Sepsis is defined as the systemic, dysregulated host immune response to an infection that leads to injury to host organ systems and, often, death. Complex interactions between pathogens and their hosts elicit microcirculatory dysfunction. Neutrophil myeloperoxidase (MPO) is critical for combating pathogens, but MPO-derived hypochlorous acid (HOCl) can react with host molecular species as well. Plasmalogens are targeted by HOCl, leading to the production of 2-chlorofatty acids (2-CLFAs). 2-CLFAs are associated with human sepsis mortality, decrease in vitro endothelial barrier function, and activate human neutrophil extracellular trap formation. Here, we sought to examine 2-CLFAs in an in vivo rat sepsis model. Intraperitoneal cecal slurry sepsis with clinically relevant rescue therapies led to ∼73% mortality and evidence of microcirculatory dysfunction. Plasma concentrations of 2-CLFAs assessed 8 h after sepsis induction were lower in rats that survived sepsis than in nonsurvivors. 2-CLFA levels were elevated in kidney, liver, spleen, lung, colon, and ileum in septic animals. In vivo, exogenous 2-CLFA treatments increased kidney permeability, and in in vitro experiments, 2-CLFA also increased epithelial surface expression of vascular cell adhesion molecule 1 and decreased epithelial barrier function. Collectively, these studies support a role of free 2-CLFAs as biomarkers of sepsis mortality, potentially mediated, in part, by 2-CLFA-elicited endothelial and epithelial barrier dysfunction.

High incidence of bleeding events remains a key risk for patients taking anticoagulants, especially those in need of long-term combination therapy with antiplatelet agents. As a consequence, patients may not receive clinically indicated combination antithrombotic therapy. Here, we report on VE-1902, a member of a novel class of precision oral anticoagulants (PROACs) that combines effective anticoagulation with reduced bleeding in preclinical testing.

The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Specifically, it is unclear whether the free protease is zymogen-like and shifts to its mature form upon a ligand-induced fit or exists in multiple conformations in equilibrium from which the ligand selects the optimal fit via conformational selection. Here we report the results of F NMR measurements that reveal the conformational properties of a protease and its zymogen precursor in the free form. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding. Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. In this scenario, prethrombin-2 is more dynamic and exists predominantly in the E* form, whereas thrombin is more rigid and exists predominantly in the E form. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation. In summary, these results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*-E equilibrium and providing no evidence that free thrombin is zymogen-like.

We previously reported that the highly reactive cell-free heme (CFH) is increased in the plasma of patients with chronic lung injury and causes pulmonary edema in animal model of acute respiratory distress syndrome (ARDS) post inhalation of halogen gas. However, the mechanisms by which CFH causes pulmonary edema are unclear. Herein we report for the first time that CFH and chlorinated lipids (formed by the interaction of halogen gas, Cl, with plasmalogens) are increased in the plasma of patients exposed to Cl gas. Ex vivo incubation of red blood cells (RBC) with halogenated lipids caused oxidative damage to RBC cytoskeletal protein spectrin, resulting in hemolysis and release of CFH. Patch clamp and short circuit current measurements revealed that CFH inhibited the activity of amiloride-sensitive epithelial Na channel (ENaC) and cation sodium (Na) channels in mouse alveolar cells and trans-epithelial Na transport across human airway cells with EC of 125 nM and 500 nM, respectively. Molecular modeling identified 22 putative heme-docking sites on ENaC (energy of binding range: 86-1563 kJ/mol) with at least 2 sites within its narrow transmembrane pore, potentially capable of blocking Na transport across the channel. A single intramuscular injection of the heme-scavenging protein, hemopexin (4 μg/kg body weight), one hour post halogen gas exposure, decreased plasma CFH and improved lung ENaC activity in mice. In conclusion, results suggested that CFH mediated inhibition of ENaC activity may be responsible for pulmonary edema post inhalation injury.

β-glycoprotein I (βGPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a life-threatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound βGPI to that in solution. βGPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, βGPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and O-circular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant βGPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular nor twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound βGPI arises from the ability of its pre-existing J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of βGPI may provide a strategy to block pathogenic aPLs in APS.

