Background

Activated neutrophils and neutrophil extracellular traps (NETs) were observed to play pivotal roles in thromboinflammatory diseases. Increasing evidence suggests that platelets (PLTs) activated by neutrophils amplify different prothrombotic disorders. However, the contribution of different PLT subpopulations in activating and modulating neutrophil response remains elusive. In this study, we aim to characterize the impact of distinct PLT phenotypes on PLT-neutrophil interplay.

Methods

PLTs were stimulated with different agonists (TRAP-6, thrombin, convulxin and combinations) to induce different PLT phenotypes including activated P-selectin single pos. [CD62p⁺] and CD62p⁺/phosphatidylserine [PS]⁺ procoagulant PLTs, and incubated with isolated neutrophils. Afterwards, samples were labelled with anti-CD41-APC and anti-CD16-PE for flow cytometric detection of PLT-neutrophil aggregates (PNAs). Using an immunofluorescence microscopy-based assay, the impact of different PLT phenotypes on NET formation was investigated. Briefly, neutrophils were allowed to attach on fibrinogen and incubated with PLTs which was followed by staining with Sytox green and anti-CD41-APC for the detection of extracellular DNA and PLTs, respectively.

Results

Treatment of PLTs with TRAP-6 and thrombin resulted in the formation of activated CD62p⁺ (mean percentage [%]±SEM: 79.0±11.3 vs. 80.4±10.7, respectively) while procoagulant [CD62p⁺/PS⁺] PLTs were only induced by convulxin, and dual TRAP-6/convulxin or thrombin/convulxin stimulation: 41.4±9.5 vs. 44.4%±8.6; and vs. 56.3±12.9, respectively. Interestingly, PNA formation seems to show agonist dependency as TRAP-6 activated PLTs induced only a low amount of PNA. Contrary, thrombin activated and procoagulant PLTs induced a significant increase in PNA formation in vitro. A finding that was further confirmed as higher amounts of extracellular DNA were induced by thrombin activated and procoagulant PLTs compared to TRAP-6 and buffer treated PLTs.

Conclusion

Our findings indicate that different PLT subpopulations harbor the potential to induce increased PLT-neutrophil interplay with subsequent formation of PNAs as well as NETs. Further dissection of the precise mechanisms in PLT-neutrophil crosstalk and identification of potential pharmaceutical targets could help to decipher the role of different PLT surface proteins and their relevance in PLT-neutrophil interactions.

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