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Regulation of Syk Activity in GPVI-Mediated Platelet Activation

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Regulation of Syk Activity in GPVI-Mediated Platelet Activation Regulation of Syk activity in GPVI-mediated platelet activation A Dissertation Submitted to The Temple University Graduate Board In Partial Fulfillment Of the Requirements for the Degree DOCTOR OF PHILOSOPHY by Dafydd Thomas August 2010 Dissertation Examining Committee: James L. Daniel, Pharmacology Alexander Tsygankov, Microbiology & Immunology Satya Kunapuli, Physiology Barrie Ashby, Pharmacology Hong Wang, Pharmacology Wolfgang Bergmeier, External Reader, Thomas Jefferson University ABSTRACT REGULATION OF SYK ACTIVITY IN GPVI-MEDIATED PLATELET ACTIVATION Dafydd Thomas Doctor of Philosophy Temple University School of Medicine, 2010 Doctoral Advisory Committee Chair: James L. Daniel, Ph.D. Activation of platelets is essential for hemostasis. Following damage to the vascular endothelium collagen is exposed, to which platelets stably adhere. After adhesion on collagen, a signaling cascade is initiated, mediated by Glycoprotein VI (GPVI), which results in platelet activation. A major signaling protein in GPVI signaling is Spleen tyrosine kinase (Syk). It undergoes phosphorylation and activation following GPVI stimulation. Syk's central role in this physiological process suggests regulation of its activity is required to maintain the platelets response to collagen within physiological limits. The regulation of Syk activation is the focus of this work. Previously published reports implicate the phosphatases SHP-1, SHP-2 and TULA-2 in the negative regulation of Syk. Therefore, we tested these phosphatases possible role in platelets. We show that SHP-1 can dephosphorylate Syk in vitro , but is unable to bind Syk. Also, Syk is hypophosphorylated in GPVI-stimulated SHP-1 deficient platelets and platelet functional responses are minimally affected compared to wild-type platelets. ii SHP-2 is unable to bind Syk and Syk is not a good substrate for SHP-2 in vitro . TULA-2 dephosphorylated Syk in vitro and associated with Syk in platelets. In TULA-2 deficient platelets, Syk and PLC γ2 were hyperphosphorylated compared to wild-type platelets. Deletion of TULA-2 resulted in enhanced GPVI-dependent platelet functional responses and a prothrombotic phenotype. c-Cbl has been shown to be a negative regulator of GPVI signaling, possibly by regulating Syk phosphorylation. Thus, SHP-1, SHP-2 and TULA-2’s role in c-Cbl regulation of GPVI was also investigated. We show that TULA-2 is able to bind c-Cbl in platelets. SHP-1 and SHP-2 do not. Furthermore, we show a striking similarity between the phenotype of TULA-2 and c-Cbl deficient platelets. However, in vitro binding studies show TULA-2 is able to bind Syk independently of c-Cbl. Thus, the exact role of c-Cbl in regulating Syk dephosphorylation is unclear. In conclusion, we show SHP-1 and SHP-2 are probably not involved in the negative regulation of Syk. However, TULA-2 is the major phosphatase responsible for the negative regulation of Syk in GPVI signaling. This serves to negatively regulate GPVI- mediated platelet function and prevent uncontrolled platelet activation that could lead to thrombosis. iii DEDICATION This dissertation is dedicated to my wife, Meredith, and my daughter, Natalie. iv ACKNOWLEDGEMENTS I would first like to thank my mentor, Dr. Daniel, for opening his laboratory to me and for giving me the chance to learn about platelets during the last five years. I am truly grateful for his guidance in all aspects of my graduate studies and his willingness to always help me and discuss things whenever it was needed. His dedication has provided me a firm scientific foundation on which I can build a career. I would like to thank my committee members: Dr. Alexander Tsygankov, Dr. Satya Kunapuli, Dr. Barrie Ashby, Dr. Hong Wang and Dr. Nick Carpino. Their feedback during my studies and for their time in reviewing my progress and thesis is greatly appreciated. I would especially like to thank Dr. Kunapuli for his support during my time at Temple, Dr. Tsygankov for leading us to TULA-2 and Dr. Ashby for his help with all things non-science related. In the laboratory I am particularly indebted to Carol Dangelmaier. I would like to thank her for her friendship during the last five years and for her invaluable training in platelets and laboratory skills. The technical lessons and skills she has taught me I will carry through the rest of my scientific career. I would like to thank the faculty and staff in Department of Pharmacology for their warmth and friendship during my time and Temple, all the members of Dr. Kunapuli’s laboratory for their support and all the students past and present for their friendship. I v would particularly like to thank Todd Getz and Neil Lamarre for providing some comic relief during the day and their friendship throughout my time at Temple. Finally, I would like to thank my family. I would like to thank my Mum and Dad and the rest of the Thomas family for giving me the best chance possible to reach my goals throughout my childhood and into adulthood. I would like to thank the Trapp family and Heinz family for their support during my time in USA and welcoming me into their family. Last, but by no means least, I would like to thank my wife and daughter. To Meredith, for supporting me throughout this whole process and giving me the drive to keep going when things got tough. To Natalie, for being such a source of inspiration and love and such a bundle of joy. I love you guys. vi TABLE OF CONTENTS Page ABSTRACT ...................................................................................................................... II DEDICATION................................................................................................................. IV ACKNOWLEDGEMENTS ............................................................................................ V LIST OF FIGURES ........................................................................................................ IX LIST OF ABBREVIATIONS ........................................................................................ XI INTRODUCTION............................................................................................................. 1 Platelets and Hemostasis .................................................................................................................. 1 Platelet Receptors ............................................................................................................................. 3 Spleen Tyrosine Kinase (Syk) .........................................................................................................12 Tyrosine Phosphatases ....................................................................................................................14 Casitas B-lineage Lymphoma (Cbl) Family Proteins ......................................................................21 MATERIALS AND METHODS ................................................................................... 26 Materials..........................................................................................................................................26 Synthesis and Purification of GST-TULA-2 and GST-TULA-2 H380A ........................................27 Sf9 Cells and Protein Production ....................................................................................................28 In vitro Phosphatase Assays ............................................................................................................31 In vitro Association Assays .............................................................................................................31 Human Platelet Isolation and Preparation .......................................................................................32 Murine Platelet Isolation and Preparation .......................................................................................32 Murine Platelet Preparation for Ca 2+ mobilization ..........................................................................33 Platelet Activation ...........................................................................................................................34 Platelet Dense Granule Secretion Measurements ............................................................................34 Lysate Preparation ...........................................................................................................................34 vii GST-pulldown Assay ......................................................................................................................35 Coimmunoprecipitation ...................................................................................................................35 Immunoblotting ...............................................................................................................................36 In vitro Kinase Assay ......................................................................................................................36 FeCl 3-induced in vivo Thrombosis Injury Model............................................................................37 Statistical Analysis ..........................................................................................................................38 RESULTS ........................................................................................................................ 39 SH2-domain Containing Phosphatases ...........................................................................................39 Summary of SH2 Domain Containing
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