Together, red blood cells (RBCs) 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 oxygen (O 2 ) and carbon dioxide (CO 2 ) transport.
RBCs are subject to intense biochemical, biomechanical, and physiologic stress during repeated circulatory transit. As such, they possess unique properties and robust energetic and antioxidant systems to maintain functionality for a 3- to 4-month lifetime.
RBCs actively regulate blood flow volume and distribution to maintain coupling between O 2 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 O 2 delivery homeostasis has emerged based on coordinated gas transport and vascular signaling by RBCs. By coordinating vascular signaling in a fashion that links O 2 and NO flux, RBCs couple vessel caliber (and thus blood flow) to O 2 need in tissue. Malfunction of this signaling system is implicated in a wide array of pathophysiologies and may explain, in part, the dysoxia frequently encountered in the critical care setting.
The erythron includes red blood cells (RBCs) at all stages of development, from progenitors to senescent forms, and is the organ (composed of anucleated cells in suspension) responsible for oxygen (O 2 ) transport from lungs to tissue. This role is newly appreciated to include active vasoregulation (by RBCs) that links regional blood flow to O 2 availability in the lung and to consumption in the periphery. Considerable energy is devoted to maintaining a robust RBC population (20–30 trillion cells circulate in the average adult—approximately 25% 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 an RBC travels 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, by limiting O 2 delivery, influence organ failure progression and outcome.
Moreover, it is essential to note that in the setting of insufficient O 2 delivery, blood flow (rather than content) is the focus of O 2 delivery regulation: O 2 content is relatively fixed, whereas flow can be increased by several orders of magnitude. Thus, blood flow volume and distribution are actively regulated to maintain coupling between O 2 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. This chapter reviews conventional RBC physiology influencing O 2 delivery (O 2 affinity and rheology) and introduces a new paradigm for O 2 delivery homeostasis based on coordinated gas transport and vascular signaling by RBCs. By coordinating vascular signaling in a fashion that links O 2 and NO flux, RBCs couple vessel caliber (and thus blood flow) to O 2 need in tissue. Malfunction of this signaling system is implicated in a wide array of pathophysiology and may explain, in part, the dysoxia frequently encountered in the critical care setting.
Hb is formed by two α-globin and two β-globin chains, each carrying a heme prosthetic group, composed of a porphyrin ring bearing a ferrous atom that can reversibly bind an O 2 molecule. In the deoxygenated state, the Hb tetramer is electrostatically held in a tense (T) conformation. Binding of the first O 2 molecule leads to mechanical disruption of these bonds, an increase in free energy, and transition to the relaxed (R) conformation. Each successive O 2 captured by T-state Hb shifts the Hb tetramer closer to the R state, which has an estimated 500-fold increase in O 2 affinity. This concept of thermodynamically coupled “cooperativity” in O 2 binding was first described by Perutz and explains the sigmoidal appearance of the O 2 -Hb binding curve, also known as the oxyhemoglobin dissociation curve (ODC). Moreover, understanding of allosteric influence of protein function by “heterotropic effectors” is essential. For example, for Hb, O 2 , which binds to the heme “active” site, is the homotropic ligand; all other molecules influencing the Hb-O 2 binding relationship are termed heterotropic effectors. In addition to the homotropic effects of ligand binding on quaternary conformational changes (e.g., cooperativity), primary ligand binding affinity (O 2 ) is also affected by multiple heterotropic effectors of significant physiologic relevance. The major heterotropic effectors that influence Hb-O 2 affinity are hydrogen ion (H + ), chloride ion (Cl − ), carbon dioxide (CO 2 ), and 2,3-diphosphoglycerate (DPG).
