Christine C. Helms

Mortality increases after massive exchange transfusion with older stored blood in canines with experimental pneumonia

Two-year-old purpose-bred beagles (n = 24) infected with Staphylococcus aureus pneumonia were randomized in a blinded fashion for exchange transfusion with either 7- or 42-day-old canine universal donor blood (80 mL/kg in 4 divided doses). Older blood increased mortality (P = .0005), the arterial alveolar oxygen gradient (24-48 hours after infection; P ≤ .01), systemic and pulmonary pressures during transfusion (4-16 hours) and pulmonary pressures for ~ 10 hours afterward (all P ≤ .02). Further, older blood caused more severe lung damage, evidenced by increased necrosis, hemorrhage, and thrombosis (P = .03) noted at the infection site postmortem. Plasma cell–free hemoglobin and nitric oxide (NO) consumption capability were elevated and haptoglobin levels were decreased with older blood during and for 32 hours after transfusion (all P ≤ .03). The low haptoglobin (r = 0.61; P = .003) and high NO consumption levels at 24 hours (r = −0.76; P < .0001) were associated with poor survival. Plasma nontransferrin-bound and labile iron were significantly elevated only during transfusion (both P = .03) and not associated with survival (P = NS). These data from canines indicate that older blood after transfusion has a propensity to hemolyze in vivo, releases vasoconstrictive cell-free hemoglobin over days, worsens pulmonary hypertension, gas exchange, and ischemic vascular damage in the infected lung, and thereby increases the risk of death from transfusion.

Mechanisms of hemolysis-associated platelet activation.

Intravascular hemolysis occurs after blood transfusion, in hemolytic anemias, and in other conditions, and is associated with hypercoagulable states. Hemolysis has been shown to potently activate platelets in vitro and in vivo, and several mechanisms have been suggested to account for this, including: (i) direct activation by hemoglobin (Hb); (ii) increase in reactive oxygen species (ROS); (iii) scavenging of nitric oxide (NO) by released Hb; and (iv) release of intraerythrocytic ADP.

Dietary nitrate supplementation improves exercise performance and decreases blood pressure in COPD patients.

Dietary nitrate (NO3(-)) supplementation via beetroot juice has been shown to increase the exercise capacity of younger and older adults. The purpose of this study was to investigate the effects of acute NO3(-) ingestion on the submaximal constant work rate exercise capacity of COPD patients. Fifteen patients were assigned in a randomized, single-blind, crossover design to receive one of two treatments (beetroot juice then placebo or placebo then beetroot juice). Submaximal constant work rate exercise time at 75% of the patients maximal work capacity was the primary outcome. Secondary outcomes included plasma NO3(-) and nitrite (NO2(-)) levels, blood pressure, heart rate, oxygen consumption (VO2), dynamic hyperinflation, dyspnea and leg discomfort. Relative to placebo, beetroot ingestion increased plasma NO3(-) by 938% and NO2(-) by 379%. Median (+interquartile range) exercise time was significantly longer (p = 0.031) following the ingestion of beetroot versus placebo (375.0 + 257.0 vs. 346.2 + 148.0 s, respectively). Compared with placebo, beetroot ingestion significantly reduced iso-time (p = 0.001) and end exercise (p = 0.008) diastolic blood pressures by 6.4 and 5.6 mmHg, respectively. Resting systolic blood pressure was significantly reduced (p = 0.019) by 8.2 mmHg for the beetroot versus the placebo trial. No other variables were significantly different between the beetroot and placebo trials. These results indicate that acute dietary NO3(-) supplementation can elevate plasma NO3(-) and NO2(-) concentrations, improve exercise performance, and reduce blood pressure in COPD patients.

Mechanisms of Human Erythrocytic Bioactivation of Nitrite

Background: Erythrocytes contribute to nitrite-mediated NO signaling, but the mechanism is unclear. Results: Deoxyhemoglobin accounts for virtually all NO made from nitrite by erythrocytes with no contributions from other proposed pathways. Conclusion: Deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions. Significance: Reduction by deoxyhemoglobin accounts for nitrite-mediated NO signaling in blood mediating vessel tone and platelet function. Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO2 are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood.

α−α Cross-Links Increase Fibrin Fiber Elasticity and Stiffness

Fibrin fibers, which are ~100 nm in diameter, are the major structural component of a blood clot. The mechanical properties of single fibrin fibers determine the behavior of a blood clot and, thus, have a critical influence on heart attacks, strokes, and embolisms. Cross-linking is thought to fortify blood clots; though, the role of α-α cross-links in fibrin fiber assembly and their effect on the mechanical properties of single fibrin fibers are poorly understood. To address this knowledge gap, we used a combined fluorescence and atomic force microscope technique to determine the stiffness (modulus), extensibility, and elasticity of individual, uncross-linked, exclusively α-α cross-linked (γQ398N/Q399N/K406R fibrinogen variant), and completely cross-linked fibrin fibers. Exclusive α-α cross-linking results in 2.5× stiffer and 1.5× more elastic fibers, whereas full cross-linking results in 3.75× stiffer, 1.2× more elastic, but 1.2× less extensible fibers, as compared to uncross-linked fibers. On the basis of these results and data from the literature, we propose a model in which the α-C region plays a significant role in inter- and intralinking of fibrin molecules and protofibrils, endowing fibrin fibers with increased stiffness and elasticity.

