Abstract
Acute organ failure is a major clinical concern in sickle cell disease (SCD). However, the mechanism responsible for this potentially lethal complication is poorly understood. We tested the hypothesis that extracellular hemin liberates an intracellular danger molecule that promotes acute organ failure in SCD. Transgenic homozygous SCD (SS), sickle-trait (AS) and normal human hemoglobin (Hb) AA mice were infused with purified hemin (35 µmoles/kg), which raised total plasma hemin by ∼0.45 mM (equivalent to 0.72 g/dl Hb) within 5 min in all three groups of mice. In agreement with our previous results, SS but not AA and AS mice (n= 6 for each genotype) developed cardiopulmonary depression at 30 min evident by reductions in oxygen saturation (99.88±0.23% to 92.1±1.3%, p<0.001), breath rate (175.4±20.6 to 77.36±2.25, p<0.001, breath per min), heart rate (574.5±22.7 to 361.9±23.25 beats per min, p<0.001) and pulse distension (512.8±18.7 to 238.8±17.6 µm, p<0.001), and ∼70% of these animals died within 2 hours. Markedly raised lung wet/dry weight ratio in SS mice that succumbed to hemin suggests that the cardiopulmonary depression was secondary to a severe pulmonary edema. To identify biological correlates for the acute adverse effects in the SS mice, cohorts of both sickle and control mice were challenged with the same dose of hemin, blood samples were drawn at baseline (i.e. time=0 min), and 5 and 30 min after the hemin infusion and analyzed for markers of oxidative stress, tissue damage, plasma scavengers and high mobility group box-1 (HMGB-1), a prototypical danger molecule. Plasma hemopexin decreased by ∼80% at 5 min compared to baseline values in all three groups of mice regardless of the Hb genotype. The catabolism of hemopexin was associated with clearance of ∼50% of the hemin infusion from the circulation of AS and AA mice at 30 min. Paradoxically, the plasma concentration of hemin in the SS mice during this same time interval increased by ∼0.2 mM (p<0.001, n=6). The magnitude of this increase was dependent on the dose of hemin administered exogenously. We discovered that the de novo hemin release in the SS mice was preceded by acute intravascular hemolysis (mean decrease in total Hb: ∼1.4 g/dl, p<0.001, n=9, mean increase in cell-free Hb: 1.0 g/dl, p=0.001, n=9), oxidation of oxyHbS to metHbS (mean increase: 12%, p<0.001, n=6) and persistence of metHbS. It is noteworthy that de novo hemin release did not occur in AS mice suggesting that this phenomenon is dominantly influenced by sickle erythrocytes and not by the presence of intracellular HbS per se. Auto-amplification of hemin may help to explain an observation made nearly fifty years ago that SCD patient plasma contains more hemin than the plasma of patients with more severe intravascular hemolysis involving normal adult Hb (e.g. paroxysmal nocturnal hemoglobinuria), who have higher plasma Hb. To determine whether this phenomenon is critical to the cardiopulmonary depression in the SS mice, recombinant human hemopexin was administered 5 min after the infusion to sequester the endogenous hemin release. In hemin challenged SS mice with respiratory distress, intravenous recombinant human hemopexin rapidly halted the decline in oxygen saturation and breath rate and averted inevitable respiratory failure. In conclusion, we have identified a phenomenon of extracellular hemin auto-amplification that appears to be unique to SCD, and may play a critical role in propagating tissue injury in this disorder. Factors that inhibit erythrocyte lysis and accelerate metHb reduction may help to limit extracellular hemin amplification and preserve organ function during episodes of acute exacerbations in SCD.
Disclosures:
No relevant conflicts of interest to declare.
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