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

Abstract

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