Objective

Wan et al (Proc Natl Acad Sci USA, 105, 2008, 16432) demonstrated that RBCs rapidly and transiently release a spike of 300% more ATP shortly downstream from a short microfluidic constriction where the cells experience a sudden increase in shear stress. More recent work by Cinar et al (Proc Natl Acad Sci USA, 112, 2015, 11783), however, yielded no evidence for a similar spike in ATP release downstream of the constriction. Our aim was to determine whether a transient spike in mechanotransduction is the typical response of RBCs to the sudden onset of increased shear.

Methods

We investigate ATP release downstream of a microfluidic constriction for 15 participants using a luciferase-based photoluminescent assay.

Results

While we observe mechanotransductive ATP release from blood drawn from all donors, we find evidence of a spike in ATP concentration after the microfluidic constriction for only 2 of 15 participants. No clear trends in ATP release are found with respect to the magnitude of the applied shear stress, or to the gender, age, or physical activity (Baecke) index of the donor.

Conclusions

In aggregate, all data acquired to date suggest that a spike in mechanotransductive ATP due to a suddenly applied increase in shear stress occurs in blood drawn from only 14% of the population.

Centrifugation is the primary preparation step for isolating red blood cells (RBCs) from whole blood, including for use in studies focused on transduction of adenosine triphosphate (ATP), an important vasodilatory signaling molecule. Despite the wide use of centrifugation, little work has focused on how the centrifugation itself affects release of ATP from RBCs prior to subsequent experimentation. Here we report that both the centrifugation force and duration have a pronounced impact on the concentration of ATP present in the packed RBCs following centrifugation. Multiple subsequent centrifugations yield extracellular ATP concentrations comparable to the amount released during the initial centrifugation, suggesting this effect is cumulative. Pairwise measurements of hemoglobin and ATP suggest the presence of ATP is primarily due to an increase in centrifugation-induced hemolysis. These results indicate that common centrifugation parameters, within the ranges explored here, can release ATP in quantities comparable to the low end of the range of values measured in typical ATP transduction experiments, potentially complicating experimental interpretation of those results.

Abdominal obesity and metabolic syndrome (MS) are multifactorial conditions associated with increased risk of cardiovascular disease and type II diabetes mellitus. Previous work has demonstrated that the hemorheological profile is altered in patients with abdominal obesity and MS, as evidenced for example by increased whole blood viscosity. To date, however, no studies have examined red blood cell (RBC) deformability of blood from individuals with obesity or metabolic abnormalities under typical physiological flow conditions. In this study, we pumped RBCs through a constriction in a microfluidic device and used high speed video to visualize and track the mechanical behavior of ~8,000 RBCs obtained from either healthy individuals (n = 5) or obese participants with metabolic abnormalities (OMA) (n = 4). We demonstrate that the OMA+ cells stretched on average about 25% less than the healthy controls. Furthermore, we examined the effects of ingesting a high-fat meal on RBC mechanical dynamics, and found that the postprandial period has only a weak effect on the stretching dynamics exhibited by OMA+ cells. The results suggest that chronic rigidification of RBCs plays a key role in the increased blood pressure and increased whole blood viscosity observed in OMA individuals and was independent of an acute response triggered by consumption of a high-fat meal.