Background: Platelets are identified to be the primary cells regulating hemostasis and thrombosis. Therefore, they play a critical role in many cardiovascular disease and antiplatelet therapy is seen as a cornerstone in treatment of patients with acute organ ischemia. However common antiplatelet drugs lead to a higher risk of bleeding which remains the most frequent complication of antiplatelet therapy. To reduce the risk of complications for patients, identification of pharmaceutical targets which do not increase bleeding is crucial. One of these potential targets is the atypical chemokine receptor 3 (ACKR3), a non classical seven transmembrane-spanning Receptor. This receptor has been postulated to regulate platelet function and thrombus formation. We were able to show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo.

Methods: We developed a number of potential agonists via in silico screening with a 3D model of ACKR3 which is based on protein structures of similar crystallized receptors. After repeatedly testing compounds and reevaluating the biological data, which were constantly used to refine the 3D model of ACKR3, we chose lead compounds to be further investigated.

Platelet survival was measured by flow cytometric analysis of phosphatidylserine expression on platelets by Annexin V staining and mitochondrial membrane potential changes using tetramethylrhodamine, ethyl ester. Apoptosis of platelets was induced with CRP or ABT-737. Platelet activation was measured by analysis of P-Selectin and Integrin alpha(IIb)beta(3) expression. In vitro thrombus formation was observed in flow chamber experiments with agonist pretreated whole blood on immobilized collagen. Binding of agonists to the receptor was measured by ß-arrestin Pathhunter^R assay (eurofins DiscoverX). Cell viability was monitored using RealTime-Glo^TM cell viability assay (Promega). To test the specificity of the ACKR3 agonists, we made use of a knock-out mouse strain with megakaryocyte/platelet-specific genetic deletion of ACKR3 and tested for effects on platelet activation and platelet-mediated thrombus formation.

Results: Herein we could show that activation of ACKR3 by our new lead agonists results in increased platelet activation by approximately 70%. In vitro thrombus formation is reduced by nearly 50%. Number of apoptotic platelets was reduced by 60% when pretreated with ACKR3 agonists.

After several rounds of screening, we were able to improve the specific ACKR3 binding affinity of our compounds from the first lead structures 257 µM to 99.6 µM in the second round and finally to an EC50 of 5.9 µM, which means we reached an approximately 40 times higher binding efficacy compared to the first lead structure. The tested compounds also do not show increased toxicity compared to a commercially available ACKR3 agonist (VUF11207, Calbiochem).

Platelets or blood, respectively, obtained from Ackr3^-/- mice did not show a reduction of CRP-induced P-selectin expression nor reduced thrombus formation compared to wildtype littermates.

Conclusion: The present work shows, that in silico developed ACKR3 agonists with their improved metabolic stability and binding affinity represent promising lead compounds for development of functional therapeutics which may modulate thrombosis without enhancing hemostasis and thus bleeding.