Hemin-induced platelet activation is regulated by the ACKR3 chemokine surface receptor and has implications for passivation of vulnerable atherosclerotic plaques.

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 were used. Hemin induces platelet aggregation and ex vivo platelet-dependent thrombus formation on immobilized collagen under a low shear rate of 500 s^-1, indicating that free hemin is a strong activator of 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 (indometacin), 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 (CD42b^pos/PAC-1^neg/AnnexinV^pos), aggregatory (CD42b^pos/PAC-1^pos/AnnexinV^neg), and inflammatory (CD42b^pos/CXCR4^pos/ACKR3^pos/AnnexinV^pos) platelet subpopulations. Treatment with ACKR3 agonists significantly decreased the formation of procoagulant and ACKR3^pos 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 using specific agonists may offer a therapeutic strategy to regulate the vulnerability of atherosclerotic plaques in areas of IPH.