Red Blood Cell Dysfunction in Critical Illness

Introduction: the Erythron

Recently, the red blood cell (RBC) series, from progenitor cells to mature erythrocytes, has, collectively, been termed the Erythron. The erythron comprises RBCs at all stages of development and is the organ (primarily composed of anucleated cells in suspension) responsible for oxygen (O₂) transport from lungs to tissue¹. This role is newly appreciated to include active (by RBCs) vasoregulation that links regional blood flow to O₂ availability in the lung and to consumption in the periphery². A considerable portion of our nutritional and energy budget is devoted to maintaining a robust RBC population (20–30 trillion cells circulate in the average adult - approximately 85% of the cells in the body are RBCs.); 1.4 million RBCs are released into the circulation per second, replacing ~ 1% of the circulating mass per day. Mature RBCs have a life span of ~ 4 months, the majority of which is spent traversing the microcirculation. It is estimated that RBCs travel approximately 400 km during this interval, having made 170,000 circuits through the vascular tree. Circulating RBCs demonstrate unique physiology and are adapted to withstand significant biomechanical and biochemical stress. As RBCs age, energy and antioxidant systems fail; key proteins (including hemoglobin (Hb) and lipids) suffer oxidative injury, negatively impacting performance (rheology, adhesion, gas transport, vascular signaling). Such cells acquire marks of senescence and are cleared by the spleen or undergo eryptosis (a process unique to RBCs, similar to apoptosis). Of importance, this process may be accelerated in the course of critical illness and thereby, by limiting O₂ delivery, influence organ failure progression and outcome.

Moreover, it is essential to note that in the setting of insufficient O₂ delivery, blood flow (rather than content) is the focus of O₂ delivery regulation: O₂ content is relatively fixed, whereas flow is modulated by several orders of magnitude. Thus, blood flow volume and distribution are the physiologic parameters most actively regulated to maintain coupling between O₂ delivery and demand. Specifically, the trapping, processing and delivery of nitric oxide (NO) by RBCs has emerged as a conserved mechanism through which regional blood flow is linked to biochemical cues of perfusion sufficiency. By coordinating vascular signaling in a fashion that links O₂ and NO flux, RBCs couple vessel caliber (and thus blood flow) to O₂ need in tissue. Malfunction of this signaling system is implicated in a wide array of pathophysiologies and may be explanatory in part for the dysoxia frequently encountered in the critical care setting.

Capture and Release of Oxygen by RBCs

Hemoglobin (Hb) is formed of 2 α and 2 β polypeptide chains each carrying a heme prosthetic group, comprised of a porphyrin ring bearing a ferrous atom that can reversibly bind an oxygen (O₂) molecule. In the deoxygenated state, the Hb tetramer is electrostatically held in a tense (T) conformation. Binding of the first O₂ molecule leads to mechanical disruption of these bonds, an increase in free energy and transition to the relaxed (R) conformation. Each successive O₂ captured by T-state Hb shifts the Hb tetramer closer to the R state, which has an estimated 500-fold increase in O₂ affinity³. This concept of thermodynamically coupled “cooperativity” in O₂ binding was first described by Bohr⁴ and explains the sigmoidal appearance of the O₂-Hb binding curve, also known as the oxy-Hemoglobin dissociation curve (ODC) (Figure 1). Moreover, understanding of allosteric influence of protein function by ‘heterotropic effectors’ (e.g. For Hb, O₂, which binds to the ‘active’ site (heme) is the homotropic ligand and all other molecules influencing the Hb~O₂ binding relationship are termed heterotropic effectors.) was first achieved following description of the variation in Hb~O₂ affinity⁵. In addition to the homotropic effects of ligand binding on quaternary conformational changes (e.g. cooperativity), primary ligand binding affinity (O₂) is also affected by multiple heterotropic effectors of significant physiologic relevance. The major heterotropic effectors that influence Hb O₂ affinity are hydrogen ion (H⁺), chloride ion (Cl⁻), carbon dioxide (CO₂) and 2,3-diphosphoglycerate (DPG)³.

Figure 1.

The normal whole blood oxygen equilibrium curve (OEC). P₅₀ is the pO₂ at which hemoglobin is half-saturated with O₂. The principal effectors that alter the position and shape of the curve under physiological conditions are indicated.

From Winslow RM. The role of hemoglobin oxygen affinity in oxygen transport at high altitude. Respir Physiol Neurobiol 2007; 158:121–127; with permission.

