Exploring the fundamentals of platelet granular storage and secretion at the single cell level

Abstract

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, &#945;- and &#948;- 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 &#948;-granule secretion by using carbon fiber microelectrode amperometry (CFMA) enabled real-time monitoring secretion of serotonin from platelet &#948;-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.</DISS_para> <DISS_para>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.