Browsing by Subject "Platelet"

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    Electrochemical studies of blood platelet exocytosis.
    (2011-06) Ge, Shencheng
    Platelets play an essential role in hemostasis and thrombosis in the bloodstream. Their critical physiological behavior is intimately related to their cellular function as secretory cells. Platelets contain populous secretory granules and release a large array of chemical messengers ranging from ions to small molecules to large protein-based macromolecules. Until now, the dynamic secretion behavior of platelet secretory granules has been poorly understood, due largely to the lack of tools to probe the small-sized platelets. To address this important biological problem and bridge the current knowledge gap, this thesis work develops and employs state-of-the-art electrochemical techniques to study single platelet exocytosis with a focus on platelet dense-body granules, the prominent secretory granules responsible for releasing clot-promoting small molecule chemical messengers. As a result, the work reveals important insights into the inner-workings of platelet secretory behavior and expands the knowledge basis on platelets for therapeutic purposes. Chapter One reviews state-of-the-art methodologies for single-cell studies of exocytosis. Based on the small-sized platelets, electrochemical methods are uniquely suited to study exocytosis on a platelet-by-platelet basis. Chapters Two and Three develop and employ carbon-fiber microelectrode fast-scan cyclic voltammetry and amperometry methods to measure real-time dense-body granule exocytosis in platelet suspensions and single platelets. The experimental results represent the first dynamic evidence of platelet quantal secretion behavior, i.e. granule content secretion via exocytosis. Chapters Four to Seven systematically examine the fundamental quantal secretion behavior of platelet dense-body granules by combining real-time electrochemical measurement tools and a wide range of chemical and pharmacological manipulation strategies. Chapter Four examines the effects of variations in temperature, extracellular pH and osmolarity on platelet dense-body granule secretion, and reveals common and distinct quantal secretion behavior of platelets among secretory cell types studied to date. Chapter Five investigates the impact of variations in granule size following pharmacological manipulation of granule content on platelet dense-body granule secretion, and reveals the dynamic interplay between granule content and the granule membrane in modulating platelet quantal secretion. Chapter Six examines the variations in natural membrane cholesterol and substituted unnatural epicholesterol content on platelet dense-body granule quantal secretion, and reveals a critical biophysical role for membrane cholesterol in regulating exocytosis. Chapter Seven studies the effects of pharmacologically manipulated cytoskeletal F-actin and microtubule integrity on platelet dense-body granule secretion, and reveals that F-actin, but not microtubule, regulates platelet dense-body granule secretion.
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    Exploring the fundamentals of platelet granular storage and secretion at the single cell level
    (2013-01) Koseoglu, Secil
    Platelets are critical cells in hemostasis and thrombosis, but they are also involved in many important physiological events including inflammation, host defense, wound healing and malignancy. Platelets pursue their physiological functions mainly as secretory cells. The three distinct platelets granules, α- and δ- granules and lysosomes, serve as storage units for critical biological mediators. Upon action of a stimulus, platelets release their granules through a conserved mechanism called exocytosis. Because blood platelets are quite small (2-3 µm in diameter) and activate easily, until the recent work in the Haynes group, measurements were limited largely to morphological studies (electron/light/fluorescence microscopy) and ensemble aggregation and release assays, missing fundamental dynamics about chemical messenger delivery. The measurements of platelet δ-granule secretion by using carbon fiber microelectrode amperometry (CFMA) enabled real-time monitoring secretion of serotonin from platelet δ-granules with sub-ms time resolution. The aim of this thesis is to exploit these single cell measurements to study the fundamentals of platelet secretion behavior and advance our current understanding of platelet exocytosis. A brief introduction to the platelet biology and single cell platelet measurements are given in Chapter 1. Since the major step involved in platelet granular secretion is fusion of the granules to either the membrane tubular system known as the open canalicular system (OCS) or the plasma membrane, Chapter 2 investigates the difference in the secretory behavior of the platelets of different species that has OCS or does not have an OCS. CFMA measurements performed on mouse, rabbit and cow platelets indicate that OCS is necessary for an efficient secretory function. Fusion of the granular membrane with the plasma membrane is mediated by both membrane protein and lipid components. While membrane proteins anchor granular membrane with plasma membrane and facilitate fusion, membrane lipids not only regulate the membrane fluidity and curvature they also mediate localization of the fusion proteins on the site of the fusion. Although dynamin-related protein1 (Drp1) is best known as a mediator of membrane fission, recent work showed that it also contributes to granule exocytosis by mediating fusion pore expansion in chromaffin cells. However, there was not any information on the role of Drp1 on platelet granule secretion. To assess whether Drp1 functions in platelet exocytosis, we tested the effect of mdivi-1, a Drp1 inhibitor, on the release of dense granules by using single-cell amperometry (Chapter 3). The results demonstrate the role of Drp-1 in fusion pore dynamics, and indicate that regulation of platelet fusion pore expansion can be used to control thrombus formation in vivo. Phospholipids are the major components of the plasma and granule membrane, and in addition to their structural importance as a cellular barrier that separates the intracellular and extracellular environment, they are also dynamically involved in and regulate many cellular processes. However, there is not much known about how the different phospholipids regulate platelet behavior. Chapter 4 examines the effect of membrane phospholipids on the major platelet functions of aggregation and exocytosis and demonstrates that different phospholipids can act on different aspects of platelet function.Besides their physiological importance, platelets can serve as an ideal model system for studying exocytosis. In the last part of the thesis, we compare the effect of cholesterol on chromaffin cells, a well-studied model cell for neural secretion, and platelets. Chromaffin cell exocytosis at altered cellular cholesterol levels was measured at single cell level and results were compared to the previously published work by Ge et al on cholesterol effects on platelet secretion. This work demonstrated that the effect of cholesterol on each cell type was different which is likely due to the fact that, unlike platelets, chromaffin cells have a nucleus and a significant synthesis capacity that enables them to tightly regulate various cell functions.Overall, the experiments performed herein expand our current understanding of the mechanism of platelet secretion and demonstrate that studying platelet secretion at the molecular level is essential to control platelet function for therapeutic purposes.
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    A Study of the Regulation Mechanisms of Platelet Activation
    (2016-01) Finkenstaedt-Quinn, Solaire
    Platelets play an important role in maintaining hemostasis in the body. As they circulate through the blood stream, platelets receive signals from other cells that call them to sites of vascular damage. When platelets reach a damaged area, they go through the processes of activation and aggregation. During activation, platelets begin to change shape and release their granular contents via exocytosis. During exocytosis, a series of small molecules and proteins are released that serve to propagate the platelet activation signal and initiate wound healing. Overall, this work explores the different aspects of platelet activation using microscopy and single cell methods. Chapter One reviews the different components of the cytoskeleton and the current advances in microscopy that are being applied to study it. While light microscopy is a useful technique for studying cellular dynamics, super-resolution imaging allows for more in depth exploration of the many regulatory roles that the cytoskeleton plays in cells. Chapter Two focuses on work performed to investigate the toxicity of mesoporous silica nanoparticles on platelets. In Chapters Three and Four, the role that the cytoskeleton and plasma membrane play in the shape change dynamics of platelet activation is detailed, with a focus on using microscopy to visualize these changes. Chapter Five analyzes platelet activation to characterize fusion pore dynamics during exocytosis. Lastly, Chapter Six provides an overview of three different chemical education projects I have worked on. The first two projects involved the development and implementation of outreach events at a local community center to teach students about the scientific process and the chemistry behind climate change. The third project is in progress and aims to develop labs for use in the high school chemistry classroom that combine literature with general chemistry concepts to promote student engagement and interdisciplinary focus.