Bayrak, A. | bioRxiv Science

bioRxiv2024

Hemin-induced platelet activation is regulated via ACKR3 chemokine surface receptor - implications for passivation of vulnerable atherosclerotic plaque

In vulnerable atherosclerotic plaques intraplaque hemorrhages (IPH) result in hemolysis of red blood cells and release of hemoglobin and free hemin. Hemin activates platelets and leads to thrombosis. Agonism of the inhibitory platelet receptor ACKR3 inhibits hemin-dependent platelet activation and thrombus formation. To characterize the effect of hemin and ACKR3 agonism on isolated human platelets, multi-color flow cytometry and classical experimental setup such as light transmission aggregometry and a flow chamber assay have been used. Hemin induces platelet aggregation and ex vivo platelet-dependent thrombus formation on immobilized collagen under low shear rate 500 s-1 indicating that free hemin is a strong activator for platelet-dependent thrombosis. Recently, we described that ACKR3 is a prominent inhibitory receptor of platelet activation. Specific ACKR3 agonists but not conventional antiplatelet compounds such as COX-1 inhibitor (indomethacin), ADP-receptor blocker (cangrelor), or PAR1 inhibitor (ML161) inhibit both hemin-dependent aggregation and thrombus formation. To further characterize the effect of hemin on platelet subpopulations we established a multi-color flow cytometry assay. We found that hemin induces procoagulant (CD42bpos/ PAC-1neg/ AnnexinVpos), aggregatory (CD42bpos / PAC-1pos / AnnexinVneg) and inflammatory (CD42bpos/ CXCR4pos/ACKR3pos/ AnnexinVpos) platelet subpopulations. Treatment with ACKR3 agonists significantly decrease the formation of procoagulant and ACKR3pos platelets in response to hemin. We conclude that hemin is a strong activator for the formation of procoagulant platelets and thrombus formation which is dependent on the function of ACKR3. Activation of ACKR3 through specific agonists may offer a therapeutic strategy to control vulnerability of atherosclerotic plaques in areas of IPH. Graphical abstractIntraplaque hemorrhages (IPH) results in hemolysis and liberation of iron containing heme and its oxidized metabolite hemin. Hemin activates platelets and has strong pro-thrombotic activity. Agonism of the atypical chemokine receptor 3 (ACKR3) inhibits hemin-induced platelet activation. O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/593847v2_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@1167f94org.highwire.dtl.DTLVardef@11ff58org.highwire.dtl.DTLVardef@192f098org.highwire.dtl.DTLVardef@1f1c568_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

ACKR3 agonism induces heterodimerization with chemokine receptor CXCR4 and attenuates platelet function

BackgroundPlatelet receptors ACKR3 and CXCR4 play a crucial role in a variety of cardio-vascular diseases. Like most chemokine receptors, CXCR4 is a G protein coupled receptor that induces platelet activation. In contrast, the atypical chemokine receptor 3 (ACKR3) lacks the ability to activate heterotrimeric G proteins and its activation leads to platelet inhibition and attenuates thrombus formation. In nucleated cells, heterodimerization of ACKR3 with CXCR4 regulates CXCL12-dependent signaling. The aim of our study was to investigate the formation of ACKR3/CXCR4 heterodimers in platelets and the subsequent consequences for platelet function. Methods and resultsUsing a microscopy proximity ligation assay (PLA, Duolink(R)) to screen for CXCR4/ACKR3 heterodimerization inducing compounds, we found that ACKR3 agonism but not conventional platelet agonists or endogen ligands lead to heterodimer formation. To further characterize the formation of ACKR3/CXCR4 heterodimers, we studied the CXCL12-dependent platelet activation via CXCR4. Both, CXCL12-dependent platelet aggregation and collagen-dependent ex vivo thrombus formation were significantly downregulated by ACKR3 agonism. Moreover, platelet intracellular calcium and Akt signaling were increased by CXCL12 and again suppressed by ACKR3-specific agonists. Previously, CXCL12 was shown to decrease platelet cAMP levels via CXCR4. Treatment with a specific ACKR3 agonist counteracted this CXCL12/CXCR4-dependent cAMP decrease. ConclusionOur results reveal that the formation of platelet ACKR3/CXCR4 heterodimers is dependent on ACKR3 rather than CXCR4. Furthermore, ACKR3 agonism induced heterodimerization is associated with mitigating CXCL12/CXCR4-dependent platelet activation possibly by modulating CXCR4-dependent G protein signaling. Our results indicate possible ACKR3 agonist functions and reinforce the potential therapeutic applications of ACKR3 agonists. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/593491v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@15a8b22org.highwire.dtl.DTLVardef@1647a63org.highwire.dtl.DTLVardef@19f36d1org.highwire.dtl.DTLVardef@1c80776_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO CXCR4/ACKR3 heterodimerization in platelets. C_FIG

physiology↗