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.

Background: Redox activity of hemoglobin (Hb) is augmented by lipopolysaccharide (LPS) to boost immune defense. Results: Computational analysis of Hb identified LPS-binding hot spots, which were further defined via peptide-based binding assays and confirmed by mutagenesis of Hb subunits. Conclusion: Regions of Hb that interact with LPS have been delineated. Significance: Knowledge of LPS-binding residues on Hb may be exploited for designing antimicrobial peptides.

biodegradation. Their paramount role in the degradation of toxic substances is the main interest in studying their mechanism of action. Pp 1,2-CCD belongs to the intradiol dioxygenase family, whose structures of its members have been only recently determined. In all those structures, an unexpected and interesting feature came up: electron density for two phospholipid molecules was found at the dimer interface, bringing up issues related to the relevance of those amphipatic molecules to enzyme catalysis. In this work, we present a series of results from our group focused on the characterization of the phospholipid binding and also on its role in enzyme function. We investigate, using a combination of experimental techniques (ESR, DSC, ITC, CD, UV-Vis absorbance), Pseudomonas putida chlorocatechol 1,2-dioxygenase (Pp 1,2-CCD) before and after delipidation procedures. Our results show that: (1) Fe(III) ion is converted to Fe(II) during catalysis and that this iron site in not influenced by the presence/absence of the phospholipid molecules; (2) Pp 1,2-CCD does not interact with several lipid model membranes; (3) delipidated Pp 1,2-CCD presents a cooperative kinetics, thus differing from lipid-containing Pp 1,2-CCD, which follows a usual Michaelis-Menten kinetics; (4) the product of the catalysis is capable of inhibiting the reaction; (5) delipidation leads to a somewhat more stable enzyme, with higher thermal-transition temperature and enthalpy. We discuss those findings in terms of Pp 1,2-CCD crystal structure that we have recently determined. These results, although regarding more fundamental aspects of the activity, shed light on features of Pp 1,2-CCD function that can be relevant towards its use in bioremediation/biotechnological processes.