Stroke-related thrombo-inflammatory processes are amplified by excessive DKK1 production and release

Background and purpose Incomplete microvascular reperfusion remains a major challenge in current recanalization strategies after ischemic stroke. Despite arterial reopening, persistent microvascular obstruction promotes secondary brain injury and worsens neurological outcomes. Thrombo-inflammation, characterized by endothelial cell activation, platelet-neutrophil interactions, and neutrophil extracellular trap (NETs) formation represents a key driver of secondary injury progression. Elevated levels of Dickkopf-1 (DKK1) in blood circulation are associated with poor clinical outcomes in stroke patients. It remains to be elucidated whether DKK1 elevated circulating levels affect stroke-induced thrombo-inflammation. Methods We used transgenic male mice that allow conditional induction of DKK1 expression, which were subjected to experimental ischemic stroke via the transient middle cerebral artery occlusion (MCAo). We evaluated the thrombo-inflammatory profile at baseline, as well as at 6 and 24 h after stroke, with additional experimental groups receiving recombinant tissue plasminogen activator (rtPA) at 3 h post-stroke. Circulating thrombo-inflammatory markers and adhesion molecules were assessed using multiplex cytokine array. Brain microvascular integrity was analyzed using ​immunofluorescence and proteomic analyses. Flow cytometry was used to quantify neutrophils, platelets and neutrophil-platelet aggregates, while NETs​-related events were evaluated using immunofluorescence analysis. Finally, platelet response was assessed after ex vivo stimulation with brain homogenates harvested from wild-type mice 6 h after MCAo. Results Elevated DKK1 levels induced a rapid pro-inflammatory and pro-coagulant state, associated with increased adhesion molecule release, neutrophil accumulation, and coagulation dysregulation. Proteomic analysis revealed altered expression of proteins involved in cytoskeletal organization and inflammation, indicating impaired microvascular integrity. Consistently, DKK1 induction enhanced NETosis-associated processes and platelet activation in the injured brain. Notably, rtPA failed to improve reperfusion in presence of DKK1, outlining the establishment of a pro-thrombotic microenvironment within the microvasculature. Conclusion Our findings suggest that DKK1 elevated levels act as a pathological mediator driving microvascular alterations, inflammation, and platelet activation in the acute phase of ischemic stroke. Therefore, individuals presenting elevated levels of DKK1 in circulation may benefit from strategies aiming to neutralize its biological activity to attenuate thrombo-inflammation, thereby improving structural and functional outcomes.