Extracellular haemoglobin upregulates and binds to tissue factor on macrophages: Implications for coagulation and oxidative stress

Abstract

The mechanisms of crosstalk between haemolysis, coagulation and innate immunity are evolutionarily conserved from the invertebrate haemocyanin to the vertebrate haemoglobin (Hb). In vertebrates, extracellular Hb resulting from haemolytic infections binds bacterial lipopolysaccharide (LPS) to unleash the antimicrobial redox activity of Hb. Because bacterial invasion also upregulates tissue factor (TF), the vertebrate coagulation initiator, we asked whether there may be functional interplay between the redox activity of Hb and the procoagulant activity of TF. Using real-time PCR, TF-specific ELISA, flow cytometry and TF activity assay, we found that Hb upregulated the expression of functional TF in macrophages. ELISA, flow cytometry and immunofluorescence microscopy showed binding between Hb and TF, in isolation and in situ. Bioinformatic analysis of Hb and TF protein sequences showed co-evolution across species, suggesting that Hbβ binds TF. Empirically, TF suppressed the LPS-induced activation of Hb redox activity. Furthermore, Hb desensitised TF to the effects of antioxidants like glutathione or serum. This bi-directional regulation between Hb and TF constitutes a novel link between coagulation and innate immunity. In addition, induction of TF by Hb is a potentially central mechanism for haemolysis to trigger coagulation.

The effect of ventricular assist device‐associated biomaterials on human blood leukocytes

“…A time course was performed by sampling at 2, 4, and 8 h; the 2 h samples were deemed most appropriate to continue with for further analysis as longer incubation showed high levels of cell death and platelet activation/aggregation with little difference between biomaterials. As plasma‐free haemoglobin (pfHB) can upregulate tissue factor expression on macrophages haemolysis in all the samples was measured using the Harboe assay to check the condition of the red blood cells before progressing to further experiments.…”

New perspectives on the thrombotic complications of haemolysis

“…Stimulating macrophages with increasing concentration of free haemoglobin up-regulates tissue factor expression and increases pro-coagulant activity in a dose-dependent manner (Bahl et al, 2014). Antioxidant treatments decrease ROS levels in macrophages and significantly inhibit tissue factor up-regulation, suggesting that the latter is partly mediated by ROS.…”

“…Antioxidant treatments decrease ROS levels in macrophages and significantly inhibit tissue factor up-regulation, suggesting that the latter is partly mediated by ROS. Surprisingly, there is both computational evolutionary evidence and in vitro evidence for an interaction between free haemoglobin and tissue factor (Bahl et al, 2014). Although the significance of this interaction in vivo remains to be determined, in vitro evidence suggests that, under certain circumstances, this interaction may alter the redox sensitivity of tissue factor to help limit oxidative damage to the host while maintaining pro-coagulant function (Bahl et al, 2014).…”

Immunological properties of oxygen-transport proteins: hemoglobin, hemocyanin and hemerythrin

“…Having said that, plasma Hb is an important warning to white blood cells (WBCs) that homeostasis has been compromised, and recently, Bahl et al outlined a novel role for Hb in blood coagulation [84]. Macrophages responded to the presence of cell-free Hb by triggering the expression of the vertebrate pro-clotting initiator, tissue factor.…”

“…glutathione), and reciprocally, the pro-oxidative potential of Hb was suppressed to mitigate collateral damage to the host’s cells. Infection-induced hemolysis and the liberation of Hb promote downstream pro-inflammatory and pro-clotting reactions [84] (Fig. 2).…”