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Background: Platelet receptors ACKR3 and CXCR4 play a crucial role in a variety of cardiovascular 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 signalling. 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 Results: Using a proximity ligation assay (PLA, Duolink®) 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 signalling 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., Conclusion: Our 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 signalling. Our results indicate possible ACKR3 agonist functions and reinforce the potential therapeutic applications of ACKR3 agonists., (© 2024 The Author(s). European Journal of Clinical Investigation published by John Wiley & Sons Ltd on behalf of Stichting European Society for Clinical Investigation Journal Foundation.)

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., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Hemolysis is a known risk factor for thrombosis resulting in critical limb ischemia and microcirculatory disturbance and organ failure. Intravasal hemolysis may lead to life-threatening complications due to uncontrolled thrombo-inflammation. Until now, conventional antithrombotic therapies failed to control development and progression of these thrombotic events. Thus, the pathophysiology of these thrombotic events needs to be investigated to unravel underlying pathways and thereby identify targets for novel treatment strategies. Here we used classical experimental set-ups as well as high-end flow cytometry, metabolomics and lipidomic analysis to in-depth analyze the effects of hemin on platelet physiology and morphology. Hemin does strongly and swiftly induce platelet activation and this process is modulated by the sGC-cGMP-cGKI signaling axis. cGMP modulation also reduced the pro-aggregatory potential of plasma derived from patients with hemolysis. Furthermore, hemin-induced platelet death evokes distinct platelet subpopulations. Typical cell death markers, such as ROS, were induced by hemin-stimulation and the platelet lipidome was specifically altered by high hemin concentration. Specifically, arachidonic acid derivates, such as PGE 2 , TXB 2 or 12-HHT, were significantly increased. Balancing the cGMP levels by modulation of the sGC-cGMP-cGKI axis diminished the ferroptotic effect of hemin. We found that cGMP modulates hemin-induced platelet activation and thrombus formation in vitro and cGMP effects hemin-mediated platelet death and changes in the platelet lipidome. Thus, it is tempting to speculate that modulating platelet cGMP levels may be a novel strategy to control thrombosis and critical limb ischemia in patients with hemolytic crisis. Graphical presentation of the signaling pathways effected by hemin (red arrows) and modulated by NO/riociguat (blue arrows) and their involvement on platelet activation, degranulation and aggregation as well as hemin-mediated cell death. [Display omitted] • cGMP modulates hemin-induced platelet activation and thrombus formation in vitro. • Hemin induces platelet death. • Free hemin causes changes in the platelet lipidome. • Hemin-induced platelet death and lipidome changes are inhibited by cGMP modulation. [ABSTRACT FROM AUTHOR]