Nitric Oxide Scavenging by Red Blood Cell Microparticles and Cell-Free Hemoglobin as a Mechanism for the Red Cell Storage Lesion

Abstract

Background Intravascular red cell hemolysis impairs NO-redox homeostasis, producing endothelial dysfunction, platelet activation and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of exocytic microvesicles or “microparticles” and hemolysis, which we hypothesized could impair vascular function and contribute to the putative “storage lesion” of banked blood. Methods and Results We now find that storage of human red blood cells under standard blood banking conditions results in the accumulation of cell free and microparticle-encapsulated hemoglobin which, despite 39 days of storage, remains in the reduced ferrous oxyhemoglobin redox state and stoichiometrically reacts with and scavenges the vasodilator nitric oxide (NO). Using stopped-flow spectroscopy and laser triggered NO release from a caged NO compound we found that both free hemoglobin and microparticles react with NO about 1000 times faster than with intact erythrocytes. In complementary in vivo studies we show that hemoglobin, even at concentrations below 10 μM (in heme), produces potent vasoconstriction when infused into the rat circulation, while controlled infusions of methemoglobin and cyanomethemoglobin, which do not consume NO, have substantially reduced vasoconstrictor effects. Infusion of the plasma from stored human red cell units into the rat circulation produces significant vasoconstriction related to the magnitude of storage related hemolysis. Conclusions The results of these studies suggest new mechanisms for endothelial injury and impaired vascular function associated with the most fundamental of storage lesions, hemolysis.

Mechanisms of Slower Nitric Oxide Uptake by Red Blood Cells and Other Hemoglobin-containing Vesicles

“…This also occurs during administration of some hemoglobin-based oxygen carriers (34 -48). Transfusion of banked blood of more than 2 weeks of age may show similar effects in vivo due to release of hemoglobin as the red cells degrade (49,50).…”

“…In addition, we have simulated our own kinetics measurements of NO uptake by microparticles derived from aged red blood cells under aerobic conditions (50). Unlike phospholipid vesicles, these microparticles still have lipid raft proteins and other components characteristic of red cells in their membrane (56).…”

“…This also occurs during administration of some hemoglobin-based oxygen carriers (34 -48). Transfusion of banked blood of more than 2 weeks of age may show similar effects in vivo due to release of hemoglobin as the red cells degrade (49,50).It is desirable to design blood substitutes that would not have the detrimental effects of cell-free hemoglobin. A potential candidate is hemoglobin encapsulated within phospholipid vesicles that are a few hundreds of nanometers in diameter (51-55).…”

Cell-Free Hemoglobin and Its Scavenger Proteins: New Disease Models Leading the Way to Targeted Therapies

“…These antagonist experiments proved that free Hb was a contributor to the observed adverse effects. Additionally, animal studies focused on the acute hemodynamic effects of stored blood have suggested that NO donors could be a therapeutic option to limit transfusion-related and hemolysis-induced pulmonary and/or systemic hypertensive responses in mice, rats, and sheep www.perspectivesinmedicine.org (Yu et al 2008(Yu et al , 2009Donadee et al 2011;Baron et al 2012). One of these studies identified Hb and Hb-containing RBC membrane microparticles released in vitro before transfusion as an additional factor that may enhance the adverse posttransfusion effects (Donadee et al 2011).…”

“…Additionally, animal studies focused on the acute hemodynamic effects of stored blood have suggested that NO donors could be a therapeutic option to limit transfusion-related and hemolysis-induced pulmonary and/or systemic hypertensive responses in mice, rats, and sheep www.perspectivesinmedicine.org (Yu et al 2008(Yu et al , 2009Donadee et al 2011;Baron et al 2012). One of these studies identified Hb and Hb-containing RBC membrane microparticles released in vitro before transfusion as an additional factor that may enhance the adverse posttransfusion effects (Donadee et al 2011). Blood transfusion leading to stimulation of the heme breakdown pathway and accumulation of heme catabolites was associated with anti-inflammatory and immunosuppressive effects in multiple animal models, and such effects could contribute to the immunecompromised state of severely ill and massively transfused patients (Yazdanbakhsh et al 2011).…”