Probing the role of calcium in erythrocyte microvesicle formation during short exposure to high shear stress.

Erythrocyte-derived microvesicles have been reported to be elevated in patients exposed to supraphysiological shear stress in the context of blood-contacting medical devices, and they have also been implicated in thrombosis and inflammation. Intracellular calcium has been proposed as a mediator of erythrocyte vesiculation. However, whether intracellular calcium accumulation is necessary for microvesicle formation under the short millisecond duration, high-intensity shear conditions present clinically remains unclear. In this study, we investigated whether calcium accumulation is necessary for shear-induced microvesicle formation using two distinct experimental setups that contain supraphysiological shear stresses. Human RBCs were exposed to transient (0 ms to 10 ms), supraphysiological shear rates (100,000 s-1) in microfluidic devices that have been previously shown to generate microvesicles. The proportion of post-shear, calcium-positive RBCs was assessed using Fluo-4, AM via flow cytometry. A second experiment included bovine erythrocytes circulating through a CentriMag blood pump for six hours in calcium-containing and calcium-free media. The pump imposed multiple exposures to supraphysiological shear stress of extremely short durations. Generated microvesicle release was quantified using carboxyfluorescein succinimidyl ester labeling. Despite exposure to extreme shear, microfluidic experiments revealed minimal calcium-positive RBCs following shear exposure. No detectable sustained calcium in the erythrocytes was observed. In the mock loop, CFSE-positive MV concentrations increased in both calcium media and calcium-free media with six hours of circulation time, independent of calcium presence. These findings suggest that, under the conditions of these experiments, erythrocyte vesiculation can occur in the absence of detectable calcium accumulation and independently of extracellular calcium availability.