We developed 2 canine models of intravascular hemolysis in order to obtain mechanistic insight into the relationship among cell-free plasma Hb, NO bioavailability, and the systemic manifestations that can occur with Hb-mediated NO consumption. Importantly, in the first model, the total intravascular Hb level is unchanged; only the compartmentalization of Hb within the erythrocyte membrane is disrupted. These experiments demonstrated that hemolysis produces dose-dependent vasoconstriction and impaired renal function secondary to the stoichiometric oxidation of endogenous and exogenous NO by cell-free plasma oxyhemoglobin. Importantly, 80 ppm inhaled NO gas oxidized 85–90% of the plasma oxyhemoglobin to methemoglobin, which inhibited endogenous NO scavenging, prevented systemic vasoconstriction, and restored responsiveness to systemically infused NO donors. This observation confirms that the observed physiological effects of hemolysis are directly mediated by the dioxygenation reaction of ferrous oxyhemoglobin and NO (Reaction 1). These studies provide evidence for the existence of a syndrome of hemolysis-associated endothelial dysfunction and suggest a potential therapeutic role of inhaled NO for iatrogenic, acquired, and hereditary hemolytic diseases. Specifically, the consumption and loss of NO activity during intravascular hemolysis may contribute to clinical signs and symptoms attributable to vasomotor instability that may be ameliorated by NO gas inhalation.
The levels of plasma Hb produced in the present study (with concentration expressed in terms of heme group) are within the ranges observed in human disease states. For example, in patients with sickle cell disease, the levels of plasma oxyheme range from 2 to 20 μM, with mean levels of 4 μM (18
). During vasoocclusive crisis, levels can rise to 20–40 μM (27
). Intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria produces abdominal pain, gastric and esophageal dystonia, and erectile dysfunction, with levels of plasma Hb ranging from 30 to 120 μM (in heme concentration) and up to 600 μM during paroxysms of hemolysis (28
). During cardiopulmonary bypass, the levels of plasma oxyheme can rise to 150 μM (29
). The present studies suggest that these pathologically relevant levels of hemoglobinemia may be associated with endogenous NO inhibition, a vasopressor effect, and impaired organ function.
The biochemical and physiologic disturbances that occur during intravascular hemolysis are related to the disruption of diffusional barriers that typically regulate the reaction of intra-erythrocytic Hb with NO (11
). Cell-free plasma Hb can react with endothelial cell–produced NO at nearly diffusion-limited rates, resulting in the rapid dioxygenation of NO by Hb with the formation of nitrate and methemoglobin (6
). These rapid reactions greatly limit the diffusion of NO from endothelium to smooth muscle. Consequently, smooth muscle guanylyl cyclase is not activated, and vascular relaxation and vasodilation are inhibited (33
). Additionally, cell-free plasma Hb can rapidly extravasate into the spaces between endothelial and smooth muscles cells and further scavenge NO, thereby prohibiting it from diffusing into smooth muscle cells (10
). This mechanism is supported by the observed decrease in hypertensive effects of the Hb-based blood substitutes as molecular weight increases (7
) and the decrease in NO scavenging effects of the high-molecular-weight haptoglobin-Hb complex, compared with non–haptoglobin-bound Hb (34
). We have proposed that the formation of Hb-haptoglobin high-molecular-weight multimers serves to limit such extravasation during physiologic intravascular hemolysis (35
NO scavenging by cell-free plasma Hb may have contributed to the increased morbidity and mortality observed in studies of stroma-free Hb artificial blood substitute solutions (7
). The administration of the blood substitute solutions in preclinical and clinical trials led to pulmonary and systemic hypertension, increased systemic vascular resistance, decreased organ perfusion, gastrointestinal spasm and dysmotility, and death (7
). While studies of new generation Hb-based blood substitutes with heme pocket mutations designed to decrease the heme reactivity with NO have shown reduced vasopressor effects (6
), other investigators have suggested that the vasoconstrictor effects of Hb-based blood substitutes occur secondarily to premature oxygen unloading at the precapillary sphincter (24
). The current study provides additional evidence that NO consumption by oxyhemoglobin is a mechanism of cell-free Hb–mediated vasoconstriction and may contribute to the adverse effects observed in the blood substitute trials and during intravascular hemolysis in human disease.
In chronic hereditary hemolytic diseases such as sickle cell anemia, clinical complications such as pulmonary hypertension, priapism, leg ulcerations, and stroke may be in part related to repeated episodes of intravascular hemolysis with consequent increased NO scavenging, vasoconstriction, and end-organ hypoperfusion (18
). In patients with sickle cell disease, chronic hemolysis leads to the destruction of approximately 10% of the circulating erythrocytes every 24 hours, with 30% of this hemolysis estimated to be intravascular (18
). The Hb scavenging system in these patients becomes saturated, as indicated by undetectable plasma haptoglobin levels, and cell-free plasma Hb accumulates, as has been demonstrated by increased levels of plasma Hb in patients with sickle cell disease compared with normal patients (18
). The plasma of these patients consumes significantly more NO than the plasma of normal patients, and the amount of NO consumption correlates with the plasma heme levels (18
). Consistent with an NO-scavenging effect of increased cell-free plasma Hb, the vasodilatory responses to nitroprusside, nitroglycerin, and other NO donors are significantly blunted in sickle cell patients and in sickle cell transgenic mouse models (18
). Scavenging of NO by cell-free plasma Hb may be involved in the pathophysiologic vasculopathy and prothrombotic state that occur in many chronic hereditary hemolytic diseases such as sickle cell disease and thalassemia (18
) and acute hemolytic disease states, such as prolonged cardiopulmonary bypass, thrombotic thrombocytopenic purpura, and malaria. These studies provide further evidence that inhaled NO therapy may attenuate the NO-scavenging effects of cell-free plasma Hb and may be able to block the pathophysiologic changes that occur during iatrogenic (cardiopulmonary bypass) or disease-specific (sickle cell pain crisis, malaria, etc.) intravascular hemolysis in many human diseases. Further research is required to assess the contribution of hemolysis and therapeutic utility of inhaled NO therapy in hereditary and iatrogenic hemolytic disorders.
In conclusion, these data provide controlled in vivo evidence that NO scavenging by cell-free plasma Hb during intravascular hemolysis disrupts endothelial NO-dependent vasomotor function and produces systemic physiologic changes and organ dysfunction, which are attenuated by inhaled NO therapy. These biochemical and physiological studies support the existence of a syndrome of hemolysis-associated endothelial dysfunction, which may contribute to the vasculopathy of hereditary, acquired, and iatrogenic hemolytic states. Furthermore, these studies support a potential therapeutic role for NO donor agents in preventing the end-organ injury associated with these disease states.