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Rationale:Cell-free hemoglobin (CFH) levels are elevated in septic shock and higher in non-survivors. Whether CFH is only a marker of sepsis severity or is involved in pathogenesis is unknown. Objective:To investigate whether CFH worsens sepsis-associated injuries and to determine potential mechanisms of harm. Methods&Results:Fifty-one, 10-12kg purpose-bred beagles were randomized to receive Staphylococcus aureus intrapulmonary challenges or saline followed by CFH infusions (oxyhemoglobin >80%) or placebo. Animals received antibiotics, and intensive care-support for 96h. CFH significantly increased mean pulmonary arterial pressures and right ventricular afterload in both septic and non-septic animals, effects that were significantly greater in non-survivors. These findings are consistent with CFH-associated nitric oxide (NO) scavenging and were associated with significantly depressed cardiac function, and worsened shock, lactate levels, metabolic acidosis and multi-organ failure. In septic animals only, CFH administration significantly increased mean alveolar-arterial oxygenation gradients, also to a significantly greater degree in non-survivors. CFH associated iron levels were significantly suppressed in infected animals, suggesting that bacterial iron uptake worsened the pneumonia. Notably, cytokine levels were similar in survivors and non-survivors and not predictive of outcome. Conclusions:In the absence and presence of infection, CFH infusions resulted in pulmonary hypertension, cardiogenic shock and multi-organ failure, likely through NO scavenging. In the presence of infection alone, CFH infusions worsened oxygen exchange and lung injury, presumably by supplying iron that promoted bacterial growth. CFH elevation, a known consequence of clinical septic shock, adversely impacts sepsis outcomes through more than one mechanism and is a biologically plausible, non-antibiotic, non-cytokine target for therapeutic intervention.

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Conflict of interestDrs. Rose, Wang, McTiernan, Tejero and Gladwin are shareholders in Globin Solutions, Inc. Dr. DeMartino is a consultant of Globin Solutions, Inc. Drs Rose, Xu, DeMartino, Dent, Tejero and Gladwin are coinventors of provisional, pending and granted patents for the use of recombinant neuroglobin and other heme-based molecules as antidotes for carbon monoxide poisoning (US 2019/0290739 A1, US 2022/0185867 A1). Drs. Rose and Tejero are officers and directors of Globin Solutions, Inc. Dr. Gladwin is a director and advisor of Globin Solutions, Inc. Globin Solutions, Inc. has licensed this technology. Globin Solutions had an option to technology directed at using hydroxycobalamin for carbon monoxide poisoning from Virginia Commonwealth University that has expired over 12 months ago. Dr. Gladwin is a coinventor on patents (US 2010/0247682 A1) directed to the use of nitrite salts in cardiovascular diseases licensed to Globin Solutions, Inc. Dr. Gladwin is a coinvestigator in a research collaboration with Bayer Pharmaceuticals to evaluate riociguat as a treatment for patients with sickle cell disease. Dr. Rose is a co-founder of Omnibus Medical Devices, LLC. and serves as an expert witness to medical legal cases not related to CO poisoning. The remaining authors have disclosed that they do not have any potential conflicts of interest. The financial conflicts of interest of JJR, JT, QX, LW, AWD, CFM and MTG were managed by the University of Pittsburgh Conflict of Interest Committee and a data stewardship committee.

Nitric oxide (NO) is an endogenously produced physiological signaling molecule that regulates blood flow and platelet activation. However, both the intracellular and intravascular diffusion of NO is severely limited by scavenging reactions with hemoglobin, myoglobin, and other hemoproteins, raising unanswered questions as to how free NO can signal in hemoprotein-rich environments, like blood and cardiomyocytes. We explored the hypothesis that NO could be stabilized as a ferrous heme-nitrosyl complex (Fe2+-NO, NO-ferroheme) either in solution within membranes or bound to albumin. Unexpectedly, we observed a rapid reaction of NO with free ferric heme (Fe3+) and a reduced thiol under physiological conditions to yield NO-ferroheme and a thiyl radical. This thiol-catalyzed reductive nitrosylation reaction occurs readily when the hemin is solubilized in lipophilic environments, such as red blood cell membranes, or bound to serum albumin. NO-ferroheme albumin is stable, even in the presence of excess oxyhemoglobin, and potently inhibits platelet activation. NO-ferroheme-albumin administered intravenously to mice dose-dependently vasodilates at low- to mid-nanomolar concentrations. In conclusion, we report the fastest rate of reductive nitrosylation observed to date to generate a NO-ferroheme molecule that resists oxidative inactivation, is soluble in cell membranes, and is transported intravascularly by albumin to promote potent vasodilation.