Original Article Extracellular Cyclophilin A Activates Platelets Via EMMPRIN (CD147) and PI3K/Akt Signaling, Which Promotes Platelet Adhesion and Thrombus Formation In Vitro and In Vivo Peter Seizer, Saskia N.I.v. Ungern-Sternberg, Tanja Schönberger, Oliver Borst, Patrick Münzer, Eva-Maria Schmidt, Andreas F. Mack, David Heinzmann, Madhumita Chatterjee, Harald Langer, Miroslav Malešević, Florian Lang, Meinrad Gawaz, Gunter Fischer, Andreas E. May Objective—Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. Approach and Results—Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride–induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA−/− platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dosedependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA−/− platelets in CyPA+/+ mice and by infusing CyPA+/+ platelets in CyPA−/− mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1−/− platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca2+-signaling. Conclusions—Extracellular CyPA activates platelets via cluster of differentiation 147–mediated phosphoinositid-3kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA. (Arterioscler Thromb Vasc Biol. 2015;35:00-00. DOI: 10.1161/ ATVBAHA.114.305112.) Key Words: antigens, CD147 ◼ blood platelets ◼ cyclophilin A
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latelets play a critical role in regulating thrombosis and inflammation at the sites of vascular and tissue injury.1,2 Mediators of inflammation, including chemokines, such as C–X–C motif chemokine 123 or C–X–C motif chemokine 16, have been described to propagate platelet-dependent thrombus formation in the microenvironment of inflammation.4–6 Cyclophilin A (CyPA) is a cytosolic protein and is expressed in a wide variety of cell types and tissues.7 CyPA is a peptidylprolyl-cis/trans isomerase that binds to the immunosuppressant cyclosporin A.8 The cyclosporin–CyPA complex inhibits
calcineurin and thereby acts as an immunosuppressor, attenuating organ rejection.9 Besides its intracellular role, CyPA is a proinflammatory cytokine with a predicted molecular weight of 18 kDa that is secreted in response to various inflammatory stimuli.10,11 Inflammatory processes are thought to be mediated both by extra and intracellular fractions of CyPA, which has been reported in various cardiovascular animal models, including atherosclerosis, inflammatory cardiomyopathy, ischemia, and reperfusion, as well as abdominal aortic aneurysms.12–15 In patients with myocardial infarction, treatment
Received on: March 16, 2014; final version accepted on: December 17, 2014. From the Medizinische Klinik III, Kardiologie und Kreislauferkrankungen (P.S., S.N.I.v.U.-S., T.S., O.B., D.H., M.C., H.L., M.G., A.E.M.), Institute of Physiology (P.M., E.-M.S., F.L.), and Institute of Anatomy (A.F.M.), Eberhard Karls-University Tübingen, Tübingen, Germany; Institute of Biochemistry, Abteilung Enzymology, Martin-Luther-University Halle-Wittenberg, Halle, Germany (M.M.); and Max-Planck-Institute für Biophysikalische Chemie Göttingen, BO Halle (Saale), Germany (G.F.). The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.305112/-/DC1. Correspondence to Peter Seizer, MD, Medizinische Klinik III, Universitätsklinikum Tübingen, Otfried-Müller-Straße 10, 72076 Tübingen, Germany. E-mail [email protected] © 2014 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.114.305112
Downloaded from http://atvb.ahajournals.org/ at CONS CALIFORNIA DIG LIB on February 28, 2015 1
2 Arterioscler Thromb Vasc Biol March 2015
Nonstandard Abbreviations and Acronyms CD CyPA mAb SOCE
cluster of differentiation cyclophilin A monoclonal antibody store-operated calcium entry
with cyclosporin A on top of the current state of the art therapy reduced infarct size.16 Extracellular CyPA affects inflammation, virus elimination, and myocardial fibrosis in coxsackievirus B3–induced myocarditis,14 propagates myocardial dysfunction after ischemia,15 and promotes atherosclerosis via binding to the extracellular matrix metalloproteinase inducer (cluster of differentiation [CD]147) on macrophages and foam cells, which is an essential step during atherosclerotic plaque formation.14,17 In monocytes and macrophages, extracellular interactions of CyPA with CD147 cause inflammation by inducing migration and activating extracellular matrix metalloproteinases.17,18 Recently, we could describe CD147 as a novel platelet surface receptor, which activates platelets via CD147–CD147 interactions.19 The role of extracellular CyPA for platelet function and thrombosis is undefined. In platelets, intracellular CyPA triggers calcium signaling and platelet activation.20,21 CyPAdeficient mice are protected from ferric chloride–induced thrombus formation, suggesting a pathophysiological relevance of CyPA in platelets.20,22 Moreover, intracellular CyPA has been reported to be involved in platelet integrin activation.22 To date, because of the lack of specific inhibitors of extracellular cyclophilins, the specific role of extracellular CyPA for platelet function has not been investigated yet. Recently, a novel group of nonpermeable cyclophilin inhibitors has been described acting only extracellularly.23–25 Thus, the aim of this study was to evaluate the specific influence of extracellular CyPA on platelet activation and function.
Materials and Methods Materials and Methods are available in the online-only Data Supplement.
