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Heterogeneity in Platelet Exocytosis

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University of Kentucky UKnowledge Theses and Dissertations--Molecular and Cellular Biochemistry Molecular and Cellular Biochemistry 2013 HETEROGENEITY IN PLATELET EXOCYTOSIS Deepa Jonnalagadda 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 Jonnalagadda, Deepa, "HETEROGENEITY IN PLATELET EXOCYTOSIS" (2013). Theses and Dissertations-- Molecular and Cellular Biochemistry. 8. https://uknowledge.uky.edu/biochem_etds/8 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 and attached hereto needed written permission statements(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). I hereby grant to The University of Kentucky and its agents the non-exclusive 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 a preapproved 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 dissertation including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Deepa Jonnalagadda, Student Dr. Sidney W. Whiteheart, Major Professor Dr. Michael Mendenhall, Director of Graduate Studies HETEROGENEITY IN PLATELET EXOCYTOSIS 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 Deepa Jonnalagadda Lexington, Kentucky Director: Dr. Sidney W. Whiteheart, Professor of Molecular and Cellular Biochemistry Lexington, Kentucky 2013 Copyright © Deepa Jonnalagadda 2013 HETEROGENEITY IN PLATELET EXOCYTOSIS 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 Deepa Jonnalagadda Lexington, Kentucky Director: Dr. Sidney W. Whiteheart, Professor of Molecular and Cellular Biochemistry Lexington, Kentucky 2013 Copyright © Deepa Jonnalagadda 2013 ABSTRACT OF DISSERTATION HETEROGENEITY IN PLATELET EXOCYTOSIS Platelet exocytosis is essential for hemostasis and for many of its sequelae. Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context‐specific pattern or via a stochastic, kinetically‐ controlled process? My work describes platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (i.e. thrombin, PAR1‐agonist, PAR4‐agonist, and convulxin) and micro‐ELISA arrays were used to quantify the release of 28 distinct α‐granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4‐agonist induced slower release of fewer molecules while thrombin rapidly induced the greatest release. Cargo with opposing actions (e.g. pro‐ and anti‐angiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time‐course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with three classes of release events, differing in their rates. The distribution of cargo into these three classes was heterogeneous suggesting that platelet secretion is a stochastic process potentially controlled by several factors such as cargo solubility, granule shape, and/or granule‐plasma membrane fusion routes. Sphingosine 1 phosphate (S1P) is a bioactive lipid that is stored in platelets. S1P is essential for embryonic development, vascular integrity, and inflammation. Platelets are an abundant source of S1P due to the absence of the enzymes that degrade it. Platelets release S1P upon stimulation. My work attempts to determine how this bioactive lipid is released from platelets. Washed platelets were stimulated with agonists for defined periods of time and the supernatant and pellet fractions were separated by centrifugation. Lipids were separated by liquid phase extraction and S1P was quantified with a triple quadrapole mass spectrometer. A carrier molecule (BSA) is required to detect release of S1P. Further, there is a dose‐dependent increase in total S1P with increasing BSA. S1P release shows characteristics similar to other platelet granule cargo e.g. platelet factor IV (PF4). Platelets from Unc13‐d Jinx mice and VAMP8‐/‐ mice, which are secretion‐deficient (dense granule, alpha granule and lysosome), were utilized to understand the process of S1P release. S1P release was more affected in Unc13‐d Jinx mice mirroring their dense granule secretion defect. Fluorescence microscopy and sub‐cellular fractionation were used to examine localization of S1P in platelets. S1P was observed to be enriched in a granule population. These studies indicate the existence of two pools of S1P, a readily extractable agranular pool, sensitive to BSA, and a granular pool that requires the secretion machinery for release. The secretion machinery of platelets in addition to being involved in the release of normal granule cargo is thus proved to be involved in the release of bioactive lipid molecules like S1P. KEYWORDS: Platelet Exocytosis, Agonist‐Potency, Micro‐ELISA arrays, S1P, BSA Deepa Jonnalagadda 04/30/2013 HETEROGENEITY IN PLATELET EXOCYTOSIS By Deepa Jonnalagadda Dr. Sidney W. Whiteheart Director of Dissertation Dr. Michael Mendenhall Director of Graduate Studies 04/30/2013 To my parents ACKNOWLEDGEMENTS I would start my long list of acknowledgements by thanking my parents for their unconditional love and support in raising me into what I am today. If not for my mentor, Dr. Sidney W. Whiteheart, I could not imagine myself completing this pre‐doctoral training program successfully. I joined his research group envisioning a challenging yet, ambient environment for my graduate career. Major part of my thesis entailed mathematical modeling. Dealing with such subject by a person trained as a Biochemist is certainly challenging. I did not fear these new concepts being introduced, but was enthusiastic in knowing them and I’m positive that this was possible only due to encouraging atmosphere in the laboratory. Both of my aforementioned goals for my graduate career were already fulfilled. In spite of a terrible unexpected car accident that disturbed my life, my mentor’s support and encouragement helped me believe that I am doing okay. Designing a good experiment and being a good scientist are the two things I attempted to learn from him. The second part of my thesis stood as an example for this. He encouraged me to pursue future research in my field of interest. Many of my friends are envious of me having such an intelligent, kind and reasonable human being as my mentor. It is certainly a sign of me being fortunate. I owe him my successful and peaceful graduate training, so I thank him a ton. Not to make him sound old, but he is a father‐ like figure to me. Dr. Matveeva (Elena), originally from Russia, was very organized, helpful, encouraging and above all, she was always concerned of my well‐being. Dr. Qiensheng Ren (Jason), a past member of the Whiteheart lab has been my teacher following my mentor. He was very helpful and encouraging. I was also fortunate to have him as my bench‐mate. I used to enjoy all the scientific conversations we had. Also, he donated blood for most of my experiments. Dr. Michael C. Chicka (Mike), another past member, helped me train in protein work. Even though, this piece of work was not successful and did not end up in my thesis, this phase of my graduate training allowed me to learn planning a meaningful experiment. Not only did he enjoy the Indian accent that I had but also used to teach me the slang used here. Dr. Choi (Wangsun) is a former Korean member of our lab. He was a clear scientific thinker and a good organizer. I enjoyed the lunch time, as people used to share the table with him and he used to speak about a new entertaining topic daily. Dr. Zhao (Chunxia) has been very helpful and encouraging irrespective of her leaving the lab for a post‐doc position after graduation. Both Jason and Chunxia were from China. Dr. Al‐ Hawas (Rania) helped me maintain my spirits high all through and have always enjoyed her bubbly personality. I thank all of them, who are geographically from five different parts of the world to expose me to different cultures and could learn at least one good quality from each of them, if not more. iii Dr. Karim (Zubair) is a senior member in our lab who helped me through thick and thin. I owe my gratitude to him and his family. Dr. Ye (Shaojing) has been very encouraging and helpful all through my stay here. My fellow graduate students in decreasing order of seniority – Yunjie Huang, Jinchao Zhang, Meenakshi Banerjee and Smita Joshi have been very helpful and have made an enjoyable environment for working in the lab.
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  • Expression of Dual Nucleotidescysteinylleukotrienes

