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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 (CD42bpos/PAC‐1neg/AnnexinVpos), aggregatory (CD42bpos/PAC‐1pos/AnnexinVneg), and inflammatory (CD42bpos/CXCR4pos/ACKR3pos/AnnexinVpos) platelet subpopulations. Treatment with ACKR3 agonists significantly decreased 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 using specific agonists may offer a therapeutic strategy to regulate the vulnerability of atherosclerotic plaques in areas of IPH. [ABSTRACT FROM AUTHOR]

Aims Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. Methods and results Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager −/− mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager −/− mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager −/− mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. Conclusion We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA–RAGE interaction thus represents a novel mechanism in thrombo-inflammation. [ABSTRACT FROM AUTHOR]

Platelet activation plays a critical role in thrombosis and hemostasis. Several pathophysiological situations lead to hemolysis, resulting in the liberation of free ferric iron‐containing hemin. Hemin has been shown to activate platelets and induce thrombo‐inflammation. Classical antiplatelet therapy failed to prevent hemin‐induced platelet activation. Thus, the aim of the present study was to characterize the mechanism of hemin‐induced platelet death (ferroptosis). We evaluated the in vitro effect of hemin on platelet activation, signaling, oxylipins, and plasma membrane destruction using light transmission aggregometry, ex vivo thrombus formation, multiparametric flow cytometry, micro‐UHPLC mass spectrometry for oxylipin profiling, and scanning ion conductance microscopy (SICM). We found that hemin induces platelet cell death indicated by increased ROS levels, phosphatidyl serine (PS) exposure, and loss of mitochondrial membrane potential (ΔΨm). Further, hemin causes lipid peroxidation and generation of distinct oxylipins, which strongly affects plasma membrane integrity leading to generation of platelet‐derived microvesicles. Interestingly, hemin‐dependent platelet death (ferroptosis) is specifically regulated by the subtilisin‐like proprotein convertase furin. In summary, platelet undergo a non‐apoptotic cell death mediated by furin. Inhibition of furin may offer a therapeutic strategy to control hemin‐induced thrombosis and thrombo‐inflammation at a site of hemolysis. [ABSTRACT FROM AUTHOR]