Activation of platelet chemokine receptor ACKR3 induces heterodimerization with CXCR4 and regulates platelet function

Valerie Dicenta-Baunach (Tübingen)1, A.-K. Rohlfing (Tübingen)1, M. Gawaz (Tübingen)1

1Universitätsklinikum Tübingen Innere Medizin III, Kardiologie und Angiologie Tübingen, Deutschland


Common antithrombotic drugs targeting platelet activation also affect the physiological properties of platelets, resulting in an enhanced risk of bleeding complications. Thus, understanding especially platelet inhibiting signaling pathways is vital to develop new therapeutic strategies. In this context, platelet atypical chemokine receptor 3 (ACKR3, formerly CXCR7) is an interesting target. Platelet-ACKR3 surface expression correlates with a more favorable outcome for all-cause mortality in CAD patients. Furthermore, ACKR3 activation leads to platelet inhibition and attenuates thrombus formation. The purpose of our study was to further understand the role of ACKR3 and its interaction partner CXCR4 for platelet inhibition functions and possible interactions with the platelet inhibitors cAMP and cGMP.

Recent studies showed, that in other cell types than platelets, CXCR4 and ACKR3 have the potential to heterodimerize which results in a change in the receptors functions. Thus, to investigate the role of CXCR4-ACKR3 heterodimerization for ACKR3 and platelet function, we used a microscopy proximity ligation assay (PLA, Duolink® Sigma Aldrich) to screen for ACKR3-CXCR4 heterodimerization inducing compounds in platelets. We found, that compared to an untreated resting control, ACKR3 specific agonists (100 µM VUF11207 and in-house agonist) promote ACKR3-CXCR4 heterodimerization, but not CXCL12, a mutual endogenous ligand of CXCR4 and ACKR3, and the CXCR4 specific ligand CXCL14 (PLA signal / platelet signal, mean±SD: 0.3655±0.3017 untreated vs. 1.029±0,6764 VUF11207, p= 0.0322 and untreated vs. 2.525±0.7687 in-house agonist, p= 0.0032). This indicates that the formation of ACKR3-CXCR4 heterodimers is an ACKR3 dependent process. Previously it was shown, that CXCL12 induces platelet activation via CXCR4 but not ACKR3. Thus, to further understand the function of ACKR3-CXCR4 dimers, we investigated the role of heterodimers for CXCL12 promoted platelet activation. Using a light transmission aggregometry assay we found, that CXCL12 induced platelet aggregation in a concentration dependent manner. Further, CXCL12 activated platelets indicated in an immunoblotting experiment by the phosphorylation of Akt. Both, platelet aggregation as well as Akt phosphorylation were attenuated in the presence of 100 µM ACKR3 agonists (max. aggregation %, mean±SD: 59.07±27,87 CXCL12 vs. 10.44±1,786 CXCL12 + VUF11207, p= 0.0433 and CXCL12 vs. 8.215±1.682 CXCL12 + in-house agonist, p= 0.0366; pAkt/total Akt signal, mean±SD: 1.399±0.4576 CXCL12 vs. 0.3969±0.2252 CXCL12 + VUF11207, p= 0,0060). To further disclose possible underlying signaling, we tested the ACKR3 agonists for VASP phosphorylation indicating a connection to either cAMP or cGMP. We found that VUF11207 induced VASP phosphorylation even in the presence of CXCL12 (pVASP/total VASP signal, mean±SD: 0.8560±0.2037 untreated vs. 1.252±0.2216 VUF11207, p= 0.0015 and 0.9300±0.1306 CXCL12 vs. 1.272±0.2279 VUF11207 + CXCL12, p= 0.0375).

We conclude that activation of the platelet receptor ACKR3 with specific agonists induces heterodimerization with the platelet activation and chemotaxis receptor CXCR4, which results in inhibition of especially CXCR4 dependent platelet activation. This may offer new antithrombotic strategies. ACKR3 agonist-dependent VASP phosphorylation suggests that platelet inhibition is cAMP or cGMP dependent.
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