P 50 , the oxygen tension at which 50% of Hb binding sites are saturated, is the standard metric employed to quantify change in Hb-O 2 affinity and is inversely related to the binding affinity of Hb for O 2 . , Elevated levels of H + , Cl − , and CO 2 reduce O 2 binding affinity (e.g., raise P 50 ). This allosteric shift in O 2 affinity, called the Bohr effect, arises from the interactions among the heterotropic effectors mentioned earlier bound to different sites on Hb, all of which serve to stabilize the low-energy, low-affinity, T-state Hb conformation. This effect is achieved by complex interactions between carbonic anhydrase (CA) and the anion exchange protein 1 (AE1—also known as the band 3 [B3] membrane protein ; Fig. 87.1 ). Specifically, CA generates H + and HCO 3 − from CO 2 encountered in the microcirculation; HCO 3 − then exchanges for Cl − across the RBC membrane through AE1. As a consequence, extra erythrocytic CO 2 is converted into intraerythrocytic HCl by the CA-AE1 complex, acidifying RBC cytoplasm and raising P 50 (lowering affinity, also termed right shifting the ODC). Additionally, through the Haldane effect, CO 2 more directly lowers O 2 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 the ODC; see Fig. 87.1 ). This set of reactions is reversed in the alkaline (and low CO 2 ) milieu in the pulmonary circulation, leading to increased Hb-O 2 binding affinity (lower P 50 ). This physiology vastly improves O 2 transport efficiency by enhancing gas capture in the lung and release to tissue and does so in proportion to perfusion sufficiency (impaired perfusion, acidosis, and hypercapnea improve O 2 release). In the setting of impaired O 2 delivery that results in anaerobic glycolysis, lactate diffusion into capillary blood (by lowering pH) further increases O 2 dissociation and facilitates O 2 delivery. Of note, this tightly regulated modulation of O 2 affinity requires coordinated interaction of a complex intraerythrocytic protein network and may fail consequent to acquired RBC injury in the setting of critical illness. This partially explains the dysoxia commonly observed in this setting.
Less acute modulation of P 50 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 O 2 affinity and elevating P 50 . DPG binding also releases protons, lowering intracellular pH and further reinforcing the Bohr effect. DPG in RBCs increases whenever O 2 availability is diminished (as in hypoxia or anemia) or when glycolytic flux is stimulated. Last, temperature significantly influences Hb-O 2 affinity. As body temperature increases, affinity lessens (P 50 increases, the ODC shifts right); the reverse happens in hypothermia. This feature is of physiologic 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 overestimation of true Hb-O 2 saturation (HbSO 2 ) percentage values and blood O 2 tension.
Carbon dioxide transport
As a lipophilic molecule, CO 2 produced from tissue respiration readily diffuses into capillary blood and through the RBC membrane. A minority (5%–10%) of diffused CO 2 binds to the α-amino terminus of Hb globin chains as carbaminohemoglobin, lowering O 2 affinity (raising P 50 ); this P 50 shift is termed the Haldane effect. , The majority of CO 2 is hydrated by the two intraerythrocytic CA isoforms, CA I (low-turnover rate) and CA II (high-turnover rate). This conversion into bicarbonate (and a proton, see earlier discussion) is essential for efficient CO 2 transport and for maintaining the venous-arterial difference in total CO 2 . The released protons stabilize the T conformation of Hb, facilitating O 2 offloading in proportion to CO 2 genesis (and thus regional tissue respiration). Carbaminohemoglobin also acts to directly stabilize T-state Hb. The process reverses during pulmonary transit (since the lungs act as a CO 2 sink), raising intraerythrocytic pH and enhancing O 2 affinity in proportion to regional alveolar ventilation (CO 2 removal).
