Search

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. [ABSTRACT FROM AUTHOR]

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]

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]