Flow cytometric kinetic assay of calcium mobilization in whole blood platelets of bottlenose dolphins (Tursiops truncatus)

Abstract

Marine mammals may suffer alterations in platelet function and hemostasia due to multiple pathologies, environmental conditions (including stress) or exposure to different contaminants that induce platelet activation. Detecting early alterations in platelet function in these animals could be an especially relevant diagnostic tool in these species because they typically do not show signs of weakness or disease until the pathology is in advanced state, in order to avoid attracting predators in natural conditions. The study of early markers of platelet activation is relevant for the detection, monitoring and therapy of inflammation and hemostasis disorders. Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular Ca2+, using sensitive fluorescent indicators that can be loaded into intact cells. In order to study intraplatelet Ca2+ mobilization in marine mammals, we have adapted a kinetic assay of human platelet activation to study platelet activation in whole-blood samples of bottlenose dolphins (Tursiops truncatus) using the Ca2+-sensitive dye Fluo-4AM and a clone of the platelet-specific antibody CD41-PE that recognizes dolphin platelets. This no-wash, no-lyse protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signaltransduction responses to platelet agonists under near-physiological conditions. The adaptation of this technique to marine mammals represents a methodological advance for basic and clinical veterinary applications but also for general environmental studies on these species.