Biophysical factors influencing gas transport
Disease-based variation in blood fluidity has been recognized since the early 20th century, and there is substantive evidence that this property strongly influences tissue perfusion. 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 nonnewtonian suspension (fluidity cannot be described by a single viscosity value). Whole blood fluidity is determined by combined rheologic 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 to 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, RBCs 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 O 2 delivery during low flow (e.g., low shear) states (such as in critical illness) in the microcirculation. RBC biomechanics and aggregation impact blood viscosity, strongly influencing the volume and distribution of O 2 delivery (again, more so in the low-shear microcirculation or when vessel tone is abnormal). This hemorheologic physiology is perturbed by oxidative stress (common in critical illness) , and in sepsis. This has been attributed to increased intracellular 2,3-DPG concentration, intracellular free Ca 2 + , and decreased intraerythrocytic adenosine triphosphate (ATP) with subsequent decreased sialic acid content in RBC membranes. Both increased direct contact between RBCs and white blood cells (WBCs) and reactive oxygen species (ROS) released during sepsis have also been shown to alter RBC membrane properties. ,
Red blood cell aggregation and adhesion
As noted earlier, 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 aggregation. Pathophysiologic conditions, such as sepsis and ischemia-reperfusion injury, have been shown to alter RBC surface proteins and increase RBC “aggregability.” Activated WBCs are also thought to cause structural changes in the RBC glycocalyx and increase RBC aggregability.
Under normal conditions, RBC adherence to endothelial cells (ECs) is insignificant, and RBC deformability permits efficient passage through the microcirculation. Again, under normal conditions, enhanced EC adherence plays a role in removal of senescent RBCs in the spleen. However, during critical illness, RBC-endothelial interactions are altered by RBC injuries associated with sepsis , , , and/or oxidative stress. This is more prominent with activated endothelium, as frequently occurs in critical illness. Such RBC-endothelial aggregates create a physiologically significant increase in apparent blood viscosity. Moreover, RBC adhesion directly damages endothelium and augments leukocyte adhesion, 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.
Red blood cell 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 to 8 μm in thickness, which act like droplets that deform reversibly under the shear encountered during circulatory transit. , Unique RBC geometry and deformability arises from cytoplasmic viscosity and specific interactions between the plasma membrane and underlying protein skeleton. Cytoplasmic viscosity is mainly determined by Hb concentration, which varies with intraerythrocytic hydration that is actively regulated by ATP-dependent cation pumps. The integral transmembrane membrane proteins AE-1 (AKA B3) and glycophorins are reversibly anchored to a submembrane filamentous protein mesh composed of spectrin, actin, and protein 4.1. Linear extensibility of this mesh defines the limits of RBC deformability. Maintenance of membrane-mesh interactions and robust RBC mechanical behavior is dependent on ATP-dependent ion pumps and support from nicotinamide adenine dinucleotide phosphate (NADPH)-dependent antioxidant systems. The sole energy source in RBCs is anaerobic glycolysis, which is discussed in detail later. RBC geometric and mechanical alterations secondary to impaired metabolism (leading to RBC dehydration, elevated intraerythrocytic calcium, and ATP/NADPH depletion) is a well-described consequence in blood stored for prolonged periods and in RBCs subjected to significant metabolic stress during critical illness. ,
Regulation of blood flow distribution by red blood cells
Microcirculatory blood flow is physiologically regulated to instantaneously match O 2 delivery to metabolic demand. This extraordinarily sensitive programmed response to tissue hypoperfusion is termed hypoxic vasodilation (HVD). This process involves the detection of point-to-point variations in arteriolar O 2 content with the subsequent initiation of signaling mechanism(s) capable of immediate modulation of vascular tone.
Over 30 years ago, intracellular RBC Hb was identified as a potential circulating O 2 sensor, following identification that, in severe hypoxia, O 2 content was more important than partial pressure of O 2 (PO 2 ) in the maintenance of regional O 2 supply. It was later demonstrated in vivo that HbSO 2 was independent of plasma or tissue PO 2 but was directly correlated with blood flow. These findings implicated a role for RBCs in the regulation of O 2 supply, given the following evidence: (1) the Hb molecule within the RBC is the only component in the O 2 transport pathway directly influenced by O 2 content, and (2) the level of O 2 content of the RBC at a particular point in the circulation is linked to the level of O 2 utilization.
