Electrochemical studies of blood platelet exocytosis.

Abstract

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.