Platelet-derived chemokines regulating neutrophil activation stages in arterial thrombus formation

Objective Platelets and neutrophils are among the first cells to appear at the site of thrombus formation and contribute to the pathology of thrombotic events. Platelet-neutrophil interactions are mediated through multiple receptor-ligand interactions and chemokines. However, it remains unknown how these interactions arise and are regulated at the site of a growing thrombus. We aimed to unravel the underlying mechanisms in the setting of arterial thrombus formation.

Material and Methods In vitro thrombus formation was induced by perfusion of human whole blood over collagen at arterial shear in a parallel plate flow chamber. Neutrophil adhesion, movement, Ca^2+-fluxes and surface activation markers were assessed in real-time by fluorescence microscopy, as well as the activation status of the platelets in the thrombi (PS exposure, CD62P and CD63).

Results Platelets in consolidated thrombi attract neutrophils depending on shear conditions. Neutrophil adhesion was mediated through a panel of receptor interactions. Following adhesion, the neutrophils stained positively for activation markers including CD11b and produced reactive oxygen species. Markedly, even after prolonged time, thrombi-interacting neutrophils did not form neutrophil extracellular traps (NETs), despite their activation state. About 65 % of thrombus-interacting neutrophils displayed repetitive intracellular Ca^2+ spiking, which was partly related to their movement at and around thrombi. Suppressing platelet activation by post-treatment of the thrombus with the prostacyclin analogue iloprost severely reduced the Ca^2+ spikes in neutrophils, suggesting a continuous release of neutrophil-activating substances by the platelets. Inhibition of the platelet-derived chemokines CXCL7 (NAP-2), CXCL4 (PF4) or CCL5 (RANTES) resulted in decreased Ca^2+-fluxes in the neutrophils without affecting their adhesive behaviour. In addition, stimulation of isolated neutrophils with CXCL7, CXCL4 and/or CCL5 induced specific patterns of Ca^2+-fluxes. On the other hand, the agonist thrombin only caused Ca^2+ responses in neutrophils in the presence of platelets.

Conclusion Neutrophil-activating effects of platelets in a thrombus are mediated by the continuous release of at least three chemokines. Together, our results suggest a central role of G protein-coupled chemokine receptors in regulating neutrophil activation and Ca^2+ signaling during arterial thrombus formation.

Publication History

Article published online:
18 June 2021