With the vascular O 2 sensor identified, the mechanism involved in mediating the vasoactive response has remained in debate. To date, three HbSO 2 -dependent RBC-derived signaling mechanisms have been proposed, the first two linked to the vasoactive effector NO and the third to RBC ATP: (1) formation and export of S-nitrosothiols (SNOs), “catalyzed” by Hb (SNOHb hypothesis) ; (2) reduction of nitrite (NO 2 − ) to NO by deoxygenated Hb (nitrite hypothesis) ; and (3) hypoxia-responsive release of ATP (ATP hypothesis). , Each of these hypotheses is addressed in the next section.
Role of red blood cell–nitric oxide interactions in vasoregulation
Interest in the free radical NO began with the identification of endothelium-derived relaxing factor (EDRF), first reported in 1980, 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. , Identification of EDRF consequently led to a race to discover its chemical identity. It was not until 7 years later that two groups simultaneously published definitive studies characterizing and identifying EDRF as NO. , However, the means by which NO exerted its physiologic effects remained unknown, and efforts focused on identifying the “NO receptor(s).” These efforts 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 effects. 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. , Because of O 2 substrate limitation, NO production by endothelial nitric oxide synthase (eNOS) is most likely attenuated by hypoxia. , In fact, NO derived from eNOS (and perhaps other NOS isoforms and/or nitrite ) is taken up by RBCs, transported, and subsequently dispensed in proportion to regional O 2 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. ,
Metabolism of endothelium-derived nitric oxide by red blood cells: Historical view
In the original NO paradigm, NO derived from eNOS was felt to play a purely paracrine role in the circulation, acting within the vicinity of its release. 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 (Fe 2 + ) of sGC and (2) in the vessel lumen, with the heme group (Fe 2 + ) 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 O 2 (the resultant autoxidation reaction), and/or O 2 -derived free radicals, including superoxide (O 2 − ), hydrogen peroxide (H 2 O 2 ), or hydroxyl radicals (OH − ). Several “barriers” were presumed to retard NO diffusion into the blood vessel lumen, where it would react avidly with the abundant Hb, including (1) the RBC membrane and the submembrane protein matrix, (2) an unstirred layer around the RBCs, , and (3) an RBC-free zone adjacent to endothelium that results from laminar flow streaming. These barriers were thought to limit these luminal reactions, 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 sGC.
Metabolism of endothelium-derived nitric oxide by red blood cells: Contemporary view
A much broader biological chemistry of endothelial NO has been elucidated. Most notable is the covalent binding of NO + to cysteine thiols, forming SNOs. This paradigm developed following the discovery that endogenously produced NO circulated in human plasma primarily complexed to the protein albumin (S-nitrosoalbumin ), 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 synthesis. The SNO hypothesis was extended to include a reactive thiol of Hb (Cysβ93) that was demonstrated to undergo S-nitrosylation and sustain bioactivity under oxygenated conditions and NO release under low O 2 conditions (see HbSNO hypothesis).
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. , 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 eNOS activation have been identified. ,
Processing and export of S-nitrosothiols by red blood cells
Hb S-nitrosylation (HbSNO), which has been characterized by both mass spectrometry and X-ray crystallography, provides an explanation as to how NO circumvents terminal reactions with Hb, enabling RBCs to conserve NO bioactivity and transport it throughout the circulation , (see Fig. 87.1 ). The formation and export of NO groups by Hb is governed by the transition in Hb conformation that occurs in the course of O 2 loading/unloading during arteriovenous (AV) 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. ,
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-β93-SNO). , 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 lung. Once in R state, the Cys-β93-SNO is protected through confinement to a hydrophobic pocket. NO group export from Cys-β93-SNO occurs when steep O 2 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 solvent. 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) and extra-erythrocytic thiols. , Resultant plasma or other cellular SNOs then become vasoactive at low nM concentrations. , Importantly, all NO transfers in this process involve NO + , , which protects bioactivity from Fe 2 + heme recapture and/or inactivation. S-nitrosothiols are the only known endogenous NO compounds that retain bioactivity in the presence of Hb. , ,
Extensive evidence supports SNO-Hb biology, whereby RBCs exert graded vasodilator and vasoconstrictor responses across the physiologic microcirculatory O 2 gradient. RBCs dilate preconstricted aortic rings at low PO 2 (1% O 2 ), while constricting at high PO 2 (95% O 2 ). , The vasodilatory response at low O 2 is enhanced following the addition of NO (or SNO) to RBCs commensurate with SNO-Hb formation. , , , Additionally, the vasodilatory response is enhanced in the presence of extracellular free thiol, occurs in the absence of endothelium , (which is consistent with in vivo observation that HVD is endothelium independent ), and transpires in the time frame of circulatory transit, as confirmed by measurements of AV gradients in SNO-Hb. , , , In addition to these ex vivo experiments, numerous groups have also demonstrated bioactivity of inhaled NO commensurate with SNO-Hb formation.
