Platelets and Megakaryocytes_ Volume 2_ Functional Assays - Ebook Pdf

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Platelets and Megakaryocytes_ Volume 2_ Functional Assays

Part II of Volume 1 focuses on approaches used to study megakaryocyte function, including the development of specialized structures for future production of platelets (e.g., the demarcation membrane system), the appearance of platelet-specific surface receptors, and the increase in ploidy. The source of megakaryocytes is often a complex issue facing many researchers owing to the extremely low density (<1%) of this cell type in its primary location, the marrow. Techniques to purify megakaryocytes from marrow based on their unique size and surface markers are described in Chapter 22, vol. 1, along with approaches to maintain these cells in culture and monitor formation of platelet-generating proplatelet structures. An alternative approach to generating megakaryocytes is to grow them in culture from precursor cells as detailed in Chapter 23, vol. 1. This requires the presence of thrombopoietin (Chapter 26, vol. 1) acting through its receptor, c-Mpl, and normally other cytokines. The availability of systems to generate megakaryocytes in vitro provides a promising avenue to generate genetically modified platelets. Although there is no doubt that continuous megakaryocyte cell lines are useful for some studies of signaling in these cells, they have their limitations and the pros and cons are discussed in Chapter 27, vol. 1. Many basic and advanced techniques for the general study of cell signaling have been applied in studies to characterize the mechanisms of regulation of platelet function. These include ligand binding assays, the study of protein and lipid kinases and phosphatases, the analysis of lipid rafts in the regulation of cell signaling, the measurement of intracellular calcium levels, electrophysiological techniques, nitric oxide signaling, the use of venom proteins, and the internalization of proteins into platelets through permeabilization. These techniques and more are presented in Part II of Volume 2. In many respects the megakaryocyte is a giant platelet. Differences do occur in the arrangement of cellular organelles and cytoskeleton in the two cells, however megakaryocytes respond to platelet agonists such as ADP with full downstream functional responses (discussed in Chapters 1 and 16, vol. 2). Therefore, despite differences in ultrastructure, the megakaryocyte has earned its place as a sufficient, if not comparable model of platelet signaling. Many of the signaling techniques are therefore beginning to be applied to the megakaryocyte which, because of its size, is proving to be an extremely interesting model for platelet signaling, particularly using single cell approaches such as imaging and electrophysiology (see Chapters 16 and 17, vol. 2). Part III of Volume 2 is dedicated to recent advances in molecular techniques and post-genomic techniques and how they may be applied to the study of platelets and megakaryocytes. This section includes descriptions of how retroviruses may be used to express genes in primary megakaryocytes, the use of GFP-fusion proteins to study signaling in live cells, two-dimensional electrophoresis for platelet proteomics, the production of platelet cDNA libraries and the use of gene array technology. Although the main aim of the book is to include practical approaches to the study of platelets and megakaryocytes, a series of perspectives chapters are included (Part I, vol. 2). These chapters review the current understanding of platelet and megakaryocyte biology in addition to their discussions of important new developments and ex-perimental strategies. Many of the methods chapters also include further discussion and background on specific techniques. This book has only been made possible by the efforts of many international experts in the field. We are grateful to them for their willingness to contribute their knowledge, in particular their tricks of the trade, which have resulted from many years of dedicated hands-on work. We also wish to thank our colleagues within the Department of Physiology at Cambridge and the School of Animal and Microbial Sciences at Reading for helpful discussion during the course of the editing work, in particular Peter Wooding on electron microscopy and Gwen Tolhurst on molecular techniques. We are also grateful to Margaret Bardy and Karen Parr for considerable secretarial assistance. We are also grateful to the following companies for supporting the cost of color reproduction: Eli Lilly and Company, Cairn Research Ltd., Bio Rad Laboratories Ltd., and Sysmex UK Ltd. Jonathan M. Gibbins Martyn P. Mahaut-Smith.

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