P₅₀, the oxygen tension at which 50% of Hb binding sites are saturated, is used as a standard means to quantify change in Hb~O₂ affinity and is inversely related to the binding affinity of Hb for O₂⁶. Elevated levels of H⁺, Cl⁻ and CO₂ reduce O₂ binding affinity (e.g. raise P₅₀). This allosteric shift in O₂ affinity, called the Bohr effect⁷, arises from the interactions among the above heterotropic effectors bound to different sites on hemoglobin – all of which serve to stabilize the low energy, low affinity, T-state Hb conformation⁸. This effect is achieved by complex interactions amongst carbonic anhydrase (CA) and the B3 membrane protein (also known as anion exchange protein 1, AE1). Specifically, CA generates H⁺ and HCO₃⁻ from CO₂ encountered in the microcirculation; HCO₃⁻ then exchanges for Cl⁻ across the RBC membrane through AE1. As a consequence, extra erythrocytic CO₂ is converted into intra-erythrocytic HCl by the CA-AE1 complex, thus acidifying RBC cytoplasm and raising p50 (lowering affinity, also termed ‘right’ shifting the ODC). Additionally, through the Haldane effect, CO₂ more directly lowers O₂ affinity (by binding to the N-terminus of the globin chains to form a carbamino, further stabilizing T-state Hb); carbamino formation also releases another hydrogen ion (further reinforcing the ‘right shift’ in ODC)³ (Figure 2). This set of reactions is reversed in the alkaline (and low CO₂) milieu in the pulmonary circulation, leading to increased Hb~O₂ binding affinity (lower P₅₀). In sum, this physiology vastly improves O₂ transport efficiency by enhancing gas capture in the lung and release to tissue – and does so in proportion to perfusion sufficiency (in the setting of perfusion lack, acidosis and hypercpanea improve O₂ release). Of note, this tightly regulated modulation of O₂ affinity may become impaired in the setting of critical illness^9–12 and may, in part explain the dysoxia commonly observed in this setting.

Figure 2.

The quantitative behavior of the Carbaminohemoglobin (HbCO₂) dissociation curves at various oxygen tension levels.

From Dash RK, Bassingthwaighte JB. Erratum to: Blood HbO2 and HbCO2 dissociation curves at varied O2, CO2, pH, 2,3-DPG and temperature levels. Annals of biomedical engineering 2010; 38:1683–1701; with permission.

Less acute modulation of P₅₀ is achieved by DPG, a glycolytic intermediate that binds in an electrically charged pocket between the β chains of hemoglobin, which stabilizes the T conformation, decreasing O₂ affinity and elevating P₅₀. DPG binding also releases protons, lowering intracellular pH and further reinforcing the Bohr effect. DPG in RBCs increases whenever O₂ availability is diminished (as in hypoxia or anemia) or when glycolytic flux is stimulated¹³. Lastly, temperature significantly influences Hb~O₂ affinity. As body temperature increases, affinity lessens (P₅₀ increases, ODC shifts right); the reverse happens in hypothermia. This feature is of physiological importance during heavy exercise, fever or induced hypothermia. It should be noted that clinical co-oximetry results and blood gas values are reported at 37°C and not at true in vivo temperature and can lead to either under or over estimation of true HbSO₂% values and blood O₂ tension¹⁴.

RBC Biophysical factors Influencing tissue perfusion

Blood Rheology

Disease-based variation in blood fluidity has been recognized since the early 20^th century¹⁵ and there is substantive evidence that this property strongly influences tissue perfusion¹⁶. Plasma is a newtonian fluid (viscosity is independent of shear rate); its viscosity is closely related to protein content and in critical illness, physiologically significant changes in viscosity may vary with concentration of acute phase reactants. Whole blood, however, is considered a non-newtonian suspension (fluidity cannot be described by a single viscosity value); whole blood fluidity is determined by combined rheological properties of plasma and the cellular components.

The cellular components of blood, particularly RBCs, influence blood viscosity as a function of both number and deformability. RBC concentration in plasma (hematocrit) has an exponential relationship with viscosity and meaningfully diminishing tissue perfusion when Hct exceeds ~ 60–65. RBC deformability, or behavior under shear stress, also strongly influences blood fluidity. Normal RBCs behave like fluid drops under most conditions, are highly deformable under shear and orient with flow streamlines. However, during inflammatory stress, RBC tend to aggregate into linear arrays like a stack of coins (rouleaux); fibrinogen and other acute phase reactants in plasma stabilize such aggregates, significantly increasing blood viscosity. Such a change in viscosity most impacts O₂ delivery during low flow (e.g. low shear) states (such as in critical illness) in the microcirculation¹⁷. RBC biomechanics and aggregation impact blood viscosity, strongly influencing the volume and distribution of O₂ delivery (again more so, in the low-shear microcirculation, or when vessel tone is abnormal)¹⁸. This hemorheologic physiology is perturbed by oxidative stress (common in critical illness)¹⁹ and in sepsis²⁰. This has been attributed to increased intracellular 2,3-DPG concentration²¹, intracellular free Ca²⁺²² and decreased intra-erythrocytic ATP with subsequent decreased sialic acid content in RBC membranes²³. Both increased direct contact between RBCs and WBCs and reactive oxygen species released during sepsis have also been shown to alter RBC membrane properties²⁴.

RBC aggregation and adhesion

As noted above, in the absence of shear, RBCs suspended in autologous plasma stack in large aggregates, known as rouleaux. Acute phase reactants especially fibrinogen, C-reactive protein, serum amyloid A, haptoglobin and ceruloplasmin have been shown to increase RBC aggregation²⁵. Pathophysiological conditions as sepsis and ischemia-reperfusion injury have been shown to alter RBC surface proteins and increase RBC “aggregability”¹⁹. Activated white blood cells (WBC) are also thought to cause structural changes in the RBC glycocalyx and increase RBC aggregability²⁶.