    Expression of Dual Nucleotidescysteinylleukotrienes

    J. Cell. Mol. Med. Vol 18, No 9, 2014 pp. 1785-1796 Expression of dual Nucleotides/Cysteinyl-Leukotrienes Receptor GPR17 in early trafficking of cardiac stromal cells after myocardial infarction Simona Cosentino a, #, Laura Castiglioni b, #, Francesca Colazzo a, #, Elena Nobili a, Elena Tremoli b, Patrizia Rosa c, Maria P. Abbracchio b, #, Luigi Sironi b, #, Maurizio Pesce d, #, * a Laboratorio di Biologia e Biochimica dell’Aterotrombosi, Centro Cardiologico Monzino, IRCCS, Milan, Italy b Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita di Milano, Milan, Italy c Dipartimento di Biotecnologie Mediche e Medicina Traslazionale (BIOMETRA), Istituto di Neuroscienze, Milan, Italy d Laboratorio di Ingegneria Tissutale Cardiovascolare, Centro Cardiologico Monzino, IRCCS, Milan, Italy Received: November 8, 2013; Accepted: March 25, 2014 Abstract GPR17 is a Gi-coupled dual receptor activated by uracil-nucleotides and cysteinyl-leukotrienes. These mediators are massively released into hypoxic tissues. In the normal heart, GPR17 expression has been reported. By contrast, its role in myocardial ischaemia has not yet been assessed. In the present report, the expression of GPR17 was investigated in mice before and at early stages after myocardial infarction by using immunofluorescence, flow cytometry and RT-PCR. Before induction of ischaemia, results indicated the presence of the receptor in a pop- ulation of stromal cells expressing the stem-cell antigen-1 (Sca-1). At early stages after ligation of the coronary artery, the receptor was expressed in Sca-1+ cells, and cells stained with Isolectin-B4 and anti-CD45 antibody. GPR17+ cells also expressed mesenchymal marker CD44. GPR17 function was investigated in vitro in a Sca-1+/CD31À cell line derived from normal hearts.