Release of Erythrocyte-Derived ATP, a Recognized Stimulus of Nitric Oxide Production, Is Increased upon Incubation of Erythrocytes with C-Peptide.
Authors: Spence Dana M. , Meyer Jennifer A. 
Published: October 2019

Abstract
Red blood cells (RBCs), when traversing microvascular beds, are subjected to mechanical deformation. It is established that RBCs release nanomolar to micromolar amounts of adenosine triphosphate (ATP), a recognized stimulus of nitric oxide (NO) production in vivo. The finding that ATP is released from RBCs in response to mechanical deformation, suggests that the RBC may be an important determinant of NO production as these cells traverse the intact circulation. An example of this potential importance of the RBC exists in diabetic complications. It is well established that patients with Type I diabetes mellitus, when compared to non-diabetics, have RBCs that are less deformable and more susceptible to oxidant stress. In conjunction with these findings, it has recently been reported that RBCs obtained from Type II diabetics release less ATP (91 +/− 10 nM) upon deformation than subjects without the disease (190 +/− 8 nM). These findings suggest that a membrane flexibilizer capable of increasing RBC-derived ATP in response to mechanical deformation may prove beneficial in improving microvascular flow. C-peptide, a 31 amino acid peptide, that connects the A and B chains of insulin, has been thought to have no significant biological role in vivo. Recently, it has been reported that C-peptide increases the deformability of erythrocytes obtained from the whole blood of patients with Type I diabetes mellitus. However, there has been no previous study to determine if C-peptide will actually increase the deformation-induced release of ATP from RBCs. Here, data is presented showing that adding nanomolar concentrations of C-peptide RBCs obtained from rabbits increases the ability of these cells to release ATP over time. When a 7% hematocrit of RBCs was incubated with a solution containing 6.6 nM C-peptide and pumped through tubing having an inside diameter that approximates resistance vessels in vivo (i.e., ~50 μm), the amount of ATP released from the RBCs increased from 0.246 ± 0.033 μM to 0.692 ± 0.140 μM (n = 10 rabbits, p<0.001) over a period of 8 hours. In the absence of the peptide, an aliquot from the same sample resulted in no significant increase in ATP release. Under conditions of non-flow, RBCs incubated with the peptide showed no statistically significant increase in ATP release. Additionally, RBCs that were incubated in the peptide containing 1 mM glybenclamide, a proven inhibitor of RBC-derived ATP, released 50% less ATP than a solution of RBCs in the absence of the inhibitor. Our results suggest that adding C-peptide to rabbit RBCs increases their ability to release ATP. Subsequent studies have shown the activity of C-peptide is depleted within 3–4 days after the solution is prepared. However, spectra obtained from a MALDI-TOF spectrometer clearly indicate that the peptide is still present in the solution exactly as it was on the day it was prepared. This suggests that there may be an activated form of the peptide that is responsible for the increase in RBC deformability and deformation-induced ATP release from these cells. In conclusion, these results suggest that C-peptide should not be considered as a biomarker alone and may have important interactions with RBCs in vivo.