Under normal conditions, RBC adherence to endothelial cells (EC) is insignificant and RBC deformability permits efficient passage through the microcirculation. Again, under normal conditions, enhanced EC adherence plays a role in the removal of senescent RBCs in the spleen. However, during critical illness, RBC~endothelial interactions are altered by RBC injuries associated with sepsis^27,28 and/or oxidative stress¹⁹. This is more prominent, with ‘activated’ endothelium, as frequently occurs in critical illness)^29,30.

Such RBC~endothelial aggregates create a physiologically significant increase in apparent blood viscosity¹⁸. Moreover, RBC adhesion directly damages the endothelium^31,32 and augments leukocyte adhesion^33–35 further impairing apparent viscosity and microcirculatory flow. This phenomenon is commonly appreciated in the pathophysiology of vaso-occlusive crises in sickle cell disease patients, malaria, diabetic vasculopathy, polycythemia vera and central retinal vein thrombosis, but may be more widespread than originally appreciated.

RBC Deformability

Tissue deformation can be defined as the relative displacement of specific points within a cell or structure. Mature RBCs are biconcave disks ranging from 2–8 μm in thickness, which act like droplets that deform reversibly under the shear encountered during circulatory transit¹⁸. Unique RBC geometry and deformability arises from (a) cytoplasmic viscosity and (b) specific interactions between the plasma membrane and underlying protein skeleton²³ (Figure 3). Cytoplasmic viscosity is mainly determined by hemoglobin concentration, which varies with intra-erythrocytic hydration, which is actively regulated by ATP-dependent cation pumps³⁶. The integral transmembrane membrane proteins AE-1 (AKA B3) and glycophorins are reversibly anchored to a submembrane filamentous protein mesh comprised of spectrin, actin, and protein 4.1. Linear extensibility of this mesh defines the limits of RBC deformability³⁷. Maintenance of membrane-mesh interactions and robust RBC mechanical behavior is dependent on ATP-dependent ion pumps as well as support from NADPH-dependent antioxidant systems³⁶. The sole energy source in RBCs is anaerobic glycolysis, which is discussed in detail below. RBC geometric and mechanical alterations secondary to impaired metabolism (leading to RBC dehydration, elevated intra-erythrocytic calcium and ATP/NADPH depletion) is a well-described consequence in blood stored for prolonged periods³⁸ and in RBCs subjected to significant metabolic stress during critical illness^39,40.

Figure 3.

The RBC membrane is composed of a phospholipid membrane bilayer and transmembrane proteins including glycophorin A and Band 3 proteins. Glycophorin A is the major sialoglycoprotein of the RBC. Sialic acid (SA) bound to glycophorin A is responsible for the negative charge of the RBC membrane. The intracellular compartment (IC) is constituted by spectrin (α and β subunits), actin, protein 4.1, and ankyrin.

From: Piagnerelli M, et al. Red blood cell rheology in sepsis. Intensive Care Med. 2003; 29(7):1052–1061; with permission.

Regulation of Blood Flow Distribution by RBCs

Microcirculatory blood flow is physiologically regulated to instantaneously match O₂ delivery to metabolic demand. This extraordinarily sensitive programmed response to tissue hypo-perfusion is termed hypoxic vasodilation (HVD)⁴¹. This process involves the detection of point-to-point variations in arteriolar O₂ content⁴² with the subsequent initiation of signaling mechanism(s) capable of immediate modulation of vascular tone (Figure 4).

Figure 4.

Local vascular reflexes support maintenance of O₂ delivery to tissue in the setting of progressive hypoxia. In a classic paper⁴¹, Guyton demonstrated regional autoregulation of systemic blood flow in normal dogs (following spinal anesthesia) by observing variation in blood flow during constant pressure blood perfusion of the femoral artery, while reducing the hemoglobin oxygen saturation (Hb SO₂%) from 100% to 0% in the perfusing blood. (A) Stepwise reduction in Hb SO₂% caused a progressive increase in blood flow through the leg. (B) These data demonstrate that autoregulation of blood flow occurs at a local level and this regulation serves to improve oxygen supply when blood oxygen content falls. In addition, effects on blood flow were replicated by injecting partially deoxygenated versus oxygenated red blood cells into the artery, demonstrating that effects could be elicited during arteriovenous transit (<1 s).

From Ross JM, Fairchild HM, Weldy J, Guyton AC. Autoregulation of blood flow by oxygen lack. Am J Physiol. 1962;202:21–24; with permission.

Over 30 years ago, intracellular RBC Hb was identified as a potential circulating O₂ sensor, following identification that in severe hypoxia, O₂ content was more important than partial pressure of O₂ (PO₂) in the maintenance of regional O₂ supply⁴³. It was later demonstrated in vivo that Hb O₂ saturation (HbSO₂) was independent of plasma or tissue PO₂, but was directly correlated with blood flow⁴⁴. These findings implicated a role for RBCs in the regulation of O₂ supply, given the following evidence: (1) the Hb molecule within the RBC is the only component in the O₂ transport pathway directly influenced by O₂ content, and (2) the level of O₂ content of the RBC at a particular point in the circulation is linked to the level of O₂ utilization⁴⁵.

