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

Annalena Fink; Anne-Katrin Rohlfing; Valerie Dicenta; David Schaale; Marcel Kremser; Zoi Laspa; Manuel Sigle; Xiaoqing Fu; Andreas Pelzer; Melina Fischer; Patrick Münzer; Tatsiana Castor; Karin Anne Lydia Müller; Oliver Borst; Michael Lämmerhofer; Meinrad Paul Gawaz

doi:10.1055/s-0043-1768057

Thrombosis and Haemostasis, Inhaltsverzeichnis

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^*

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Anne-Katrin Rohlfing^*

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Valerie Dicenta

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

David Schaale

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Marcel Kremser

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Zoi Laspa

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Manuel Sigle

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Xiaoqing Fu

²Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, University of Tübingen, Tübingen, Germany

,

Andreas Pelzer

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Melina Fischer

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Patrick Münzer

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Tatsiana Castor

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Karin Anne Lydia Müller

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Oliver Borst

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

,

Michael Lämmerhofer

²Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, University of Tübingen, Tübingen, Germany

,

Meinrad Paul Gawaz

¹Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany

› Institutsangaben

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

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Referenzen

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