Abstract/Summary

Background Platelets are increasingly recognised for their roles beyond thrombosis and haemostasis, notably in modulating inflammation through direct interactions with innate immune cells. Connexins, a family of transmembrane proteins, form hexameric hemichannels that dock between adjacent cells to create gap junctions, enabling direct intercellular communication. Several connexins, including Cx37, Cx40, and Cx62, are expressed in human platelets, while Cx37 and Cx40 are also found in blood neutrophils, where they mediate communication with endothelial cells. Given the critical role of platelet-neutrophil interactions in inflammation, connexins may facilitate intercellular signalling between these cells. Aim and Hypothesis This study aimed to determine whether connexin-mediated gap junction intercellular communication (GJIC) facilitates platelet-neutrophil signalling and modulates inflammatory processes. It was hypothesised that connexins (Cx37, Cx40, and Cx62) enable direct intercellular communication between platelets and neutrophils, supporting bidirectional calcium signalling and promoting aggregate formation. Results The expression of Cx37, Cx40, and Cx62 in human neutrophils was confirmed by western blotting. Platelet-neutrophil interactions were evaluated using flow cytometry and confocal microscopy. Stimulation with PMA (10 ng/mL), TRAP-6 (30 μM), or fMLP (1 μM) significantly enhanced these interactions. However, treatment with the non-selective gap junction inhibitor carbenoxolone (Cbx), as well as selective Gap27 peptides targeting Cx37, Cx40, and Cx62, significantly reduced platelet-neutrophil aggregate formation. To investigate calcium signalling, neutrophils were loaded with Fura-2 and co-incubated with washed platelets, then stimulated with CRP-XL or fMLP. CRP-XL-induced platelet activation III triggered intracellular calcium mobilisation in neutrophils, a response inhibited by Cbx and each Gap27 peptide. Notably, CRP-XL alone did not stimulate neutrophil calcium signalling in the absence of platelets. Similarly, fMLP-induced neutrophil activation triggered calcium mobilisation in platelets, which was likewise inhibited by Cx-specific inhibitors. However, fMLP alone did not activate platelets. To assess hemichannel function, calcein dye transfer was monitored. Upon PMA, TRAP-6, or fMLP stimulation, fluorescence transfer from donor neutrophils to platelets was observed, indicating functional gap junction formation. This dye transfer was abolished by Cbx and each connexin-specific inhibitor, confirming the role of connexins in mediating intercellular exchange. Conclusion This study provides strong evidence that connexin-mediated GJIC plays a pivotal role in platelet-neutrophil interactions by supporting bidirectional calcium signalling and the exchange of small molecules, such as calcein. Inhibition of Cx37, Cx40, and Cx62 significantly disrupted these processes, underscoring their importance in direct intercellular communication. These findings reveal connexins as key regulators of inflammatory platelet-leukocyte crosstalk and suggest their potential as therapeutic targets in thrombo-inflammatory conditions.