With the vascular O₂ sensor identified, the mechanism involved in mediating the vasoactive response has remained in debate. To date, three HbSO₂ dependent RBC derived signaling mechanisms have been proposed, the first two linked to the vasoactive effector NO, and the third to RBC adenosine triphosphate (ATP) : (1) formation and export of S-nitrosothiols, ‘catalyzed’ by Hb (SNOHb hypothesis)^46–48; (2) reduction of nitrite (NO₂⁻) to NO by deoxygenated Hb (nitrite hypothesis)⁴⁹; and (3) hypoxia responsive release of ATP (ATP hypothesis)^45,50. Each of these hypotheses will be addressed further, below.

Role of RBC~NO interactions in vasoregulation

Interest in the free radical NO began with the identification of EDRF (endothelium derived relaxing factor), first reported in 1980⁵¹, which resolved the apparent paradox as to why acetylcholine, an agent known to be a vasodilator in vivo often caused vasoconstriction in vitro. Experiments performed with dissected segments of rabbit thoracic aorta mounted on a force transducer, demonstrated that handling of the tissue in a fashion that preserved endothelium always resulted in acetylcholine having relaxant properties. However, removal of the endothelium eradicated this action^51,52. Identification of EDRF consequently led to a race to discover its chemical identity. It was not until seven years later that two groups simultaneously published definitive studies characterizing and identifying EDRF as NO^53,54. However, the means by which NO exerted its physiological effects remained unknown and effort focused upon identifying the “NO receptor(s)”. This effort characterized the ‘classical’ signaling pathway for NO via soluble guanylate cyclase (sGC) and cyclic guanosine 3’,5’-monophosphate (cGMP) that appeared to clarify the means by which NO achieves its myriad effects⁵⁵. Over time, however, it is now appreciated that this pathway has little to do with the vasoregulation that governs regional blood flow distribution.

In terms of the HVD response (which underlies blood flow regulation) it is essential to appreciate that endothelium-derived NO plays no direct role in this reflex^44,56. Because of O₂ substrate limitation, NO production by eNOS is most likely attenuated by hypoxia^57,58. In fact, NO derived from eNOS⁴⁶ (and perhaps other NOS isoforms⁵⁹ and/or nitrite⁶⁰) is taken up by RBCs, transported, and subsequently dispensed in proportion to regional O₂ gradients to effect HVD at a time and place remote from the original site of NO synthesis. This key process enables RBCs to instantaneously modulate vascular tone in concert with cues of perfusion insufficiency, including hypoxia, hypercarbia, and acidosis^46,47.

Metabolism of endothelium derived NO by RBCs – Historical view

In the original NO paradigm, NO derived from endothelial nitric oxide synthase (eNOS) was felt to play a purely paracrine role in the circulation, acting within the vicinity of its release⁶¹. Its metabolic fate was explained by the diffusion of the “gas” in solution and its terminal reactions (1) in vascular smooth muscle cells with the ferrous heme iron (Fe²⁺) of soluble guanylate cyclase (sGC)⁶², and (2) in the vessel lumen, with the heme group (Fe²⁺) of oxyHb (the resultant oxidation reaction forming MetHb and nitrate), or deoxyHb (the resultant addition reaction forming iron nitrosyl Hb; HbNO), or in plasma with dissolved O₂ (the resultant autoxidation reaction)⁶³, and/or O₂ derived free radicals including superoxide (O₂⁻), hydrogen peroxide (H₂O₂), or hydroxyl radicals (OH⁻). Several “barriers” were presumed to retard NO diffusing into the blood vessel lumen to react avidly with the abundance of Hb, including the RBC membrane, the submembrane protein matrix, an unstirred layer around the RBC^64,65, in addition to laminar blood flow⁶⁶. These barriers were thought to limit these luminal reactions, thus allowing the local concentration of NO adjacent to endothelial cells to increase sufficiently to provide a diffusional gradient for NO to activate the underlying vascular smooth muscle sGC. Reactions of NO in the bloodstream were assumed only to scavenge/inactivate NO via the formation of metabolites unable to activate sGC⁶².

Metabolism of endothelium derived NO by RBCs – Modern view

A much broader biological chemistry of endothelial NO has been elucidated^67–69. Most notable is the covalent binding of NO⁺ to cysteine thiols, forming S-nitrosothiols (SNO). This paradigm developed following the discovery that endogenously produced NO circulated in human plasma primarily complexed to the protein albumin (S-nitrosoalbumin⁷⁰), which transformed the understanding of blood borne NO signaling. SNO proteins thus offered a means to conserve NO bioactivity, allowing the storage, transport, and potential release of NO remote from its location of synthesis⁷¹. The SNO hypothesis was extended to include a reactive thiol of Hb (Cysß93) that was demonstrated to undergo S-nitrosylation and sustain bioactivity under oxygenated conditions and NO release under low O₂ conditions (see HbSNO hypothesis)⁴⁶.

In this SNO paradigm, the NO radical must be oxidized to an NO⁺ (nitrosonium) equivalent, which can then be passed between thiols in peptides and proteins preserving NO bioactivity^67,68. S-nitrosylation then is akin to protein phosphorylation in terms of regulating protein function. SNO biochemistry offers NO a far broader signaling repertoire and has enabled awareness that the heme in sGC is not the sole, or even the principal, target of NO generated by endothelium. A wide array of alternative sGC (cyclic guanosine monophosphate)-independent reactions following endothelial NOS (eNOS) activation have been identified^69,72.