Metabolism of nitrite by red blood cells
NO 2 − , 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 a circulating pool of bioactive NO. Some have suggested that the reduction of nitrite by deoxyHb may serve as the RBC-derived signaling mechanism regulating HVD. However, this hypothesis has two major shortcomings in terms of known NO chemistry/biochemistry and HVD physiology. First, to influence vascular tone, the NO radical produced from NO 2 − must escape RBCs at low O 2 tension in order to elicit a vasodilatory response. However, experimental evidence unambiguously refutes the possibility of NO escaping RBCs as an authentic radical, especially given the proximity, high concentration, and rapid reaction kinetics (10 7 M -1 s -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 (HbFe 2 + NO) being further converted into SNO-Hb, , as HbFe 2 + NO itself acts as a vasoconstrictor rather than vasodilator. The second shortcoming relates to the fact that the NO 2 − reductase activity of deoxyHb is purportedly symmetric across the physiologic O 2 gradient, , with maximal activity occurring at the P 50 of Hb (27 mm Hg). , This reaction profile does not match the HVD response, which increases in a steadily graded fashion as PO 2 falls in the physiologic range from 100 mm Hg down to approximately 5 mm Hg (HbSO 2 1%–2%). , If RBC-based vasoactivity were maximal at Hb’s P 50 , then blood flow would be diverted away from regions with PO 2 below 27 mm Hg, where it would be needed most. Additionally, based on the symmetry of Hb nitrite reductase activity at the P 50 , RBCs traversing vascular beds with PO 2 at 25 or 75 would generate equal NO-based activity, where different blood flow demands are required.
Vasoregulation by red blood cell-derived adenosine triphosphate
Adenosine triphosphate (ATP) has long been known to act as an endothelium-dependent vasodilator in humans, binding to P 2 Y 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, , with release stimulated by conditions associated with diminished O 2 supply relative to demand, hypoxia, hypercapnia, and low pH. , O 2 offloading from membrane-associated Hb is thought to initiate RBC ATP release, stimulating heterotrimeric G protein, as a result of membrane deformation. This leads to activation of adenylyl cyclase and an increase in cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA). PKA stimulates cystic fibrosis transmembrane conductance regulator, which activates release of ATP from the RBC via pannexin 1. 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.
Despite potential as an HVD mediator, RBC-derived ATP falls short on two fronts. First, HVD is unaltered by both endothelial denudation and eNOS deletion ; however, ATP vasoactivity is endothelial dependent. Second, 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 AV 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, respectively.
