Effect of Processing and Storage on RBC function in vivo

Abstract

Red Blood Cell (RBC) transfusion is indicated to improve oxygen delivery to tissue, and for no other purpose. We have come to appreciate that donor RBCs are fundamentally altered during processing and storage, in a fashion that both impairs oxygen transport efficacy and introduces additional risk by perturbing both immune and coagulation systems. The protean biophysical and physiologic changes in RBC function arising from storage are termed the ‘storage lesion’; many have been understood for some time; for example, we know that the oxygen affinity of stored blood rises during the storage period1 and that intracellular allosteric regulators, notably 2,3-bisphosphoglyceric acid (DPG) and ATP, are depleted during storage. Our appreciation of other storage lesion features has emerged with improved understanding of coagulation, immune and vascular signaling systems. Herein we review key features of the ‘storage lesion’. Additionally, we call particular attention to the newly appreciated role of RBCs in regulating linkage between regional blood flow and regional O2 consumption by regulating the bioavailability of key vasoactive mediators in plasma, as well as discuss how processing and storage disturbs this key signaling function and impairs transfusion efficacy.

Goal of Red Blood Cell (RBC) Transfusion

Red Blood Cell (RBC) transfusion is indicated to improve oxygen delivery to tissue, and for no other purpose. We have come to appreciate that donor RBCs are fundamentally altered during processing and storage, in a fashion that both impairs oxygen transport efficacy and introduces additional risk by perturbing both immune and coagulation systems. The protean biophysical and physiologic changes in RBC function arising from storage are termed the ‘storage lesion’; many have been understood for some time; for example, we know that the oxygen affinity of stored blood rises during the storage period¹ and that intracellular allosteric regulators, notably 2,3-bisphosphoglyceric acid (DPG) and ATP, are depleted during storage. Our appreciation of other storage lesion features has emerged with improved understanding of coagulation, immune and vascular signaling systems. In this review, we will call particular attention to the newly appreciated role of RBCs in regulating regional blood flow (and thus O₂ delivery) as well as discuss how disturbance of this key signaling function (by processing and storage for transfusion) impairs transfusion efficacy (improving O₂ delivery).

Alterations to RBCs during Processing and Storage (‘the Storage Lesion’)

Many recent reports summarize the changes that occur with RBC storage^2-7 (FIGURE 1). These reports document increased potassium, lactate, and free hemoglobin with increased RBC storage time^{8, 9}. Additionally, RBCs loose deformability with increased duration of storage⁸, limiting passage through the microcirculation, which is further impaired by increased RBC aggregation and adhesion to endothelium^{8, 10-12}. As noted above, the concentration of 2,3 DPG decreases with storage time⁸, the resultant increase in oxygen affinity limits oxygen unloading from hemoglobin during systemic perfusion¹³. RBC storage also impacts recipient immune function. Stored RBCs induce alterations in multiple cytokines after incubation with plasma or whole blood samples, including increased IL-6, IL-8, phospholipase A2, and superoxide anions, and decreased TNF-alpha concentrations^{9, 14}. The clinical consequences of this phenomena was first reported in the 1970's when Opelz et al. reported improved renal allograft success in patients transfused pre-renal transplant¹⁵. Subsequently, additional reports indicating an immune suppressive effect of RBC transfusions have documented an association with increased cancer reoccurrence, live births in women with a history of spontaneous abortions, and increased postoperative infection rates^{16, 17}.

FIGURE 1.

Interaction between donor RBCs and transfusion recipients as a consequence of receiving RBCs altered by processing and storage². The figure illustrates that the storage lesion impacts overlapping pathways of oxygen delivery, RBC rheology and physiology, as well as immune modulation. MOF = multiple organ failure; NO = nitric oxide.

Of note, the genesis of RBC microparticles during RBC storage may be related to the influence of transfusion upon donor immune and coagulation systems^{18, 19}. The oxidative injury that occurs with storage leads to formation of RBC membrane microparticles and release of bioactive lipids from its membrane²⁰. Such RBC microvesicles contain CD47 antigens associated with macrophage inhibition²⁰. Other data suggests that increased generation of procoagulant phospholipids occurs during RBC storage^{21, 22}. These bioactive lipids, such as lysophosphatidylcholine, have also been demonstrated to be pro-inflammatory and are associated with an increased risk of acute lung injury²³. The immune dysfunction secondary to transfusion has been termed Transfusion Related Immune Modulation (TRIM) and has been well described^{24, 25}. Interestingly, the transfusion of RBCs of increased storage duration has also been associated with Transfusion Related Acute Lung Injury (TRALI) via pro-inflammatory mechanisms¹⁷.