Processing and export of S-nitrosothiols by RBCs

Hb S-nitrosylation (HbSNO), which has been characterized by both mass spectrometry⁷³ and X-ray crystallography⁷⁴, provides an explanation as to how NO circumvents terminal reactions with Hb, enabling RBCs to conserve NO bioactivity and transport it throughout the circulation^46,47 (Figure 5). The formation and export of NO groups by Hb is governed by the transition in Hb conformation that occurs in the course of O₂ loading/unloading during arterio-venous (A-V) transit. This is due to conformational dependent change in reactivity of the Cysβ93 residue toward NO, which is higher in the R (oxygenated) Hb state and lower in the T (deoxygenated) Hb state^46,47.

Figure 5.

RBCs transduce regional O₂ gradients in tissue to control nitric oxide (NO) bioactivity in plasma by trapping or delivering NO groups as a function of hemoglobin (Hb) O₂ saturation. (A) In this fashion, circulating NO groups are processed by Hb into the highly vasoactive (thiol-based) NO congener, S-nitrosothiol (SNO). By exporting SNOs as a function of Hb deoxygenation, RBCs precisely dispense vasodilator bioactivity in direct proportion to regional blood flow lack. (B) O₂ delivery homeostasis requires biochemical coupling of vessel tone to environmental cues that matches perfusion sufficiency to metabolic demand. Because oxy- and deoxy-Hb process NO differently (see text), allosteric transitions in Hb conformation afford context-responsive (O₂-coupled) control of NO bioavailability, thereby linking the sensor and effector arms of this system. Specifically, Hb conformation governs the equilibria among deoxy-HbFeNO (A; NO sink), SNO-oxy-Hb (B; NO store), and acceptor thiols including the membrane protein SNO-AE-1 (C; bioactive NO source). Direct SNO export from RBCs or S-transnitrosylation from RBCs to plasma thiols (D) or to endothelial cells directly (not shown) yields vasoactive SNOs, which influence resistance vessel caliber and close this signaling loop. Thus, RBCs either trap (A) or export (D) NO groups to optimize blood flow. (C) NO processing in RBCs (A and B) couples vessel tone to tissue PO₂; this system subserves hypoxic vasodilation in the arterial periphery and thereby calibrates blood flow to regional tissue hypoxia.

From Doctor A, Stamler JS. NO Transport in Blood: A third gas in the respiratory cycle. In: Comprehensive Physiology: Respiratory Physiology. Wagner P and Hlastala M, Ed’s. American Physiological Society. Compr Physiol 1:541–568, 2011; with permission.

In a tightly regulated fashion, Hb captures and binds NO at its β-hemes and then passes the NO group from the heme to a thiol (Cys-ß93-SNO)^60,75. Transfer of NO between heme and thiol requires heme-redox coupled activation of the NO group, which is controlled by its allosteric transition across the lung⁷⁶. Once in R state the Cys-ß93-SNO is protected through confinement to a hydrophobic pocket⁷⁴. NO group export from Cys-β93-SNO occurs when steep O₂ gradients are encountered in the periphery (HVD). The R to T state conformational transition that occurs on Cys-β93-SNO deoxygenation (or oxidation) results in a shift in the location of the β-chain from its hydrophobic niche toward the aqueous cytoplasmic solvent⁷⁴. This allows the Cys-β93-SNO to be “chemically available” for transfer to target thiol containing proteins, including those associated with the RBC membrane protein AE-1 (Band 3)⁷⁷ and extra-erythrocytic thiols^78,79. Resultant plasma or other cellular SNOs, then become vasoactive at low nM concentrations)^46,47. Importantly all NO transfers in this process involve NO^{+ 46,48}, which protects bioactivity from Fe²⁺ heme recapture and/or inactivation. S-nitrosothiols are the only known endogenous NO compounds that retain bioactivity in the presence of Hb^46,79,80.

Extensive evidence supports SNO-Hb biology, whereby RBCs exert graded vasodilator and vasoconstrictor responses across the physiological microcirculatory O₂ gradient. RBCs dilate pre-constricted aortic rings at low PO₂ (1% O₂), while constricting at high PO₂ (95% O₂)^47,80–82. The vasodilatory response at low O₂ is enhanced following the addition of NO (or SNO) to RBCs, commensurate with SNO-Hb formation^46,77,80,83. Additionally, the vasodilatory response is enhanced in the presence of extra cellular free thiol⁸⁰, occurs in the absence of endothelium^48,80 (which is consistent with in vivo observation that HVD is endothelium independent⁸⁴), and transpires in the time frame of circulatory transit, as confirmed by measurements of A-V gradients in SNO-Hb^46,78,81,82.

In addition to these ex vivo experiments, numerous groups have also demonstrated bioactivity of inhaled NO, commensurate with SNO-Hb formation^85–89.

