Hemolysis-Endothelial Dysfunction Syndrome: A Novel Paradigm of Disease Mechanism.
Authors: Minneci Peter C. , Deans Katherine J. , Zhi Huang , Cushenberry Katherine , Grant Peter , Banks Steven M. , Natanson Charles , Gladwin Mark T. , Solomon Steven B. 
Published: October 2019

Abstract
Background: Normal vasomotor tone is maintained by the production of nitric oxide (NO) from endothelial-derived nitric oxide synthase in excess of consumption of NO within the vasculature by intra-erythrocytic hemoglobin. NO reacts up to 600-fold less rapidly when hemoglobin is compartmentalized within the erythrocyte rather than in a cell free form (i.e. cell-free plasma hemoglobin). This implies that during intravascular hemolysis, NO consumption by hemoglobin would be greatly increased. We have proposed that NO consumption and loss of normal vasomotor tone contributes to the pathogenesis of sickle cell disease. However, the increased reactivity of cell-free plasma hemoglobin, released during hemolysis, compared to intra-erythrocytic hemoglobin and the physiologic consequences of hemolysis on nitric oxide consumption have not been definitively proven in vivo.
Methods: We have developed a canine model of free water induced intravascular hemolysis to test this mechanism and to examine whether inhaled NO can selectively oxidize the cell free plasma hemoglobin and thus attenuate any associated physiologic changes. Paired experiments were performed in each animal which included a 6-hour control infusion of 5% Dextrose (D5W) followed one week later by a 6-hour infusion of either D5W or free water with or without 80 ppm inhaled NO (full factorial design). Based on pilot experiments, infusions of free water or D5W (control) were administered at a rate of 16 ml/kg/hr. At the end of each experiment, an escalating sodium nitroprusside infusion, a direct NO donor (1 to 27 mcg/kg/min), was administered to evaluate the vascular responsiveness to NO in the presence and absence of hemolysis. In this model, total intravascular hemoglobin concentration remains constant, whereas the ratio of plasma to intracellular hemoglobin concentration increases with hemolysis.
Results: In comparison to D5W, free water infusion caused linear increases in plasma hemoglobin (25–50 mM/h) which remained ferrous (FeII) and correlated with increased plasma nitric oxide consumption (R = 0.95; p<0.001). This free water induced intravascular hemolysis caused significant increases in mean systemic arterial pressure (MAP; p=0.0003) and systemic vascular resistance index (SVRI; p=0.0003) and a significant decrease in cardiac index (CI; p=0.02) compared to an equivalent infusion of D5W. In the setting of hemolysis, concurrent inhalation of nitric oxide gas converted the cell free plasma hemoglobin to methemoglobin (50–90% of total plasma hemoglobin), inhibited NO consumption by plasma (methemoglobin does not oxidize NO to nitrate), and normalized hemodynamic parameters. Furthermore, free water induced hemolysis led to blunted hemodynamic effects of sodium nitroprusside on MAP, CI, and SVRI, which were restored with inhaled nitric oxide therapy (p=0.01, p=0.09, and p=0.003 respectively). This further supports the thesis that cell-free plasma hemoglobin consumes NO in the vasculature.
Conclusion: These data support the hypothesis that NO scavenging by cell-free plasma hemoglobin disrupts endothelial NO-dependent vasomotor function producing systemic physiologic changes which are attenuated by inhaled nitric oxide therapy. These biochemical and physiological studies support the existence of a hemolysis-endothelial dysfunction syndrome, which may contribute to the vasculopathy of hereditary, acquired and iatrogenic hemolytic states.