Red blood cell energetics and consequences of antioxidant system failure
RBCs produce ATP by glycolysis only, with two branches : the Embden Meyerhof Pathway (EMP) and the Hexose Monophosphate Pathway (HMP). Importantly, the HMP is the sole means for recycling NADPH, which powers the thiol-based antioxidant system. HMP flux is gated by protein complex assembly upon the cytoplasmic domain of the Band 3 membrane protein (cdB3 “metabolon”). HMP flux oscillates with PO 2 , as a function of Hb conformation and cdB3 phosphorylation. Of note, RBC antioxidant systems fail when HMP flux is blunted by altered cdB3 protein assembly/phosphorylation caused by aberrant Hbs or hypoxia. , Strikingly similar perturbations to cdB3 are reported in sepsis, , possibly arising from caspase 3 activation and/or direct endotoxin or complement membrane binding , (altering metabolon assembly, glycolysis, and ROS clearance; see Fig. 87.1 C). As such, it appears that sepsis (particularly in the setting of hypoxic and/or uremic/oxidative environments) disturbs cdB3-based metabolic control, leading to (1) EMP activation, (2) limited glucose-6-phosphate availability, (3) HMP flux constraint, (4) depowered NADPH/glutathione recycling, (4) antioxidant system failure, and (5) injury to proteins/lipids that are key to O 2 delivery homeostasis (SiRD). This full pattern has been reported in other settings impacting protein assembly at cdB3 , ; further, such HMP constraint has functional similarity to glucose-6-phosphate dehydrogenase (G6PD) deficiency, which amplifies vulnerability to sepsis. Moreover, hypoxia critically limits RBC energetics and depowers RBC antioxidant systems. , In health, O 2 • abundance is tightly regulated by the superoxide dismutase (SOD) family. However, overwhelming O 2 • genesis is implicated in sepsis-associated injury cascades. , Of note, sepsis-associated O 2 • excess injures RBCs, impairing O 2 delivery by altering control of O 2 affinity, NO processing, rheology, , , , , , , and adhesion. , O 2 • excess also disrupts vasoregulation via NO consumption and catecholamine inactivation in plasma. Specifically, ROS sourced directly to RBCs , , , injure vessels. , Such reciprocal injuries mutually escalate; as such, the dysoxia characteristic of septic shock (ischemia despite adequate blood O 2 content and cardiac output) may arise from SiRD-vascular interactions. , Notably, ROS excess is also a common consequence of uremia/kidney injury, particularly during sepsis. 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.
Acquired red blood cell injury, eryptosis, and clearance
After maturation to an anucleated cell furnished with the metabolic systems described earlier, the estimated normal life span of a mature RBC is 110 to 120 days. To date, clearance of normal senescent RBCs has not been clearly understood. Two mechanisms have been proposed: (1) clustering of the band 3 (B3) membrane protein and (2) externalization of membrane phosphatidylserine (PS). Both of these processes may be accelerated in the setting of critical illness, impairing O 2 transport capacity. Oxidatively modified 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 macrophages. 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, signaling RBC removal by the reticuloendothelial system. 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 Ca 2 + entry through Ca 2 + -permeable, PGE 2 -activated cation channels, by ceramide, caspases, calpain, complement, hyperosmotic shock, energy depletion, oxidative stress, and deranged activity of several kinases (e.g., AMPK, GK, PAK 2 , CK1α, JAK 3 , 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 disease. , ,
Influence of red blood cells on hemostasis
The principal impact of RBCs in clot formation in vivo is rheologic, since RBC laminar shearing promotes platelet margination, as well as RBC aggregation and deformability of RBCs, which also support clot assembly/retraction. In addition, RBCs interact directly and indirectly with ECs and platelets during thrombosis. Both the stiffness of RBCs and the extent to which they form a procoagulant surface to generate thrombin through exposure of PS appear to play an important role in both clot initiation and completion. , Moreover, RBC-derived microparticles transfused with stored RBCs or formed in various pathologic conditions associated with hemolysis have strong procoagulant potential along with prothrombotic effects of the extracellular hemoglobin and heme. Additionally, RBCs directly interact with fibrin(ogen) and affect the structure, mechanical properties, and lytic resistance of clots and thrombi. 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 healing.
Evidence is mounting in support of a causal relationship between acquired RBC dysfunction and a host of perfusion-related morbidities that complicate critical illness. , , , Recently, it has been observed that levels of SNO-Hb are altered in several disease states characterized by disordered tissue oxygenation. , , , , In addition, where examined, RBCs from such patients exhibit impaired vasodilatory capacity. , , , , 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 failure, diabetes, , pulmonary hypertension , and sickle cell disease, , all of which are conditions characterized by inflammation, oxidative stress, and dysfunctional vascular control. Moreover, known crosstalk 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.