Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w

Triggering ACKR3 and CXCR4 heterodimerization in platelets by targeting ACKR3

V. Dicenta-Baunach1, K. Kolb1, A.-K. Rohlfing1, M. Gawaz1

1Innere Medizin III, Kardiologie und Kreislauferkrankungen, Universitätsklinikum Tübingen, Tübingen;

Background: Besides their crucial role in hemostasis and thrombosis, platelets are critically involved in regulating inflammation in various disease states including for example acute myocardial and cerebral ischemia. Platelets have a variety of surface receptors which can be regulated by coagulation factors and mediators like chemokines controlling adhesion and activation. One prominent receptor is the G-protein coupled C-X-C chemokine receptor type 4 (CXCR4) promoting cell migration and autocrine platelet activation. The atypical chemokine receptor 3 (ACKR3, formerly known as CXCR7) functions as counterpart to CXCR4. Activation of ACKR3 promotes platelet survival, decreases platelet activation and attenuates thrombus formation. Since ACKR3 lacks a G-protein binding domain, it has been shown to mediate signaling via β-arrestin recruitment but also via direct phosphorylation by G protein-coupled receptor kinase 2 (GRK2). CXCR4 and ACKR3 have the potential to heterodimerize. Here we analyze the effect of receptor heterodimerization in platelets. Methods: We established the immunofluorescence proximity ligation assay Duolink® (Sigma Aldrich) for analyzing platelet ACKR3 and CXCR4 heterodimerization. Effects on platelet activation and degranulation were measured by analyzing the surface expression of activated Integrin α(IIb)β(3) and P-Selectin via flow cytometry. Platelet survival was analyzed by measuring the phosphatidylserine expression on platelets by Annexin V staining and mitochondrial membrane potential changes using TMRE (tetramethylrhodamine, ethyl ester). Furthermore, we investigated platelet aggregation using a light-transmission aggregometer and in vitro thrombus formation was observed via flow chamber experiments. Results: Herein, we used the proximity ligation assay to screen for CXCR4-ACKR3 heterodimerization modulating compounds. We found that activation of ACKR3 using a potent ACKR3 agonist significantly increases the formation of heterodimers. Interestingly, inhibition of direct ACKR3 phosphorylation by a GRK2 inhibitor also resulted in a significant increase in CXCR4-ACKR3 heterodimers. Co-incubation with both compounds enhanced heterodimer formation even further. Both, the ACKR3 agonist and the GRK2 inhibitor, revealed significant effects on platelet function. We found a decrease in platelet activation and degranulation, reduced platelet aggregation and attenuated thrombus formation in vitro. In addition, platelet survival was increased after pre-incubation with the agonist as well as the inhibitor. Conclusions: We were able to show that targeting ACKR3 using an ACKR3 agonist or inhibiting ACKR3-phophorylation triggers heterodimerization and increases platelet inhibition and therefore platelet survival. These findings indicate, that the formation of CXCR4-ACKR3-heterodimers is strongly dependent on ACKR3 and ACKR3 is able to reduce the platelet activating function of CXCR4 by heterodimerizing.

https://dgk.org/kongress_programme/jt2023/aP2229.html