Metabolism of Nitrite by RBCs

Nitrite (NO₂⁻), formed mainly via hydration reactions involving N-oxides, was long viewed as an inactive oxidation product of NO metabolism. More recently it has been proposed as circulating pool of bioactive NO⁹⁰. Some have suggested that the reduction of nitrite by deoxyHb may serve as the RBC derived signaling mechanism regulating HVD⁹¹. However, this hypothesis has two major shortcomings in terms of known NO chemistry/biochemistry and HVD physiology. Firstly, to influence vascular tone, the NO radical produced from NO₂⁻ must escape RBCs at low O₂ tension in order to elicit a vasodilatory response. Experimental evidence, however, unambiguously refutes the possibility of NO escaping RBCs as an authentic radical, especially given the proximity, high concentration, and rapid reaction kinetics (10⁷M⁻¹s⁻¹) of authentic NO with deoxyHb. The only plausible reconciliation of this would be that bioactivity from this reaction may derive from heme captured NO (HbFe²⁺NO) being further converted into SNO-Hb^60,75, as HbFe²⁺NO itself acts as a vasoconstrictor rather than vasodilator⁹². The second shortcoming relates to the fact that the NO₂⁻ reductase activity of deoxyHb is purportedly symmetrical across the physiological O₂ gradient^93,94, with maximal activity occurring at the P₅₀ of Hb (~ 27 mmHg)^93,95. This reaction profile does not match the HVD response, which increases in a steadily graded fashion as PO₂ falls in the physiological range from 100 mmHg down to approximately 5 mmHg (HbSO₂ ~ 1–2%)^41,44. If RBC based vasoactivity were maximal at Hb’s P₅₀, then blood flow would be diverted away from regions with PO₂ below 27 mmHg, where it would be needed most. Additionally, based upon the symmetry of Hb nitrite reductase activity at the P₅₀, RBCs traversing vascular beds with PO₂ at 25 or 75 would generate equal NO-based activity⁹¹, where different blood flow demands are required.

Vasoregulation by RBC-derived Adenosine Triphosphate (ATP)

ATP has long been known to act as an endothelium dependent vasodilator in humans⁴⁵, binding to P₂Y purinergic receptors to induce local and conducted vasodilation via stimulation of vasoactive signals including endothelial NO, prostaglandins, and endothelial-derived hyperpolarization factors (EDHFs). More recently, RBCs have been identified as sources of vascular ATP^45,96, with release stimulated by conditions associated with diminished O₂ supply relative to demand,, hypoxia, hypercapnia, and low pH^45,97. O₂ offloading from membrane associated Hb is thought to initiate RBC ATP release⁹⁶, stimulating heterotrimeric G protein⁹⁸, as a result of membrane deformation. This leads to activation of adenylyl cyclase and an increase in cAMP⁹⁹, which activates protein kinase A (PKA)⁹⁹. PKA stimulates cystic fibrosis transmembrane conductance regulator (CFTR)¹⁰⁰, which activates release of ATP from the RBC via pannexin 1¹⁰¹. Release of ATP via this pathway requires an increase in intracellular cAMP, which is controlled by the relative activities of adenylyl cyclase and phosphodiesterase 3 (PDE3B)⁵⁰.

Despite potential as a HVD mediator, RBC derived ATP falls short on two fronts. Firstly, HVD is unaltered by both endothelial denudation and eNOS deletion⁴⁸, however ATP vasoactivity is endothelial dependent. Secondly, blood levels of ATP rise and fall over a period of minutes, which is not commensurate with the HVD response that occurs in the course of A-V transit over a couple of seconds. Despite its shortcomings in terms of acting as a primary mediator of HVD, it is likely that Hb and ATP serve complementary vasoactive roles, in acute local and prolonged systemic hypoxia respectively⁴⁸.

RBC Energetics and Consequences of Antioxidant System Failure

RBCs produce ATP by glycolysis only, with two branches:¹⁰² the Embden Meyerhof Pathway (EMP) and the Hexose Monophosphate Pathway (HMP).¹⁰³ Importantly, the HMP is the sole means for recycling NADPH,¹⁰⁴ which powers the thiol-based antioxidant system.¹⁰⁴ HMP flux is gated by protein complex assembly upon the cytoplasmic domain of the Band 3 membrane protein (cdB3 ‘metabolon’).^105–112 HMP flux oscillates with pO₂, as a function of Hb conformation and cdB3 phosphorylation (Figure 6 A–B).^113–119 Of note, RBC antioxidant systems fail when HMP flux is blunted by altered cdB3 protein assembly/phosphorylation caused by aberrant Hbs or hypoxia.^120,121 Strikingly similar perturbations to cdB3 are reported in sepsis,^122,123 possibly arising from caspase 3 activation^124–126 and/or direct endotoxin or complement membrane binding^127–134 (altering metabolon assembly, glycolysis and ROS clearance, Figure 2c).^135–137 As such, it appears that that sepsis (particularly, in the setting of hypoxic and/or uremic/oxidative^138–145 environments) disturbs cdB3-based metabolic control (Figure 6c), leading to: 1) EMP activation, 2) limited glucose-6-phosphate availability, 3) HMP flux constraint, 4) depowered NADPH/GSH recycling, 4) antioxidant system failure, and 5) injury to proteins/lipids that are key to O₂ delivery homeostasis (SiRD). This full pattern has been reported in other settings impacting protein assembly at cdB3;^120,121 further, such HMP constraint has functional similarity to G6PD deficiency,¹²⁰ which amplifies vulnerability to sepsis.^146–148 Moreover, hypoxia critically limits RBC energetics and depowers RBC antioxidant systems^149,150. In health, O₂• abundance is tightly regulated by the superoxide dismutase (SOD) family;¹⁵¹ however, overwhelming O₂• genesis¹⁵² is implicated in sepsis-associated injury cascades^153,154 Of note, sepsis-associated O₂• excess injures RBCs, impairing O₂-delivery by altering: control of O₂ affinity,^9–12 NO processing,^155–157 rheology,^{20,23,130,131,158–160} and adhesion.^161,162 O₂• excess also disrupts vasoregulation via NO consumption and catecholamine inactivation in plasma.^163–169 Specifically, ROS sourced directly to RBCs^170–173 injure vessels.^174,175 Such reciprocal injuries mutually escalate and as such, the dysoxia characteristic of septic shock (ischemia despite adequate blood O₂ content and cardiac output),^176–179 may arise from SiRD ~ vascular interactions.^27,28 Notably, ROS excess is also a common consequence of uremia/kidney injury^138–145, particularly during sepsis^180–186. As such, the combination of lung injury (hypoxia) and kidney injury (uremia) simultaneously constrain RBC energetics and antioxidant systems and present substantive oxidant loading conditions, meaningfully increasing RBC injury risk.

