Abstract

High hydrostatic pressure inhibits platelet aggregation, whereas rapid decompression causes platelet activation in divers and in vitro platelet suspensions. Platelet activation contributes to decompression sickness in humans and other terrestrial mammals, however marine mammals are routinely exposed to pressures up to several hundred atmospheres and do not suffer from decompression-related illness. A rise in intracellular calcium is a crucial step in platelet activation, thus we monitored elephant seal and human platelet intracellular calcium ([Ca²⁺]i) levels during and following a high pressure excursion.

Elephant seal and human platelets were washed free of plasma components, incubated with the fluorescent calcium ratio dye Fura-2 AM, washed again, and calcium was added back to a final external concentration of 1mM. Platelets were then added to a specialized high pressure optical cell, placed in a spectro-fluorometer, and baseline calcium concentrations were established for each sample. Samples were pressurized to 160 atmospheres, held there for fifty minutes, and decompressed rapidly over a five-minute period. The internal calcium concentration was monitored continuously throughout the pressurization period, and for an additional 30 minutes following decompression.

Elephant seal platelet [Ca²⁺]i rose by 34% during the high pressure period, while human platelet [Ca²⁺]i rose by 93% under identical conditions. Unpressurized control platelets from both species showed a 180% rise in [Ca²⁺]i over the same time period. This rise over time is likely the result of Fura-2 binding to calcium that has leaked into the cytoplasm before it can be sequestered within the dense tubular system or pumped back out of the cells. In the absence of extracellular calcium, unpressurized elephant seal and human [Ca²⁺]i still rises by 90%, indicating leakage of calcium from intracellular stores.

We suggest that the relatively small rise of elephant seal platelet [Ca²⁺]i reflects inherent differences in membrane structure and function. Cholesterol has been shown to be an important modulator of membrane fluidity, thus the greater concentration of cholesterol present in elephant seal platelet membranes (21% by weight as opposed to human platelet cholesterol 7.5% by weight) likely helps prevent leakage of calcium ions under high pressure, allowing these cells to maintain near basal levels of calcium and remain in a resting state during diving.

Acknowledgments

We are very grateful to Frances Gulland, Martin Haulena, and the staff and volunteers of The Marine Mammal Center, Sausalito CA, and to Dan Costa (University of California- Santa Cruz) and Dan Crocker (Sonoma State University) for helping us obtain elephant seal blood samples.