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12 The average human body has on the order of 10 circulating platelets. They are crucial for hemostasis, and yet excessive platelet activation is a major cause of m- bidity and mortality in Western societies. It is therefore not surprising that platelets have become one of the most extensively investigated biological cell types. We are, however, far from understanding precisely how platelets become activated under physiological and pathophysiological conditions. In addition, there are large gaps in our knowledge of platelet production from their giant precursor cell, the megakar- cyte. Understanding megakaryocyte biology will be crucial for the development of platelet gene targeting. The aim of Platelets and Megakaryocytes is therefore to bring together established and recently developed techniques to provide a comprehensive guide to the study of both the platelet and the megakaryocyte. It consists of five s- tions split between two volumes. The more functional assays appear in Volume 1, whereas Volume 2 includes signaling techniques, postgenomic methods, and a n- ber of key perspectives chapters. Part I of Volume 1, Platelets and Megakaryocytes: Functional Assays, describes many well-established approaches to the study of platelet function, including aggregometry, secretion, arachidonic acid metabolism, procoagulant responses, pla- let adhesion under static or flow conditions, flow cytometry, and production of microparticles. Although one would ideally wish to perform experiments with human platelets, studies within the circulation using intravital microscopy require the use of animal models, which are described in Chapter 16, vol. 1.
Contents
Perspectives.- Signaling Receptors on Platelets and Megakaryocytes.- Manipulation of Gene Expression in Megakaryocytes.- In Vitro Changes of Platelet Parameters.- Pharmacological Approaches to Studying Platelet Function.- Inhibitors of Cellular Signaling Targets.- Signaling Techniques.- Ligand-Binding Assays for Collagen.- Use of Radiolabeled 2-Methylthio-ADP to Study P2Y Receptors on Platelets and Cell Lines.- Ligand-Binding Assays.- Techniques for Analysis of Proteins by SDS-Polyacrylamide Gel Electrophoresis and Western Blotting.- Study of Tyrosine Kinases and Protein Tyrosine Phosphorylation.- Protein Tyrosine Phosphatases.- The Study of Serine-Threonine Kinases.- Phosphoinositides.- Isolation and Analysis of Platelet Lipid Rafts.- Measurement and Manipulation of [Ca2+]i in Suspensions of Platelets and Cell Cultures.- Measurement and Manipulation of Intracellular Ca2+ in Single Platelets and Megakaryocytes.- Patch-Clamp Recordings of Electrophysiological Events in the Platelet and Megakaryocyte.- Evaluation of Nitrotyrosine-Containing Proteins in Blood Platelets.- Nitric Oxide Signaling in Platelets.- Snake Venom Toxins Affecting Platelet Function.- Peptide Synthesis in the Study of Collagen-Platelet Interactions.- Platelet Permeabilization.- Molecular and Post-Genomic Techniques.- Using Retroviruses to Express Genes in Primary Megakaryocyte Lineage Cells.- Use of Antisense Oligonucleotide Technology to Investigate Signaling Pathways in Megakaryocytes.- GFP Fusion Proteins to Study Signaling in Live Cells.- Two-Dimensional Polyacrylamide Gel Electrophoresis for Platelet Proteomics.- Preparation of mRNA and cDNA Libraries From Platelets and Megakaryocytes.- Platelet Receptor Structures and Polymorphisms.- Gene Array Technology and the Study of Platelets and Megakaryocytes.