Figure 6.

Simplified scheme of cdB3-based control of RBC metabolism and proposed causal path for sepsis induced red cell dysfunction: (A) Energy metabolism in RBCs proceeds through either the Embden-Meyerhof pathway (EMP, orange arrows), or the hexose monophosphate pathway (HMP, blue arrows, AKA ‘pentose shunt’). Both share glucose-6 phosphate (G6P) as initial substrate. The HMP is the sole source of NADPH in RBCs and generates fructose-6-phosphate (F6P) or glyceraldehyde-3-phosphate (G3P), which rejoin the EMP prior to glyceraldehyde-3-phosphate dehydrogenase (G3PD/GAPDH), a key regulatory point. The EMP generates NADH (utilized by metHb reductase), as well as ATP (to drive ion pumps) and 2,3-DPG (to modulate hemoglobin P₅₀). Hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻) are the principal endogenous reactive O₂ species (ROS) that are generated / encountered by RBCs. Both ROS are generated internally in the course of HbO₂ cycling.^230–232 Notably, only H₂O₂ can cross the membrane directly. O₂⁻ enters/departs RBCs via the Band 3 channel (anion exchange protein 1, or AE-1). O₂⁻ and H₂O₂ are ultimately reduced to water by catalase (CAT) or glutathione peroxidase (GPx). (B) O₂ content modulates EMP/HMP balance via reciprocal binding for cdB3 between deoxyHb and key EMP enzymes (PFK, Aldo, G3PD, PK, and LDH). In oxygenated RBCs (right half of stylized O₂ dissociation plot), EMP enzyme sequestration to cdB3 inactivates this pathway, resulting in HMP dominance and maximal NADPH (and thus GSH) recycling capacity. In deoxygenated RBCs (left half of O₂ dissociation plot), deoxyHb binding to cdB3 disperses bound EMP enzymes, activating the EMP, creating G6P substrate competition, constraining HMP flux, limiting NADPH and GSH recycling capacity and weakening resilience to ROS, such as O₂⁻. (C) In sepsis, data suggest cdB3-complex assembly may be prevented (particularly, with coincident hypoxia, see text). As in settings similarly impacting the cdB3 complex, it appears that this disturbs normal EMP/HMP balance (disfavoring HMP), depowering antioxidant systems and rendering RBCs vulnerable to oxidant attack. GSH, glutathione; GR, glutathione reductase; NADPH, nicotinamide adenine dinucleotide phosphate; PFK, phosphofructokinase; Aldo, aldolase; PK, pyruvate kinase; LDH, lactate dehydrogenase

Acquired RBC Injury, Eryptosis and Clearance

After maturation to an anucleated cell furnished with the metabolic systems described above, the estimated normal life span of a mature RBC is 110–120 days¹⁸⁷. To date, clearance of normal senescent RBC has not been clearly understood. Two mechanisms have been proposed, clustering of the band 3 (B3) membrane protein^188–191 and externalization of membrane phosphatidyl serine (PS)^192–195, both of these processes may be accelerated in the setting of critical illness, impairing oxygen transport capacity. Oxidatively modified hemoglobin (Hb) forms hemichrome aggregates, which associate with the cytoplasmic domain of the abundant membrane protein B3. Subsequent clustering of B3 exofascial domains increases affinity of naturally occurring anti-B3 autoantibodies, which activate the complement system leading to RBC uptake and destruction by macrophages¹⁹⁶. Normally, PS is asymmetrically distributed in the plasma membrane (a process regulated by flippases). Disruption of this pattern is a well-documented mark of RBC senescence^192–195, signaling RBC removal by the reticulo-endothelial system¹⁹⁵. Alternatively, RBCs may proceed through a form of ‘stimulated suicide’ similar to apoptosis (termed eryptosis), which is characterized by cell shrinkage and cell membrane scrambling, that is stimulated by Ca²⁺ entry through Ca²⁺⁻permeable, PGE₂-activated cation channels, by ceramide, caspases, calpain, complement, hyperosmotic shock, energy depletion, oxidative stress, and deranged activity of several kinases (e.g. AMPK, GK, PAK₂, CK1α, JAK₃, PKC, p38-MAPK). Eryptosis has been described in the setting of ethanol intoxication, malignancy, hepatic failure, diabetes, chronic renal insufficiency, hemolytic uremic syndrome, dehydration, phosphate depletion, fever, sepsis, mycoplasma infection, malaria, iron deficiency, sickle cell anemia, thalassemia, G6PD deficiency, and Wilson’s disease^195,197,198.

