of AA alone to neutrophils did not increase the expression of Mac-1, indicating that elevated expression of Mac-1 was related to the interaction between neutrophils and platelets (data not shown). The mRNA transcription level of Mac-1 in neutrophils did not significantly increase (p>0.05，Figure 2B). Immunofluorescence study showed that Mac-1 was uniformly distributed within neutrophils in a resting state and aggregated near the cell membrane after interaction with activated platelet (Figure 2C).

Conclusion(s): Activated platelets was the key to PNAs formation, leading to the translocation of Mac-1 from the intracellular to the membrane of neutrophils, and resulting in the increase expression of Mac-1 on the surface of neutrophils.