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ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN Αiibβ3 TRAFFICKING, PLATELET SPREADING, and CLOT RETRACTION

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University of Kentucky UKnowledge Theses and Dissertations--Molecular and Cellular Biochemistry Molecular and Cellular Biochemistry 2015 ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION Yunjie Huang University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Huang, Yunjie, "ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION" (2015). Theses and Dissertations--Molecular and Cellular Biochemistry. 20. https://uknowledge.uky.edu/biochem_etds/20 This Doctoral Dissertation is brought to you for free and open access by the Molecular and Cellular Biochemistry at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Molecular and Cellular Biochemistry by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Yunjie Huang, Student Dr. Sidney W. Whiteheart, Major Professor Dr. Michael D. Mendenhall, Director of Graduate Studies ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By Yunjie Huang Lexington, KY Director: Dr. Sidney W. Whiteheart, Professor of Molecular and Cellular Biochemistry Lexington, Kentucky 2015 Copyright © Yunjie Huang 2015 ABSTRACT OF DISSERTATION ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6-/- mice injected with biotinylated-fibrinogen, showed lower accumulation of the modified protein than did WT mice. Resting and activated αIIbβ3 levels, measured by FACS, were unchanged in Arf6-/- platelets. Arf6-/- platelets had normal agonist-induced aggregation and ATP release; however, they showed faster clot retraction and enhanced spreading, which appears due to altered αIIbβ3 trafficking since myosin light chain phosphorylation and Rac1 activation, -/- in response to thrombin, were unaffected. Arf6 mice showed no hemostasis defect in tail-bleeding or FeCl3–induced carotid injury assays. These data suggest a role for Arf6 in integrin αIIbβ3 trafficking in platelets. Additionally, the regulation of Arf6 in platelets was also investigated, focusing on integrin αIIbβ3 outside-in signaling which was suggested to be responsible for the second wave of Arf6-GTP loss. G protein-coupled receptor kinase-interacting protein 1 (GIT1), a GTPase- activating protein (GAP) toward Arf6, is suggested to be involved in αIIbβ3 downstream signaling. I found that GIT1, complex with β-PIX, was translocated to the detergent- insoluble pellet upon human platelet activation, a process that is blocked by RGDS and myrArf6 peptide treatment. Moreover, tyrosine-phosphorylation of GIT1 was impaired by treatment with both peptides or with actin polymerization inhibitors. GIT1’s role in platelets was further studied using platelet-specific, GIT1 knockout mice. GIT1-/- platelets failed to show any defect, including clot retraction or fibrinogen storage. Unlike human platelets, GIT1 expression levels were much lower in mouse platelets, suggesting that GIT2 may be the functionally relevant Arf6-GAP in mouse platelets. The data in this dissertation identify that Arf6 mediates fibrinogen storage, implying its role in integrin αIIbβ3 trafficking in platelets. KEYWORDS: Platelets, Arf6, GIT1, Integrin αIIbβ3, Trafficking Yunjie Huang March 16th, 2015 ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION By Yunjie Huang Dr. Sidney W. Whiteheart Director of Dissertation Dr. Michael D. Mendenhall Director of Graduate Studies March 16th, 2015 To Fanmuyi, Francis, Irene Acknowledgements I have to say it has been hard but invaluable experience during my graduate study. From the days I arrived in Lexington to the point where I am now, I have been showering in love from many people, including my mentor, my colleges, my friends, my family and beyond. Without these love, it is impossible for me to get to this point. The first person I would like to thank is my mentor and thesis advisor, Dr. Sidney “Wally” Whiteheart. He is absolutely a great person and scientist, and it is my greatest honor to have him as my mentor. His generous willingness gave me the opportunity to touch bench work and to science, an experience that still influences me. As many Ph.D. student may know, there are a lot of hurdles that have to be passing through during this trip, such as reading papers, writing a proposal, taking the qualify exam, giving a scientific presentation, designing experiments, analyzing data, and so on. Dr. Whiteheart is such a nice mentor who is always there for my questions with patient, knowledge, positive and optimistic view, and encouragements. More importantly, he knows what is good for me at a specific level, and feeds me what I need as I grew up in science. His impact will last for the rest of my career. It is still my honor to have Dr. Douglas A. Andres, Dr. Rebecca E. Dutch, and Dr. Susan Smyth in my thesis committee. I appreciate very much their encouragements and comments that are surely indispensable to make my projects progress. I would also thank my outside examiner, Dr. Yinan Wei for her time and comments in the final exam. Additionally, I would like to thank faculty members from the Biochemistry department for their support. Particularly, I would like to thank Dr. Louis B. Hersh (in addition to Dr. Douglas A. Andres, Dr. Susan Smyth), for his recommendation for my successful pre- doctoral fellowship application. I also thank Dr. Michael D. Mendenhall for his support during my preparation for my final exam; thank Dr. Sabire Ozcan, Dr. Craig Vander Kooi, and Dr. Daniel Noonan, as my student seminars advisors, for their comments; thank Dr. Qingjun Wang for her support during collaboration; and thank Dr. Zhenyu Li for his sharing with reagents and tools. iii I am always feeling blessed to have been working in the Whiteheart lab because it is surrounded by such a group of people who make the research life even more fun. Specially, I want to thank Dr. Wangsun Choi and Dr. Zubair Karim, the pioneers of Arf6 project, for their training and support. I also want to thank Dr. Shaojing Ye, Dr. Chunxia Zhao, Dr. Qiansheng Ren, Dr. Garland Crawford, Dr. Rania Al. Hawas, Dr. Deepa Jonnalagadda, Dr. Elena A. Matveeva, Dr. Mike C. Chicka, Jinchao Zhang, Meenakshi Banerjee, and Smita Joshi for their support and for being like a family to me. I also appreciate the Biochemistry students, postdocs and staff for their support and friendship. My Arf6 project would not be able to make progress without the help from Dr. Yasunoki Kanaho at University of Tsukuba in Japan, who provided me the Arf6-floxed mice strain, and from Dr. Richard T. Premont at Dr. Duke University, who provided me the GIT1-floxed mice strain. Their generosity is greatly appreciated. I would like to thank my friends in Lexington, particularly Brothers and Sisters in Lexington Chinese Christian Church, for their support during my living in Lexington. I would like to thank my family for their love and support. First of all, I want to express my most sincere acknowledgement to my wife Fanmuyi Yang. She is a great life partner who I could rely on to overcome each difficulty encountered in life and who bring to family the joy and a positive attitude. I really appreciate her hard work and the sacrifices that she has made for the sake of my work and career. The most importantly, I thank her for her love that leads me to become a better person. Secondly, I would like to thank my parents- in-law, Jiemin Yang and Yu Fang, for their love and great support, particularly during the times when my son and my daughter were born.
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