Platelets and Megakaryocytes

12 The average human body has in 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.

Includes supplementary material: sn.pub/extras

Platelet Functional Assays.- Effects of Anticoagulants Used During Blood Collection on Human Platelet Function.- Preparation of Washed Platelet Suspensions From Human and Rodent Blood.- Platelet Counting.- Electron Microscopy Methods for Studying Platelet Structure and Function.- Platelet Aggregation.- Platelet Aggregation in Whole Blood.- Secretion From Dense Granules.- Platelet Dense-Granule Secretion.- Analysis of the Releasable Nucleotides of Platelets.- Studies of Secretion Using Permeabilized Platelets.- Measurement of Platelet Arachidonic Acid Metabolism.- Measurement of the Platelet Procoagulant Response.- Platelet Adhesion Assays Performed Under Static Conditions.- Platelet Adhesion Assays Under Flow Using Matrix Protein-Coupled Adhesion Columns.- Techniques to Examine Platelet Adhesive Interactions Under Flow.- In Vivo Models of Platelet Function and Thrombosis.- Adhesion Between Platelets and Leukocytes or Endothelial Cells.- In Vitro Measurement of High-Shear Platelet Adhesion and Aggregation by the PFA-100®.- Flow-Cytometric Analysis of Platelet-Membrane Glycoprotein Expression and Platelet Activation.- Flow-Cytometric Analysis of Mouse Platelet Function.- Platelet Microparticles.- Megakaryocyte Functional Assays.- Isolation of Primary Megakaryocytes and Studies of Proplatelet Formation.- Isolation and Culture of Megakaryocyte Precursors.- Assays of Megakaryocyte Development.- Assays of Megakaryocyte Development.- Thrombopoietin Bioassay.- Culture of Megakaryocytic Cell Lines.