Novel implications of lost serotonin transporter function on platelet biology By Kendra Helen Oliver Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Pharmacology December, 2016 Nashville, Tennessee Approved: Date: _______________________________________________ __________________ Jonathan Schoenecker, M.D., Ph.D. (Chair) _______________________________________________ __________________ Heidi E. Hamm, Ph.D. _______________________________________________ __________________ Ana M.D. Carneiro, Ph.D. _______________________________________________ __________________ Digna R. Velez Edwards Ph.D., M.S. _______________________________________________ __________________ Vsevolod Gurevich, Ph.D. DEDICATION To my parents, Michael Cherry and Kimberlee Oliver, to my brilliant sister, Briana Oliver, and to my partner in crime, Dr. Max Joffe. ii ACKNOWLEDGEMENTS There are so many wonderful people that have I would like to thank for their support and guidance during this challenging and daunting process. Most importantly, I would like to thank my mentors, Dr. Heidi E Hamm and Dr. Ana M.D. Carneiro. Dr. Carneiro has pushed me beyond my limits and has provided my scholarly foundation. Dr. Hamm has also been an outstanding mentor and role model as a scientist. I would also like to thank my committee members for their guidance during my scientific scholarship. The direction and administrative support received from Dr. Christine Konrodi and Karen Geig, and Dr. Joey Barrett, has been extraordinary, and for that I am very thankful. Additionally, I would like to thank and acknowledge my lab members. In particular, Dr. Matthew Duvernay has helped me navigate the world of platelet biology and Dr. Ali Kali for his support and encouragement. I would also like to thank Dr. Matthew Mazalouskas and Dr. Michael Dohn. Dr. Mazalouskas has not only been there to discuss the grueling hardships of graduate studies, but he is also a role model for deep scientific rigor and inquisitiveness. I would also like to acknowledge the rest of the Hamm Lab. Additionally, there have been a number of undergraduates who have contributed to this work, notably including Cecillia Li and Cynthia Mairink. I would also like to thank Jae Maeng for assisting with animal husbandry and experiments. I would like to acknowledge the wonderful guidance and inspiration of Dr. John Wikswo, whose mentorship has meant a great deal towards my progression as a scientist. Dr. Wikswo has provided me numerous opportunity to expand my intellectual phase space, for which I am tremendously appreciative. He also enabled me with the opportunity to work with The Systems Biology and Bioengineering Undergraduate Research Experience (SyBBURE) and Dr. Christina Marasco. I would like to thank Dr. Wikswo and Dr. Marasco for allowing me to mentor a number of undergraduates and fellows through the SyBBRE program. In particular, Babtunde Bello and Attiyah Houston have been instrumental in the design and creation of the serotonin homeostasis model. Finally, I would like to thank others at Vanderbilt who have given me extraordinary opportunities to follow my interests in teaching and learning including Dr. Cynthia Brame, Danielle Picard, and Dr. Derek Bruff at the Center for Teaching. This work would not have been possible without the financial support of the American Heart Association, and the T32 Training Program in Mechanisms of Vascular Disease. iii TABLE OF CONTENTS Page DEDICATION ii ACKNOWLEDGEMENTS iii LIST OF TABLES vii LIST OF FIGURES viii Chapter 1 INTRODUCTION 2 The cardiovascular system, hemostasis and the role of platelets ........................................ 2 Role of the vasculature in hemostasis ............................................................................. 4 Role of coagulation in hemostasis ................................................................................... 6 Role of platelets in hemostasis ........................................................................................ 7 Serotonergic signaling in platelets .................................................................................... 33 Serotonin and the cardiovascular system ...................................................................... 33 Platelet function and serotonin ...................................................................................... 38 A crossroads between cardiovascular function and depression .................................... 40 Dissertation overview ....................................................................................................... 44 2 MATERIALS AND METHODS 46 Materials. .......................................................................................................................... 46 Methods............................................................................................................................. 50 3 ACUTE AND CHRONIC LOSS OF SEROTONIN TRANSPORTER FUNCTION IN PLATELETS 64 The serotonin transporter: pharmacology and protein interactions .................................. 64 The serotonin transporter (SERT) ................................................................................. 64 The acute role of the serotonin transporter in platelet function ........................................ 78 Acute SSRI treatment ....................................................................................................... 85 Abstract ......................................................................................................................... 85 Objectives ...................................................................................................................... 85 Results ........................................................................................................................... 86 iv Alternative effects of lost SERT function: Serotonylation of extracellular matrix proteins ........................................................................................................................................... 95 Abstract ......................................................................................................................... 95 Objectives ...................................................................................................................... 95 Results ........................................................................................................................... 96 Conclusion ........................................................................................................................ 97 SSRIs and their acute versus chronic effect on platelet function .................................. 97 4 ROLE OF THE 5HT2A RECEPTOR IN PLATELET ACTIVATION 103 Introduction ..................................................................................................................... 103 Serotonergic receptors ................................................................................................. 104 Loss of SERT and the consequences on 5HT2AR ....................................................... 110 Loss of serotonin transporter function alters ADP-mediated αIIbβ3 activation through dysregulation of 5HT2A receptor. .................................................................................... 113 Abstract ....................................................................................................................... 113 Objectives .................................................................................................................... 114 Results ......................................................................................................................... 115 Spreading and 5HT2AR activation .................................................................................. 127 Abstract ....................................................................................................................... 127 Objectives .................................................................................................................... 127 Results ......................................................................................................................... 128 Conclusion ...................................................................................................................... 131 The alteration in 5HT2AR surface expression with sustained inhibition of SERT and its effects on ADP-mediated αIIbβ3 activation. ............................................................... 131 5HT2AR and spreading ................................................................................................ 135 5 INTEGRIN MODULATION OF SEROTONERGIC SIGNALING 137 Introduction ..................................................................................................................... 137 PIA2 polymorphism and platelet function .................................................................... 137 Mouse model of PIA2 and platelet functional consequences ....................................... 139 Pro32Pro33 mutation in the integrin β3 domain result in αIIbβ3 priming and enhanced adhesion: Reversal of the hypercoagulability phenotype by Src inhibitor SKI-606 ...... 142 Abstract ....................................................................................................................... 142 Objectives ...................................................................................................................