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Thromb Haemost 2023; 123(07): 679-691
DOI: 10.1055/s-0043-1768057
Cellular Haemostasis and Platelets

The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI

Annalena Fink   *
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Anne-Katrin Rohlfing   *
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Valerie Dicenta
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
David Schaale
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Marcel Kremser
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Zoi Laspa
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Manuel Sigle
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Xiaoqing Fu
2   Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, University of Tübingen, Tübingen, Germany
,
Andreas Pelzer
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Melina Fischer
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Patrick Münzer
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Tatsiana Castor
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Karin Anne Lydia Müller
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Oliver Borst
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
,
Michael Lämmerhofer
2   Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, University of Tübingen, Tübingen, Germany
,
Meinrad Paul Gawaz
1   Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
› Author Affiliations Funding This investigation was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project number 374031971—TR 240 grant to Meinrad P. Gawaz and, funded by the German Research Foundation (DFG) Project number 335549539-GRK2381.
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Abstract

Introduction Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis.

Methods We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin.

Results We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions.

Conclusion In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

Keywords

hemolysis - hemin - platelets - GPVI - shedding

* A.F. and A.-K.R. are co-first authors.


Authors' Contribution

M.P.G. and A.-K.R. designed and planned the experiments. A.F., M.K., V.D., Z.L., D.S., A.P., X.F. M.F., P.M., and A.-K.R. performed experiments. All the authors participated in the interpretation and discussion of the results. A.F., M.L., O.B., and A.-K.R. analyzed the data including statistical analysis. A.F. and A.-K.R. designed the figures. A.-K.R., A.F., M.P.G., M.K., M.S., T.C., V.D., Z.L., and K.A.L.M. wrote and proof read the article.


Supplementary Material



Publication History

Received: 01 September 2022

Accepted: 04 March 2023

Article published online:
10 April 2023

© 2023. Thieme. All rights reserved.

Georg Thieme Verlag KG
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