Propensity of red blood cells to undergo P2X7 receptor–mediated phosphatidylserine exposure does not alter during in vivo or ex vivo aging

Abstract

Sluyter, R. (2015). Propensity of red blood cells to undergo P2X7 receptor-mediated phosphatidylserine exposure does not alter during in vivo or ex vivo aging. Transfusion, 55 (8), 1946Transfusion, 55 (8), -1954 Propensity of red blood cells to undergo P2X7 receptor-mediated phosphatidylserine exposure does not alter during in vivo or ex vivo aging Abstract Phosphatidylserine (PS) exposure facilitates the removal of red blood cells (RBCs) from the circulation, potentially contributing to the loss of stored RBCs after transfusion, as well as senescent RBCs. Activation of the P2X7 receptor by extracellular adenosine 5′-triphosphate (ATP) can induce PS exposure on freshly isolated human RBCs, but whether this process occurs in stored RBCs or changes during RBC aging is unknown. STUDY DESIGN AND METHODS RBCs were processed and stored according to Australian blood banking guidelines. PS exposure was determined by annexin V binding and flow cytometry. Efficacy of P2X antagonists was assessed by flow cytometric measurements of ATP-induced ethidium+ uptake in RPMI 8226 cells. Osmotic fragility was assessed by lysis in hypotonic saline. RBCs were fractionated by discontinuous density centrifugation. RESULTS ATP (1 mmol/L) induced PS exposure on RBCs stored for less than 1 week. This process was near-completely inhibited by the P2X7 antagonists A438079 and AZ10606120 and the P2X1/P2X7 antagonist MRS2159 but not the P2X1 antagonist NF499. ATP-induced PS exposure on RBCs was not dependent on K+, Na+, or Cl− fluxes. ATP did not alter the osmotic fragility of stored RBCs. ATP-induced PS exposure was similar between RBCs of different densities. ATP-induced PS exposure was also similar between RBCs stored for less than 1 week or for 6 weeks.

Properties of donated red blood cell components from patients with hereditary hemochromatosis

“…Increased levels of annexin V in the supernatant of stored RBCs have been previously reported . Our data are in agreement with those of other reports …”

Hemolysis in human erythrocytes by Clostridium perfringens epsilon toxin requires activation of P2 receptors

“…To test whether P2X7 receptors participate in ETX-induced hemolysis, we used antagonists with relative selectivity for P2X7: Brilliant Blue G (BBG), ATP-2′, 3′-dialdehyde (OxATP), MRS2159 [26], KN-62 [12], Mg 2+ [25], and A438079 [12]. BBG decreased hemolysis in a concentration-dependent manner with an EC 50 value of 90 μM (Figure 5(a)).…”

“…MRS2159, which is generally considered a P2X1 antagonist, is also a potent antagonist of P2X7 with an IC 50 of at least 1 log lower than that of A438079 [26]. MRS2159 potently inhibited hemolysis in human erythrocytes in a concentration-dependent manner (EC 50 : ~349 μM) (Figure 5(e)).…”

“…As P2X1 and P2X7 share similar inhibitor profiles for BBG, KN-62, OxATP and MRS2159 [12,25,26], we cannot exclude the possibility that P2X1 receptor is partially involved in ETX-induced hemolysis in human erythrocytes. A previous study showed that Mg 2+ ions were effective in suppressing the P2X7 current component in P2X1/7 co-expressing oocytes, whereas the P2X1 current component remained relatively unaltered [25].…”

The Proteome of the Red Blood Cell: An Auspicious Source of New Insights into Membrane-Centered Regulation of Homeostasis

“…The presence of regulatory networks, involving receptor-mediated signaling, constitutes a more complex conceptual challenge. Various receptors have been identified in the membrane fraction, as well as activated forms of kinases and secondary messengers [38,39]. Already, a combination of refined cell age separation techniques with the measurement of phosphatidylserine exposure has indicated the involvement of hitherto unsuspected signaling pathways in the recognition and removal of damaged red blood cells [40].…”