Results Inhibition of Extracellular CyPA Reduces Thrombus Formation In Vivo and In Vitro Whereas the role of intracellular CyPA in thrombus formation has been investigated intensively,20–22 the specific role of extracellular CyPA for platelet function has not been reported yet. To evaluate the role of extracellular CyPA for thrombus formation, we made use of a recently described nonpermeable CyPA inhibitor MM284.24 The effect of MM284 on thrombus formation was compared with a cell-permeable CyPA inhibitor NIM811 that inhibits both intra and extracellular CyPA. Thrombus formation was visualized in vivo by fluorescence microscopy on ferric chloride–induced mesenteric arteriole injury. In mice treated with MM284 (20 mg/kg), the time to complete vessel occlusion was significantly delayed compared with animals treated with a control vehicle (PBS; time to
vessel occlusion: 13.6±1.1 versus 21.0±2.8 minutes; P
P
latelets play a critical role in regulating thrombosis and inflammation at the sites of vascular and tissue injury.1,2 Mediators of inflammation, including chemokines, such as C–X–C motif chemokine 123 or C–X–C motif chemokine 16, have been described to propagate platelet-dependent thrombus formation in the microenvironment of inflammation.4–6 Cyclophilin A (CyPA) is a cytosolic protein and is expressed in a wide variety of cell types and tissues.7 CyPA is a peptidylprolyl-cis/trans isomerase that binds to the immunosuppressant cyclosporin A.8 The cyclosporin–CyPA complex inhibits
calcineurin and thereby acts as an immunosuppressor, attenuating organ rejection.9 Besides its intracellular role, CyPA is a proinflammatory cytokine with a predicted molecular weight of 18 kDa that is secreted in response to various inflammatory stimuli.10,11 Inflammatory processes are thought to be mediated both by extra and intracellular fractions of CyPA, which has been reported in various cardiovascular animal models, including atherosclerosis, inflammatory cardiomyopathy, ischemia, and reperfusion, as well as abdominal aortic aneurysms.12–15 In patients with myocardial infarction, treatment
Received on: March 16, 2014; final version accepted on: December 17, 2014. From the Medizinische Klinik III, Kardiologie und Kreislauferkrankungen (P.S., S.N.I.v.U.-S., T.S., O.B., D.H., M.C., H.L., M.G., A.E.M.), Institute of Physiology (P.M., E.-M.S., F.L.), and Institute of Anatomy (A.F.M.), Eberhard Karls-University Tübingen, Tübingen, Germany; Institute of Biochemistry, Abteilung Enzymology, Martin-Luther-University Halle-Wittenberg, Halle, Germany (M.M.); and Max-Planck-Institute für Biophysikalische Chemie Göttingen, BO Halle (Saale), Germany (G.F.). The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.305112/-/DC1. Correspondence to Peter Seizer, MD, Medizinische Klinik III, Universitätsklinikum Tübingen, Otfried-Müller-Straße 10, 72076 Tübingen, Germany. E-mail [email protected] © 2014 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.114.305112
Downloaded from http://atvb.ahajournals.org/ at CONS CALIFORNIA DIG LIB on February 28, 2015 1
2 Arterioscler Thromb Vasc Biol March 2015
Nonstandard Abbreviations and Acronyms CD CyPA mAb SOCE
cluster of differentiation cyclophilin A monoclonal antibody store-operated calcium entry
with cyclosporin A on top of the current state of the art therapy reduced infarct size.16 Extracellular CyPA affects inflammation, virus elimination, and myocardial fibrosis in coxsackievirus B3–induced myocarditis,14 propagates myocardial dysfunction after ischemia,15 and promotes atherosclerosis via binding to the extracellular matrix metalloproteinase inducer (cluster of differentiation [CD]147) on macrophages and foam cells, which is an essential step during atherosclerotic plaque formation.14,17 In monocytes and macrophages, extracellular interactions of CyPA with CD147 cause inflammation by inducing migration and activating extracellular matrix metalloproteinases.17,18 Recently, we could describe CD147 as a novel platelet surface receptor, which activates platelets via CD147–CD147 interactions.19 The role of extracellular CyPA for platelet function and thrombosis is undefined. In platelets, intracellular CyPA triggers calcium signaling and platelet activation.20,21 CyPAdeficient mice are protected from ferric chloride–induced thrombus formation, suggesting a pathophysiological relevance of CyPA in platelets.20,22 Moreover, intracellular CyPA has been reported to be involved in platelet integrin activation.22 To date, because of the lack of specific inhibitors of extracellular cyclophilins, the specific role of extracellular CyPA for platelet function has not been investigated yet. Recently, a novel group of nonpermeable cyclophilin inhibitors has been described acting only extracellularly.23–25 Thus, the aim of this study was to evaluate the specific influence of extracellular CyPA on platelet activation and function.
Materials and Methods Materials and Methods are available in the online-only Data Supplement.
Results Inhibition of Extracellular CyPA Reduces Thrombus Formation In Vivo and In Vitro Whereas the role of intracellular CyPA in thrombus formation has been investigated intensively,20–22 the specific role of extracellular CyPA for platelet function has not been reported yet. To evaluate the role of extracellular CyPA for thrombus formation, we made use of a recently described nonpermeable CyPA inhibitor MM284.24 The effect of MM284 on thrombus formation was compared with a cell-permeable CyPA inhibitor NIM811 that inhibits both intra and extracellular CyPA. Thrombus formation was visualized in vivo by fluorescence microscopy on ferric chloride–induced mesenteric arteriole injury. In mice treated with MM284 (20 mg/kg), the time to complete vessel occlusion was significantly delayed compared with animals treated with a control vehicle (PBS; time to
vessel occlusion: 13.6±1.1 versus 21.0±2.8 minutes; P
