Hemin-induced platelet activation is regulated via ACKR3 chemokine surface receptor

Zoi Laspa (Tübingen)1, V. Dicenta-Baunach (Tübingen)1, A.-K. Rohlfing (Tübingen)1, D. Schaale (Tübingen)1, M. Sigle (Tübingen)1, T. Harm (Tübingen)1, M. Gawaz (Tübingen)1

1Universitätsklinikum Tübingen Innere Medizin III, Kardiologie und Angiologie Tübingen, Deutschland


The hemolysis of red blood cells and muscle damage results in the release of the heme proteins hemoglobin, myoglobin, and free heme into the vasculature. Enhanced levels of hemin lead to platelet activation and intravascular thrombus formation. Conventional platelet antagonists such as acetylsalicylic acid or P2Y12 inhibitors do not significantly inhibit hemin-induced platelet activation. The purpose of our study was to further characterize the underlying mechanisms of hemin-dependent platelet activation and to disclose potential pathways to interfere with this process.

We found that hemin (6.25, 25 µM) induces platelet aggregation (light aggregometry) and ex vivo platelet-dependent thrombus formation on immobilized collagen under low shear rate (500 sec-1) indicating that free hemin is a strong activator for platelet-dependent thrombosis. In the presence of indomethacin (10 µM) and cangrelor (1 - 10 µM) neither aggregation nor thrombus formation was significantly attenuated. Recently, we described that the chemokine receptor ACKR3 (CXCR7) is a prominent inhibitory receptor of platelet activation. Thus, we asked whether ACKR3 activation modulates hemin-induced platelet activation. We found that in the presence of specific ACKR3 agonists (VUF11207, LN5651) but not in the presence of a biological inactive ACKR3 compound (C46) both hemin-dependent aggregation and thrombus formation was significantly inhibited (maximal aggregation response (%): 6.25 µM hemin versus 6.25 µM hemin + VUF11207, p = 0.0175 (Figure A), 6.25 µM hemin versus 6.25 µM hemin + LN5651, p = 0.0474 (Figure B); thrombus formation (area fraction): 6.25 µM hemin versus 6.25 µM hemin + VUF11207, p = 0.0113 (Figure C). Further, formation of platelet-derived microvesicles (flow cytometry) was significantly enhanced after treatment with hemin (6.25, 25 µM) which in turn was substantially reduced in the presence of an ACKR3 agonist (microvesicles (%): 6.25 µM hemin versus 6.25 µM hemin + VUF11207, p = 0.0045 (Figure D)). To further characterize the effect of hemin on platelet sub phenotypes we established an assay using multipanel flow cytometry (anti-CD62P, anti-CD63, anti-CD61, anti-CD41, anti-CD42b, anti-αIIbβ3 (clone: PAC-1), anti-CXCR4, anti-ACKR3, Annexin V, Zombie NIR) among other things, hemin-induced clusters were created from this (Figure E). We found that hemin preferentially induces procoagulant (Annexin V+) and ACKR3+ platelet subpopulations (Figure E). As noted for the platelet function assays, treatment of platelets with ACKR3 agonists significantly decreased the formation of procoagulant and ACKR3+ platelets in response to hemin.

We conclude that hemin is a strong activator for formation of procoagulant platelets and thrombus formation which is dependent on function of ACKR3. Activation of ACKR3 through specific agonists may offer a therapeutic strategy to interfere with uncontrolled thrombus formation in areas of tissue damage with enhanced release of heme-carrying proteins.

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