Influence of RBCs on hemostasis

The principle impact of RBCs in clot formation in vivo is rheological, since RBC laminar shearing promotes platelet margination¹⁹⁹, as well as RBC aggregation and deformability of RBCs, which also support clot assembly/retraction²⁰⁰. In addition, RBCs interact directly and indirectly with endothelial cells and platelets during thrombosis²⁰¹. Both the stiffness of RBCs and the extent to which they form a procoagulant surface to generate thrombin through exposure of phosphatidylserine appear to play an important role, both in clot initiation and completion^202,203. Moreover, RBC-derived MPs transfused with stored RBCs or formed in various pathological conditions associated with hemolysis have strong procoagulant potential along with prothrombotic effects of the extracellular hemoglobin and heme²⁰⁴. Additionally, RBCs directly interact with fibrin(ogen) and affect the structure, mechanical properties, and lytic resistance of clots and thrombi²⁰⁵. Finally, tessellated polyhedral RBCs (polyhedrocytes) are recognized to be a significant structural component of contracted clots, enabling the impermeable barrier important for hemostasis and wound healing²⁰⁶.

Summary: RBC Dysfunction disrupts of O₂ delivery during critical illness

Evidence is mounting in support of a causal relationship between acquired RBC dysfunction and a host of perfusion-related morbidities that complicate critical illness^{82,171,207–221}. Recently, it has been observed that levels of SNO-Hb are altered in several disease states characterized by disordered tissue oxygenation^{82,83,155,156,222–227}. In addition, where examined, RBCs from such patients exhibit impaired vasodilatory capacity^{78,82,83,224,226–228}. These data suggest that altered RBC-derived NO bioactivity may contribute to human pathophysiology. Specifically, alterations in thiol-based RBC NO metabolism have been reported in congestive heart failure⁸², diabetes^83,223, pulmonary hypertension^81,222 and sickle cell disease^224,229, all of which are conditions characterized by inflammation, oxidative stress and dysfunctional vascular control. Moreover, known cross-talk between SNO signaling and cellular communication via carbon monoxide, serotonin, prostanoids, catecholamines and endothelin may permit broad dispersal of signals generated by dysfunctional RBCs. Precise understanding of the roles of dysregulated RBC-based NO transport in the spread of vasomotor dysfunction from stressed vascular beds may open novel therapeutic approaches to a range of pathologies.

Synopsis.

Oxygen (O₂) delivery, the maintenance of which is fundamental to supporting those with critical illness, is a function of blood O₂ content and flow. Here, we review red blood cell (RBC) physiology and dysfunction relevant to disordered O₂ delivery in the critically ill. Flow (rather than content) is the focus of O₂ delivery regulation: O₂ content is relatively fixed, whereas flow fluctuates by several orders of magnitude. Thus, blood flow volume and distribution vary to maintain coupling between O₂ delivery and demand. The trapping, processing and delivery of vasoactive effectors (NO and ATP) by RBCs has emerged as a conserved mechanism through which regional blood flow is linked to biochemical cues of perfusion sufficiency. We will review conventional RBC physiology influencing O₂ delivery (O₂ affinity & rheology) and introduce a new paradigm for O₂ delivery homeostasis based on coordinated gas transport and vascular signaling by RBCs. By coordinating vascular signaling in a fashion that links O₂ and NO flux, RBCs couple vessel caliber (and thus blood flow) to O₂ need in tissue. Malfunction of this signaling system is implicated in a wide array of pathophysiologies and may be explanatory for the dysoxia frequently encountered in the critical care setting.

Key Points.

• Together, all red blood cells (RBC) at each stage of development, may be considered an organ (termed the erythron) now appreciated to participate in active regulation of regional blood flow distribution as well as O₂ and CO₂ transport.

• RBCs are subject to intense biochemical, biomechanical and physiologic stress during repeated circulatory transit and as such, possess unique properties and robust energetic and antioxidant systems to maintain functionality for a 3–4-month lifetime.

• RBCs actively regulate blood flow volume and distribution to maintain coupling between O₂ delivery and demand. The trapping, processing and delivery of nitric oxide (NO) by RBCs has emerged as a conserved mechanism through which regional blood flow is linked to biochemical cues of perfusion sufficiency.

• A new paradigm for O₂ delivery homeostasis has emerged, based on coordinated gas transport and vascular signaling by RBCs. By coordinating vascular signaling in a fashion that links O₂ and nitric oxide (NO) flux, RBCs couple vessel caliber (and thus blood flow) to O₂ need in tissue. Malfunction of this signaling system is implicated in a wide array of pathophysiologies and may be in part explanatory for the dysoxia frequently encountered in the critical care setting.