Examination of the Final Stages of Platelet Production Candidate. My Ph.D. thesis, under the direction of Dr. Dana Devine (Professor, University of British Columbia;Vice President, Canadian Blood Services), entailed the application of proteomics, molecular biology, and biochemistry to identify mechanisms involved in regulating the storage-related deterioration of platelets (PLTs). In Dana's lab I worked closely with Canadian Blood Services, clinicians, and investigators at the Centre for Blood Research to improve the collection, processing, and storage of blood PLTs. My post-doctoral research in Dr. Joseph Italiano's laboratory (Associate Professor, Harvard University), has added to my repertoire a number of specialized cell biology techniques including fluorescence and electron microscopy, live cell imaging, retroviral infection, cell culture, and transgenic mouse models to study PLT production. These projects have provided me with the necessary expertise to meet my career goals by familiarizing me with the cytoskeletal mechanics, contribution of environmental factors such as extracellular matrix (ECM) components and shear forces, and the signaling pathways that orchestrate PLT formation and regulate their function. Environment. Dr. Italiano's laboratory has offered unequalled access to an extensive network of exceptionally talented PLT researchers whose input and experience have helped guide my research and allowed me to markedly expand my arsenal of analytical, management, writing, and oratory skills. Dr. Italiano has also made available to me a range of highly specialized equipment, armed me with a number of molecular biology techniques that are complementary to my research goals, and provided me with dedicated mentorship that has enabled me to become an accomplished PLT biologist and microscopist. The opportunity to train at an institute that is world-renowned for its PLT work has allowed me to establish meaningful collaborative relationships with PLT researchers worldwide. My joint appointment at Harvard Medical School and Brigham and Women's Hospital has afforded me access to a multitude of courses, internal training programs, departmental seminars, and career development and educational programs that have made me a better scientist, and supported my career trajectory toward independent investigator. Research. Blood PLTs play an essential role in hemostasis and the pathophysiology of thrombosis. My interests lay in investigating the mechanisms of PLT formation for the purpose of developing targeted therapies for thrombocytopenia. The ability to control in vitro megakaryocyte (MK) expansion and maturation into PLTs will result in an important source of PLTs for infusion. My short-term goals are to investigate the final stages of PLT production, for which a research plan comprising three specific aims is proposed. MKs release long branched extensions called proPLTs into sinusoidal blood vessels that undergo repeated abcissions to yield circulating PLTs.
Aim 1 will examine the cytoskeletal mechanics by which individual PLTs are released from proPLTs. These experiments will address the microtubule, and filamentous-actin-based forces that power proPLT extension and PLT release both in live cells and within a permeabilized cell system.
Aim 2 will model how MKs establish polarity and direct proPLTs toward sinusoidal blood vessels to deliver PLTs into the circulation.
Aim 3 will resolve the contribution of environmental factors such as ECM interactions and continuous blood flow in generating functional PLTs and regulating their rate/extent of release. Research career development plan. Research on aim 1 will commence during the mentored (K99) phase, with aim 1.3 continuing into the independent (R00) phase. The goals described in aims 1.1 and 1.2 represent a mentored departure from the research aims of my primary supervisor, which will run parallel to my work and focus on protein translation/degradation pathways that initiate proPLT production, and the role of the spectrin- based membrane skeleton on PLT formation. Research on aim 2 will also commence during the mentored phase and proceed into the first year of the independent phase, to bridge the gap between the cytoskeletal and environmental determinants of proPLT extension and PLT release. The preliminary data deriving from aims 1 and 2 will allow me to launch an independent research program in year 3 to recapitulate the bone marrow and sinusoidal blood vessel microenvironments in vitro and study their role in terminal PLT production (aim 3.1 and 3.2). Research on aim 3.3 will commence in year 3, after initial biochip development, and will examine the quality of culture-derived PLTs with each new advancement in biochip design. These data will support my long-term career goals of (1) developing bio-mimetic systems to generate useable numbers of clinically viable human PLTs for infusion, and (2) establishing representative ex vivo models of human bone marrow and surrounding vasculature for the purposes of testing drugs and developing targeted therapies for thrombocytopenia. Neither the specific aims nor the research goals listed in this application overlap with those of my mentors, and I have recieved permission to take them with me to my own research lab.
- Examination of the Final Stages of Platelet Production Relevance to public health. Platelets are essential to control bleeding and platelet transfusions are widely used to treat patients with inherited or acquired thrombocytopenia (low platelet counts) resulting from immune thrombocytopenic purpura, myelodysplastic syndromes, aplastic anemia, complications during pregnancy and birth, human immunodeficiency virus infection, chemotherapy and surgery. Although more than 10 million platelet units are transfused yearly in the United States, the limited availability of donor platelets owing to their 5-day shelf life, potential to induce an immune response and risk of bacterial contamination is of serious clinical concern. A better understanding of the mechanisms of platelet formation will lead to improved therapies for thrombocytopenia and an alternative source of platelets for infusion.
|Thon, J N; Medvetz, D A; Karlsson, S M et al. (2015) Road blocks in making platelets for transfusion. J Thromb Haemost 13 Suppl 1:S55-62|
|Bender, Markus; Thon, Jonathan N; Ehrlicher, Allen J et al. (2015) Microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein. Blood 125:860-8|
|Thon, Jonathan N (2014) Nothing to lose: why early career scientists make ideal entrepreneurs. Trends Biochem Sci 39:571-3|
|Machlus, Kellie R; Thon, Jonathan N; Italiano Jr, Joseph E (2014) Interpreting the developmental dance of the megakaryocyte: a review of the cellular and molecular processes mediating platelet formation. Br J Haematol 165:227-36|
|Bordoli, Mattia R; Yum, Jina; Breitkopf, Susanne B et al. (2014) A secreted tyrosine kinase acts in the extracellular environment. Cell 158:1033-44|
|Thon, Jonathan N; Kitterman, Alice C; Italiano Jr, Joseph E (2013) Animating platelet production adds physiological context. Trends Mol Med 19:583-5|
|Hildick-Smith, Gordon J; Cooney, Jeffrey D; Garone, Caterina et al. (2013) Macrocytic anemia and mitochondriopathy resulting from a defect in sideroflexin 4. Am J Hum Genet 93:906-14|
|Gobbi, Giuliana; Mirandola, Prisco; Carubbi, Cecilia et al. (2013) Proplatelet generation in the mouse requires PKCÎµ-dependent RhoA inhibition. Blood 122:1305-11|
|Macaulay, Iain C; Thon, Jonathan N; Tijssen, Marloes R et al. (2013) Canonical Wnt signaling in megakaryocytes regulates proplatelet formation. Blood 121:188-96|