Bromine (Br) is an organohalide found in nature and is integral to many manufacturing processes. Br is toxic to living organisms and high concentrations can prove fatal. To meet industrial demand large amounts of purified Br are produced, transported, and stored worldwide providing a multitude of interfaces for potential human exposure through either accidents or terrorism. To identify the key mechanisms associated with acute Br exposure, we havesurveyed the lung proteomes of C57BL/6 male mice and human lung derived microvascular endothelial cells (HMEC's) at 24 hrs following exposure to Br in concentrations likely to be encountered in the vicinity of industrial accidents. Global discovery proteomics applications, combined with systems biology analysis identified robust and highly significant changes in proteins associated with three biological processes: 1. exosome secretion, 2. inflammation, and 3. vascular permeability. We focused on the latter, conducting physiological studies on isolated perfused lungs harvested from mice 24 hrs post Br exposure. These experiments revealed significant increases in the filtration coefficient (Kf) indicating increased permeability of the pulmonary vasculature. Similarly, confluent monolayers of Br and Br-lipid treated HMEC's exhibited differential levels of ZO-1 that found to be dissociated from cell wall localization, an increase in phosphorylation and internalization of E-cadhedrin, as well as increased actin stress fiber formation, all of which are consistent with increased permeability. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with physiological measurements of permeability, revealed both profound and novel biological changes that contribute to our current understanding of Br toxicity.

Many metabolic diseases disrupt endoplasmic reticulum (ER) homeostasis, but little is known about how metabolic activity is communicated to the ER. Here, we show in hepatocytes and other metabolically active cells that decreasing the availability of substrate for the tricarboxylic acid (TCA) cycle diminished NADPH production, elevated glutathione oxidation, led to altered oxidative maturation of ER client proteins, and attenuated ER stress. This attenuation was prevented when glutathione oxidation was disfavored. ER stress was also alleviated by inhibiting either TCA-dependent NADPH production or Glutathione Reductase. Conversely, stimulating TCA activity increased NADPH production, glutathione reduction, and ER stress. Validating these findings, deletion of the Mitochondrial Pyruvate Carrier-which is known to decrease TCA cycle activity and protect the liver from steatohepatitis-also diminished NADPH, elevated glutathione oxidation, and alleviated ER stress. Together, our results demonstrate a novel pathway by which mitochondrial metabolic activity is communicated to the ER through the relay of redox metabolites.

Endothelial activation and dysfunction are hallmarks of inflammation. Neutrophil-vascular endothelium interactions have significant effects on vascular wall physiology and pathology. Myeloperoxidase (MPO)-derived products released from activated neutrophils can mediate the inflammatory response and contribute to endothelial dysfunction. 2-Chlorofatty aldehyde (2-ClFALD) is the direct oxidation product of MPO-derived hypochlorous acid (HOCl) targeting plasmalogen phospholipids. The role of 2-ClFALD in endothelial dysfunction is poorly understood and may be dependent on the vascular bed. This study compared the role of 2-ClFALD in eliciting endothelial dysfunction in human coronary artery endothelial cells (HCAEC), human lung microvascular endothelial cells (HLMVEC), and human kidney endothelial cells (HKEC). Profound increases in selectin surface expression as well as ICAM-1 and VCAM-1 surface expression were observed in HCAEC and HLMVEC. The surface expression of these adherence molecules resulted in robust adherence of neutrophils and platelets to 2-ClFALD treated endothelial cells. In contrast to HCAEC and HLMVEC, 2-ClFALD-treated HKEC had substantially reduced adherence molecule surface expression with no resulting increase in platelet adherence. 2-ClFALD-treated HKEC did have an increase in neutrophil adherence. All three endothelial cell lines treated with 2-ClFALD displayed a time-dependent loss of barrier function. Further studies revealed 2-ClHDyA localizes to ER and Golgi when using a synthetic alkyne analog of 2-ClFALD in HCAEC and HLMVEC. These findings indicate 2-ClFALDs promote endothelial cell dysfunction with disparate degrees of responsiveness depending on the vascular bed of origin.