Aus dem Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten der Ludwig-Maximilians-Universität München Direktor: Univ.-Prof. Dr. med. Christian Weber Effect of GPVI-Fc in combination with standard antiplatelet drugs and of GPVI-Fc fused to CD39 on platelet thrombus formation Dissertation zum Erwerb des Doktorgrades der Medizin an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Ann-Katrin Mojica Muñoz, geb. Ledwig aus Donaueschingen 2019 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Prof. Dr. med. Wolfgang Siess Mitberichterstatter: Dr. Sabine Liebscher Prof. Dr. med. Andreas May Mitbetreuung durch den promovierten Mitarbeiter: Dr. rer. nat. Janina Jamasbi Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 18.07.2019 II Eidesstattliche Versicherung Mojica Muñoz, Ann-Katrin Johanna Name, Vorname Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema: Effect of GPVI-Fc in combination with standard antiplatelet drugs and of GPVI-Fc fused to CD39 on platelet thrombus formation selbstständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. Ingolstadt, 05.09.2019 Ann-Katrin Mojica Muñoz Ort, Datum Unterschrift Doktorandin III Table of Contents Abbreviations and acronyms ...................................................................................................... V List of publications ................................................................................................................... VII Introduction ................................................................................................................................ 1 Atherothrombosis and current standard antiplatelet therapy ................................................... 1 The importance of GPVI in atherothrombosis ......................................................................... 3 GPVI in hemostasis ................................................................................................................ 4 GPVI as a target in antiplatelet therapy .................................................................................. 5 GPVI targeting strategies........................................................................................................ 6 Advantages of GPVI targeting via GPVI-Fc ............................................................................ 7 Objectives .................................................................................................................................. 8 I. GPVI-Fc in combination with standard antiplatelet drugs in plaque-induced thrombus formation in vitro ..................................................................................................................... 8 II. Enhancing the antithrombotic potential of GPVI-Fc by fusion to CD39 ................................ 9 Summary ..................................................................................................................................11 Zusammenfassung ...................................................................................................................13 References ...............................................................................................................................17 Publication I ..............................................................................................................................25 Publication II .............................................................................................................................26 Acknowledgement .....................................................................................................................40 IV Abbreviations and acronyms ACS acute coronary syndromes ADP adenosine diphosphate ADAM a disintegrin and metalloproteinase AGEs advanced glycation end products AMP adenosine monophosphate ASA acetylsalicylic acid Ca2+ calcium ion CHO cells Chinese hamster ovary cells COL/ADP collagen + adenosine diphosphate COL/EPI collagen + epinephrine COX cyclooxygenase DAPT dual antiplatelet therapy Fc fragment crystallizable region of IgG FcRγ Fc receptor γ-chain GPO glycine-proline-hydroxyproline GP glycoprotein GPIIbIIIa glycoprotein IIbIIIa GPVI glycoprotein VI GPVI-Fc recombinant dimeric GPVI-Fc fusion protein “revacept®” Ig immunoglobulin ITAM immunoreceptor tyrosine-based activation motif MEA multiple electrode aggregometry Mg2+ magnesium ion MMPs matrix metalloproteinases NSTEMI non ST-elevation acute coronary syndrome Pi inorganic phosphate PCI percutaneous coronary intervention PFA-200 platelet function analyzer 200 PLCγ2 phospholipase Cγ2 STEMI STelevation myocardial infarction sCD39 soluble CD39 V sGPVI soluble GPVI TxA2 thromboxane A2 UFH unfractionated heparin VI List of publications Mojica Muñoz AK, Jamasbi J, Uhland K, Degen H, Münch G, Ungerer M, Megens R, Weber C, Lorenz R, Brandl R, Siess W. Recombinant GPVI-Fc added to single or dual anti-platelet therapy in vitro prevents plaque-induced platelet thrombus formation. Thrombosis and Haemostasis. 