Felipo Benavent, María del Mar

Platelet functionality plays a crucial role in marine mammals. Alterations in platelet function can result from stress, pathologies, or exposure to xenobiotics, among others. The early detection of platelet function abnormalities is essential in these species to prevent advanced pathology and mitigate potential risks. Our main objective was to establish a range of physiological values of platelet function in bottlenose dolphins (Tursiops truncatus), beluga whales (Delphinapterus leucas), sea lions (Otaria flavescens) and walruses (Odobenus rosmarus). Intraplatelet Ca2+ mobilization using adenosine diphosphate (ADP) as a platelet agonist was used as a platelet function biomarker, adapting the methodology previously described by us in dolphins (Felipo-Benavent et al., 2022) to the rest of the species. The assay was also adapted to a seal (Phoca vitulina). Numerical indicators of intraplatelet Ca2+ mobilization kinetics were established, and statistical analyses were performed to compare the effects of species, sex, age, aquarium and species. Significant differences were observed between species, being the platelets of the sea lions the more reactive to the agonist. This work demonstrates the usefulness of this assay in the diagnosis or monitoring of animals with hemostatic diseases, showing two clinical cases in which intraplatelet calcium mobilization values were altered in marine mammals suffering haemorrhages. This assay may also serve as a means to monitor environmental changes and their potential impact on the health of marine mammal populations.

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.