Hemopexin as an Inhibitor of Hemolysis-Induced Complement Activation.

Hemopexin is the main plasmatic scavenger of cell-free heme, released in the context of intravascular hemolysis or major cell injury. Heme is indispensable for the oxygen transport by hemoglobin but when released outside of the erythrocytes it becomes a danger-associated molecular pattern, contributing to tissue injury. One of the mechanisms of pro-inflammatory action of heme is to activate the innate immune complement cascade. Therefore, we hypothesized that injection of hemopexin will prevent hemolysis-induced complement activation. Human plasma-derived hemopexin is compatible with the heme clearance machinery of the mice. 100 or 500 mg/kg of hemopexin was injected in C57Bl/6 mice before treatment with phenylhydrazine (inducer of erythrocytes lysis) or with PBS as a control. Blood was taken at different timepoints to determine the pharmacokinetic of injected hemopexin in presence and absence of hemolysis. Complement activation was determined in plasma, by the C3 cleavage (western blot) and in the kidneys (immunofluorescence). Kidney injury was evaluated by urea and creatinine in plasma and renal NGAL and HO-1 gene expression were measured. The pharmacokinetic properties of hemopexin (mass spectrometry) in the hemolytic mice were affected by the target-mediated drug disposition phenomenon due to the high affinity of binding of hemopexin to heme. Hemolysis induced complement overactivation and signs of mild renal dysfunction at 6 h, which were prevented by hemopexin, except for the NGAL upregulation. The heme-degrading capacity of the kidney, measured by the HO-1 expression, was not affected by the treatment. These results encourage further studies of hemopexin as a therapeutic agent in models of diseases with heme overload.

In vivo fate of hemopexin and heme-hemopexin complexes in the rat.

The disposition in the rat of the plasma heme-binding protein hemopexin (Hx), as the native apoprotein and as its heme complex (HHx), has been studied using the residualizing protein label dilactitol-125I-tyramine (*I-DLT). The aim of this work was to identify the tissue sites of Hx uptake and catabolism, independent of heme binding, and to evaluate how heme loading affects Hx catabolism at these sites. *I-DLT-Hx had a circulating half-life of approximately 1.2 days and was recovered in degraded form in comparable amounts in visceral (liver, kidney, spleen) and peripheral (skin, muscle) tissues, indicating a generalized diffuse catabolism of the protein throughout the body. The plasma half-life of *I-DLT-Hx injected as a preformed heme-Hx complex was the same as that of the apoprotein; however, injection of the complex resulted in about a twofold increase in hepatic degradation of Hx. The lack of an effect of heme on overall catabolism of the preformed HHx complex was consistent with the approximately 1-h half-life of heme, injected as 14C-heme-Hx, in the circulation; however, as much as 20-fold more 14C-heme than Hx protein was recovered in liver from 14C-heme-Hx. The absolute amount of *I-DLT-Hx degraded in liver was significantly increased when heme was injected in excess of the heme binding capacity of circulating Hx, while 131I-DLT-albumin catabolism in liver was unaffected. Thus, depending on the physiological conditions studied, the data are consistent with a model in which, following hepatic uptake of heme from HHx, varying proportions of the protein are either returned to the circulation or degraded in the liver.

Expression of the haemopexin-transport system in cultured mouse hepatoma cells. Links between haemopexin and iron metabolism.

Minimal deviation hepatoma (Hepa) cells, from the mouse hepatoma B7756, synthesize and secrete haemopexin and express both the haemopexin receptor and the membrane haem-binding protein (MHBP) associated with the receptor, making this cell line the first available for detailed study of both haemopexin metabolism and hepatic transport. The 17.5 kDa MHBP was detected in Triton X-100 extracts of Hepa cells by immunoblotting with goat anti-rabbit MHBP. Scatchard-type analysis of haem-125I-haemopexin binding at 4 degrees C revealed 35,000 receptors per cell of high affinity (Kd 17 nM). Haemopexin-mediated haem transport at 37 degrees C is saturable, having an apparent Km of 160 nM and a Vmax. of 7.5 pmol of haem/10(6) cells per h during exponential growth. Haem-transport capacity is highest in the period just before the cells enter their exponential phase of growth and slowest in stationary phase. Interestingly, haem-haemopexin serves as effectively as iron-transferrin as the sole source of iron for cell growth by Hepa cells. Furthermore, depriving Hepa cells of iron by treatment with desferrioxamine (DF) increases the number of cell-surface haemopexin receptors to 65,000 per cell and consequently increases haemopexin-mediated haem transport. The effects of DF do not appear to require protein synthesis since they are not prevented by cycloheximide. Treatment of Hepa cells with hydroxyurea, an inhibitor of the iron-requiring enzyme ribonucleotide reductase that is obligatory for DNA synthesis, enhanced haemopexin-mediated haem transport. Thus, these studies provide the first evidence for regulation of haem transport by the iron status of cells and suggest a linkage between haemopexin, iron homeostasis and cell growth.