2017 Aug 1; 117(8):1651-1659. doi: 10.1160/TH16-11-0856. Epub 2017 Jun 1. Degen H, Borst O, Ziegler M, Mojica Muñoz AK, Jamasbi J, Walker B, Göbel S, Fassbender J, Adler K, Brandl R, Münch G, Lorenz R, Siess W, Gawaz M, Ungerer M. ADPase CD39 Fused to Glycoprotein VI‐Fc Boosts Local Antithrombotic Effects at Vascular Lesions. Journal of the American Heart Association. 2017; 6(8): e005991. doi: 10.1161/JAHA.117.005991. VII Introduction Atherothrombosis and current standard antiplatelet therapy Myocardial infarction and ischemic stroke are leading causes of mortality and morbidity worldwide [1]. A critical step precipitating these ischemic events is the erosion or rupture of atherosclerotic plaques that exposes thrombogenic material to the bloodstream [2, 3]. Atherosclerosis is a systemic disease characterized by intimal thickening due to cell and lipid accumulation leading to plaque formation and eventually plaque disruption [4, 5]. As a result of plaque rupture or erosion, platelets become activated, aggregate and form intravascular thrombi—a pathological process referred to as atherothrombosis [2-4]. In contrast, the physiological platelet activation during primary hemostasis is initiated in response to endothelial injury of the healthy vessel wall and prevents hemorrhage and blood loss [6]. Drug therapies aiming to prevent atherothrombotic events target platelet activation and aggregation and are commonly used to lower the vascular risk in patients suffering from coronary or cerebrovascular disease [7]. Antiplatelet therapy is generally conducted either as a single drug application of acetylsalicylic acid (ASA) alone or as dual anti-platelet therapy (DAPT) by combining the former with an antagonist of the ADP-receptor P2Y12 [8-10]. ASA inhibits the formation of the prothrombotic thromboxane A2 (TxA2) by irreversible inhibition of the platelet cyclooxygenase I (COX I) [11]. The P2Y12-receptor antagonists inhibit platelet activation by adenosine diphosphate (ADP). ADP is released from dense platelet granules at sites of platelet adhesion and aggregation accompanied by different cytokines and other biologically active substances [8, 11]. Released ADP then activates additional platelets via the P2Y12- and P2Y1-receptors and leads to further platelet aggregation and secretion via feedback amplification and, hence, to thrombus propagation [8]. The substances cangrelor and ticagrelor—the latter was used as a representative of the P2Y12-receptor antagonists in the conducted studies—act as direct and reversible P2Y12-receptor antagonist, while clopidogrel, prasugrel and ticlopidine irreversibly block the P2Y12-receptor after being transformed into their active metabolite via hepatic metabolism [8, 11]. 1 DAPT and heparin is the standard therapy in patients with acute coronary syndromes (ACS) and myocardial infarction undergoing percutaneous coronary intervention (PCI) [12-15]. In certain high risk patients undergoing PCI an even stronger antiplatelet medication with glycoprotein IIbIIIa (GPIIbIIIa) inhibitors can be indicated: GPIIbIIIa inhibitors such as abciximab, eptifibatide or tirofiban are then applied intravenously in addition to unfractionated heparin (UFH) and pretreatment with or without clopidogrel [8, 12-19]. By blocking the platelet integrin αIIbβ3, GPIIbIIIa inhibitors act as fibrinogen-receptor antagonists and thereby strongly inhibit platelet aggregation [11]. All mentioned drugs target and inhibit platelet activation and aggregation at the cost of an increased bleeding risk, ranking from minor bleeding to fatal intracranial bleeding [9, 10, 20, 21]. While the pathological thrombus formation is inhibited by these antiplatelet drugs, the physiological process of primary hemostasis that prevents hemorrhage and blood loss, is affected as well. Severe bleedings occur especially when drug combinations are applied [10]. However, the combination of antiplatelet drugs is more effective in preventing ischemic events compared to single drug therapy [9, 10]. Although