Plasma nitrate and nitrite are increased by a high-nitrate supplement but not by high-nitrate foods in older adults

Little is known about the effect of dietary nitrate on the nitrate/nitrite/nitric oxide cycle in older adults. We examined the effect of a 3-day control diet vs high-nitrate diet, with and without a high-nitrate supplement (beetroot juice), on plasma nitrate and nitrite kinetics and blood pressure using a randomized 4-period crossover controlled design. We hypothesized that the high-nitrate diet would show higher levels of plasma nitrate/nitrite and lower blood pressure compared with the control diet, which would be potentiated by the supplement. Participants were 8 normotensive older men and women (5 female, 3 male, 72.5 ± 4.7 years old) with no overt disease or medications that affect nitric oxide metabolism. Plasma nitrate and nitrite levels and blood pressure were measured before and hourly for 3 hours after each meal. The mean daily changes in plasma nitrate and nitrite were significantly different from baseline for both control diet + supplement (P < .001 and P = .017 for nitrate and nitrite, respectively) and high-nitrate diet + supplement (P = .001 and P = .002), but not for control diet (P = .713 and P = .741) or high-nitrate diet (P = .852 and P = .500). Blood pressure decreased from the morning baseline measure to the three 2-hour postmeal follow-up time points for all treatments, but there was no main effect for treatment. In healthy older adults, a high-nitrate supplement consumed at breakfast elevated plasma nitrate and nitrite levels throughout the day. This observation may have practical utility for the timing of intake of a nitrate supplement with physical activity for older adults with vascular dysfunction.

Hemoglobin-Mediated Nitric Oxide Signaling

The rate that hemoglobin reacts with nitric oxide (NO) is limited by how fast NO can diffuse into the heme pocket. The reaction is as fast as any ligand/protein reaction can be and the result, when hemoglobin is in its oxygenated form, is formation of nitrate in what is known as the dioxygenation reaction. As nitrate, at the concentrations made through the dioxygenation reaction, is biologically inert, the only role hemoglobin was once thought to play in NO signaling was to inhibit it. However, there are now several mechanisms that have been discovered by which hemoglobin may preserve, control, and even create NO activity. These mechanisms involve compartmentalization of reacting species and conversion of NO from or into other species such as nitrosothiols or nitrite which could transport NO activity. Despite the tremendous amount of work devoted to this field, major questions concerning precise mechanisms of NO activity preservation as well as if and how Hb creates NO activity remain unanswered.

Transfusion of older stored blood worsens outcomes in canines depending on the presence and severity of pneumonia

In experimental pneumonia we found that transfused older blood increased mortality and lung injury that was associated with increased in vivo hemolysis and elevated plasma cell‐free hemoglobin (CFH), non–transferrin‐bound iron (NTBI), and plasma labile iron (PLI) levels. In this study, we additionally analyze identically treated animals that received lower or higher bacterial doses.

Low NO Concentration Dependence of Reductive Nitrosylation Reaction of Hemoglobin

Background: Interactions of nitric oxide with hemoglobin are critical to NO function. Results: Reductive nitrosylation of hemoglobin is faster at low NO concentrations and sensitive to allosteric modifications. Conclusion: Hemoglobin can catalyze faster reductive nitrosylation reactions at physiological, low NO concentrations. Significance: Reductive nitrosylation may be more relevant in vivo than previously recognized. The reductive nitrosylation of ferric (met)hemoglobin is of considerable interest and remains incompletely explained. We have previously observed that at low NO concentrations the reaction with tetrameric hemoglobin occurs with an observed rate constant that is at least 5 times faster than that observed at higher concentrations. This was ascribed to a faster reaction of NO with a methemoglobin-nitrite complex. We now report detailed studies of this reaction of low NO with methemoglobin. Nitric oxide paradoxically reacts with ferric hemoglobin with faster observed rate constants at the lower NO concentration in a manner that is not affected by changes in nitrite concentration, suggesting that it is not a competition between NO and nitrite, as we previously hypothesized. By evaluation of the fast reaction in the presence of allosteric effectors and isolated β- and α-chains of hemoglobin, it appears that NO reacts with a subpopulation of β-subunit ferric hemes whose population is influenced by quaternary state, redox potential, and hemoglobin dimerization. To further characterize the role of nitrite, we developed a system that oxidizes nitrite to nitrate to eliminate nitrite contamination. Removal of nitrite does not alter reaction kinetics, but modulates reaction products, with a decrease in the formation of S-nitrosothiols. These results are consistent with the formation of NO2/N2O3 in the presence of nitrite. The observed fast reductive nitrosylation observed at low NO concentrations may function to preserve NO bioactivity via primary oxidation of NO to form nitrite or in the presence of nitrite to form N2O3 and S-nitrosothiols.

A modular fibrinogen model that captures the stress-strain behavior of fibrin fibers.

We tested what to our knowledge is a new computational model for fibrin fiber mechanical behavior. The model is composed of three distinct elements: the folded fibrinogen core as seen in the crystal structure, the unstructured α-C connector, and the partially folded α-C domain. Previous studies have highlighted the importance of all three regions and how they may contribute to fibrin fiber stress-strain behavior. Yet no molecular model has been computationally tested that takes into account the individual contributions of all these regions. Constant velocity, steered molecular dynamics studies at 0.025 Å/ps were conducted on the folded fibrinogen core and the α-C domain to determine their force-displacement behavior. A wormlike chain model with a persistence length of 0.8 nm (Kuhn length = 1.6 nm) was used to model the mechanical behavior of the unfolded α-C connector. The three components were combined to calculate the total stress-strain response, which was then compared to experimental data. The results show that the three-component model successfully captures the experimentally determined stress-strain behavior of fibrin fibers. The model evinces the key contribution of the α-C domains to fibrin fiber stress-strain behavior. However, conversion of the α-helical coiled coils to β-strands, and partial unfolding of the protein, may also contribute.