Background: Platelet activation status a is critical factor in thrombosis and thrombo-inflammation. A cornerstone of the treatment of acute organ ischemia is the inhibition of platelet activation which comes with an increased risk of bleeding for the patients. Modulation of the platelet chemokine receptors likely interferes with thrombosis and thrombo-inflammation and we hypothesized, that it can modulate ischemia/reperfusion (I/R) induced organ injury. Accordingly, we found previously that the chemokine receptor ACKR3 (formally CXCR7) inhibits platelet activation and aggregation and increases the acute and chronic I/R injury within the heart. Here, we want to analyze the effect of platelet-dependent ACKR3 depletion on the I/R injury in the brain.

Methods: We performed transient middle cerebral artery occlusion (tMCAO) in megakaryocyte/platelet-specific knock-out mice (PF4-Cre; Ackr3^-/-) and wild type siblings to assess the effect of genetic Ackr3 deficiency in platelets on brain injury. Tissue inflammation, injury and regeneration was characterized by gross pathology, histology, histochemistry and NanoString® analysis to access mRNA expression within the stroke area. 

Results: The acute ischemic injury by tMCAO was substantially enhanced after 24 h in Ackr3^−/− mice compared to Ackr3^fl/fl controls. This enhanced injury was also reflected in a decreased performance of Ackr3^−/− mice in a standard grip test compared to Ackr3^fl/fl controls. After 7 days the infract volume was still significantly enhanced in the Ackr3^−/− mice versus to control animals. Deficiency in platelet ACKR3 also results in altered cell migration into the brain following ischemia/reperfusion. Ly6G⁺ cells were significantly enhanced in the ischemic compared to non-ischemic areas of the brain. Similar to previously observed Ly6G⁺ cells migration into the ischemic myocardium.

Further, transcriptome analysis of 254 gene (NanoString®) revealed showed significantly upregulated key inflammatory mediators in ischemic brain tissue derived from Ackr3^−/− and control littermates 24 h after I/R. Nine of the tested 254 mRNAs were upregulated (CXCL5, CCL24, CHI3L3, IRF3, MMP9, PLA2G4A, RIPK1, NFATC3) in Ackr3^−/− mice compared littermate controls. Pathway enrichment analysis (KEGG pathways) of the upregulated genes included the protein translation-related categories metabolic pathways, chemokine signaling, toll-like receptor signaling, and apoptosis. Nine mRNAs were downregulated in Ackr3^−/− mice. The key regulators of the toll-like receptor/inflammasome pathway (RAC1, MAP2K4, PLCB1, GRB2, MAPK8, RAPGEF2, GRIB1) showed the strongest decrease. mRNAs from the MAPK and RAS pathway were also downregulated.

Conclusions: These findings indicate, that the loss of platelet-ACKR3 results in substantial cerebral injury and inflammation. Thereby, we demonstrate that the platelet chemokine receptor ACKR3 is a critical regulator of organ injury following ischemia/reperfusion in various tissues. Therefore, a permanent loss of platelet function by inhibition through anti-platelet therapy might have a contrary effect on organ regeneration. Consequently, the time-dependent positive and negative effects of platelet on ischemic tissues repair have to be explored in more detail.