BackgroundHemerythrins, are the non-heme, diiron binding respiratory proteins of brachiopods, priapulids and sipunculans; they are also found in annelids and bacteria, where their functions have not been fully elucidated.ResultsA search for putative Hrs in the genomes of 43 archaea, 444 bacteria and 135 eukaryotes, revealed their presence in 3 archaea, 118 bacteria, several fungi, one apicomplexan, a heterolobosan, a cnidarian and several annelids. About a fourth of the Hr sequences were identified as N- or C-terminal domains of chimeric, chemotactic gene regulators. The function of the remaining single domain bacterial Hrs remains to be determined. In addition to oxygen transport, the possible functions in annelids have been proposed to include cadmium-binding, antibacterial action and immunoprotection. A Bayesian phylogenetic tree revealed a split into two clades, one encompassing archaea, bacteria and fungi, and the other comprising the remaining eukaryotes. The annelid and sipunculan Hrs share the same intron-exon structure, different from that of the cnidarian Hr.ConclusionThe phylogenomic profile of Hrs demonstrated a limited occurrence in bacteria and archaea and a marked absence in the vast majority of multicellular organisms. Among the metazoa, Hrs have survived in a cnidarian and in a few protostome groups; hence, it appears that in metazoans the Hr gene was lost in deuterostome ancestor(s) after the radiata/bilateria split. Signal peptide sequences in several Hirudinea Hrs suggest for the first time, the possibility of extracellular localization. Since the α-helical bundle is likely to have been among the earliest protein folds, Hrs represent an ancient family of iron-binding proteins, whose primary function in bacteria may have been that of an oxygen sensor, enabling aerophilic or aerophobic responses. Although Hrs evolved to function as O2 transporters in brachiopods, priapulids and sipunculans, their function in annelids remains to be elucidated. Overall Hrs exhibit a considerable lack of evolutionary success in metazoans.

Background The resolution of deep vein thrombosis (DVT) requires an inflammatory response and mobilization of proteases, such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs), to degrade the thrombus and remodel the injured vein wall. PAI-2 is a serine protease inhibitor (serpin) with unique immunosuppressive and cell survival properties that was originally identified as an inhibitor of uPA. Objective To investigate the role of PAI-2 in venous thrombus formation and resolution. Methods Venous thrombus resolution was compared in wild type C57BL/6, PAI-2 -/- and PAI-1 -/- mice using the stasis model of DVT. Formed thrombi were harvested, thrombus weights were recorded, and tissue was analyzed for uPA, and MMP activities, PAI-1 expression, and the nature of inflammatory cell infiltration. Results We found that absence of PAI-2 enhanced venous thrombus resolution, while thrombus formation was unaffected. Enhanced venous thrombus resolution in PAI-2 -/- mice was associated with increased uPA activity and reduced levels of PAI-1, with no significant effect on MMP-2 and -9 activities. PAI-1 deficiency resulted in an increase in thrombus resolution similar to PAI-2 deficiency, but additionally reduced venous thrombus formation and altered MMP activity. PAI-2 deficient thrombi had increased levels of the neutrophil chemoattractant, CXCL2, which was associated with early enhanced neutrophil recruitment. Conclusions These data identify PAI-2 as a novel regulator of venous thrombus resolution, which modulates several pathways involving both inflammatory and uPA activity mechanisms, distinct from PAI-1. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution.

The giant extracellular hexagonal bilayer haemoglobins (HBL-Hbs), found in most terrestrial, aquatic, shallowwater and deep-sea annelids (including vestimentiferans) are complexes of globin and nonglobin linker chains, of 3.6 MDa. They represent a summit of complexity for oxygen-binding haem proteins [1,2] and a remarkable hierarchical organization, as evidenced by the crystal structure of Lumbricus Hb [3]. A model of the quaternary structure of Arenicola marina HBL-Hb has been proposed by Zal and collaborators based on electrospray ionization (ESI)-MS analysis and multiangle laser light scattering (MALLS) measurements [4]. The authors provided an inventory of the constituting polypeptide chains and identified the existence of 10 subunits (eight of which are globins), including two monomers (a 1 and a 2 ) of 15 kDa, and five disulfide-bonded trimers ( 49 kDa). The remaining two chains are linkers that are disulfide bonded to form homo-and heterodimers ( 50 kDa). These latter polypeptide chains are essential for maintaining the integrity of the HBL-Hb molecule [5,6]. Three and six copies of each of the two monomer subunits, and one copy of the trimer, form a dodecamer subunit [(a 1 ) 3 (a 2 ) 6 T], of a mean mass close to 200 kDa. The molecular mass of the dodecamer subunit has been determined, by ESI-MS, to be 204 ± 0.08 kDa [7], which is in good agreement with the model of the quaternary structure proposed by Zal and collaborators [4]. Twelve such complexes of globin chains are linked together by 42 linker chains to reach a total mass of 3648 ± 24 kDa. Therefore, each of the 12 subunits of the whole molecule is then associated to an average of 3.5 linkers, leading to the overall formula [(a 1 ) 3 (a 2 ) 6 T]L 3.5 . The extracellular haemoglobin of the marine polychaete, Arenicola marina, is a hexagonal bilayer haemoglobin of 3600 kDa, formed by the covalent and noncovalent association of many copies of both globin subunits (monomer and trimer) and nonglobin or 'linker' subunits. In order to analyse the interactions between globin and linker subunits, dissociation and reassociation experiments were carried out under whereby Arenicola hexagonal bilayer haemoglobin was exposed to urea and alkaline pH and the effect was followed by gel filtration, SDS ⁄ PAGE, UV-visible spectrophotometry, electrospray-ionization MS, multiangle laser light scattering and transmission electron microscopy. The analysis of Arenicola haemoglobin dissociation indicates a novel and complex mechanism of dissociation compared with other annelid extracellular haemoglobins studied to date. Even though the chemically induced dissociation triggers partial degradation of some subunits, spontaneous reassociation was observed, to some extent. Parallel dissociation of Lumbricus haemoglobin under similar conditions shows striking differences that allow us to propose a hypothesis on the nature of the intersubunit contacts that are essential to form and to hold such a complex quaternary structure.Abbreviations ESI, electrospray ionization; Hb, haemogl...

Globins are the most common type of oxygen‐binding protein in annelids. In this paper, we show that circulating intracellular globin (Alvinella pompejana and Glycera dibranchiata), noncirculating intracellular globin (Arenicola marina myoglobin) and extracellular globin from various annelids share a similar gene structure, with two conserved introns at canonical positions B12.2 and G7.0. Despite sequence divergence between intracellular and extracellular globins, these data strongly suggest that these three globin types are derived from a common ancestral globin‐like gene and evolved by duplication events leading to diversification of globin types and derived functions. A phylogenetic analysis shows a distinct evolutionary history of annelid extracellular hemoglobins with respect to intracellular annelid hemoglobins and mollusc and arthropod extracellular hemoglobins. In addition, dehaloperoxidase (DHP) from the annelid, Amphitrite ornata, surprisingly exhibits close phylogenetic relationships to some annelid intracellular globins. We have characterized the gene structure of A. ornata DHP to confirm assumptions about its homology with globins. It appears that it has the same intron position as in globin genes, suggesting a common ancestry with globins. In A. ornata, DHP may be a derived globin with an unusual enzymatic function.