RBC Transfusion Efficacy as a function of RBC Storage Duration

While transfusion of RBCs increases the hematocrit (Hct) and therefore increases oxygen (O₂) content, it does not necessarily follow that O₂ delivery to tissue is likewise increased. The purpose of transfusion is not to merely improve O₂ content, but instead – to improve O₂ delivery. As outlined above, fundamental changes occur to RBCs during storage that alter their biological function and their ability to deliver O₂⁷. Beyond their role as O₂ carriers, RBCs play a fundamental role in the O₂ dependent regulation of vascular tone by exporting vasodilatory signals that regulate regional blood flow. This key RBC regulatory function is modified by processing and storage, resulting in compromised RBC-dependent vasoregulation, which impairs regional O₂ delivery^{8, 26}.

In general, animal models evaluating transfusion efficacy demonstrate adverse effects of storage upon O₂ delivery^27-29. For example, in an animal shock model, human RBCs of decreased storage age improved O₂ consumption whereas older RBCs could not²⁹. In humans, several groups have measured O₂ delivery and consumption before and after RBC transfusion in critically ill patients and have reported a disconcerting lack of benefit directly attributable to RBC transfusion^30-39. Though it is difficult to ascertain the RBC transfusion efficacy and there are many difficulties with measures of global or tissue-specific cellular respiration, available evidence suggests that transfusion of stored RBCs may have adverse effects on microcirculatory flow and oxygen utilization.

Most storage lesion effects appear to worsen with increased storage duration. When older RBCs (21-42 days) have been compared to fresh RBCs (0-4 days of storage): inflammatory response, mediators of oxidative injury, and risk of hyper-coagulation appear increased^{8, 14, 21, 22, 40, 41}.

RBCs stored for a longer time also appear more likely to activate recipient neutrophils that have been primed by recipient disease (such as sepsis or multiple trauma)²³, a mechanism posited for transfusion related acute lung injury (TRALI)^{17, 42}. In a study of critically ill adults, transfusion of RBCs stored for > 14 days was associated with a decrease in tissue oxygenation, whereas transfusion of RBCs stored < 14 days did not change tissue O₂ saturation⁴³. In a study of rats in shock, human RBCs stored for 3 days increased O₂ consumption whereas human RBCs stored for 28 days did not²⁹. RBCs of increased storage age have also been associated with decreased perfusion and O₂ consumption in critically ill adults⁴. These results were not confirmed in a similar study involving 22 patients which did not reveal any improvement in perfusion or oxygen consumption with RBCs of increased or decreased age potentially because it was not adequately powered to detect a difference in O₂ consumption⁴⁴. Conflicting results have also been reported for patients with traumatic brain injury, where one study reports no improvement in the partial pressure of O₂ in brain tissue with RBCs > 19 days of storage and another found no relationship between RBC age and cerebral oxygenation^{45, 46}. Other small studies have not measured a decrease in O₂ consumption or tissue saturations with the transfusion of RBC stored for longer times⁴⁶. Of note, evaluation of blood product efficacy and safety is best informed by distinctly separating analysis of outcome in critically ill and non–critically ill populations, since the risk-benefit ratio for blood product administration is dependent on the baseline physiologic state and differs between critically ill and non–critically ill patients⁴⁷. Specifically, patients with marginally compensated organ failure are less tolerant of additional insult, including adverse effects attributable to transfusion¹⁷.

RBC Vasoactivity: Relevance to O₂ Delivery and Transfusion Efficacy

Oxygen delivery is a function of blood O₂ content and flow, but the latter (flow) is the far more important determinant. Specifically, the ability to increase O₂ content is somewhat limited (varying linearly with Hb concentration and % O₂ saturation), whereas regional blood flow (a function of vessel radius to the 4^th power) may be increased or decreased by several orders of magnitude. In fact, under most circumstances, it is principally the volume and distribution of blood flow that vary to maintain dynamic coupling between O₂ delivery and metabolic demand. Of note, classic experiments demonstrate that it is Hb O₂ saturation (rather than free plasma or tissue PO₂) that is coupled to blood flow to maintain this physiology in vivo^{48, 49}. These and other studies provide evidence that RBCs act as both O₂ sensors and transducers of vasoconstrictor and vasodilator responses^50-53. RBCs have been demonstrated to recapitulate this response in vitro, actuating graded vasodilation across the physiological O₂ gradient⁵². As such, remarkably, RBCs dilate blood vessels in concert with, and proportional to, O₂ delivery^{54, 55}. That this coupling is directly linked to Hb O₂ saturation rather than to PO₂, indicates that, in addition to service as an O₂ transport protein, Hb is a key physiological effector in resolution of tissue hypoxia^{49, 56-59}.

Control of NO transport and bioavailability is fundamental to RBC-based blood flow autoregulation and acts by effecting reflex hypoxic vasodilation (HVD) in the circulation. It should be noted, however, that endothelium-derived NO does not directly mediate the HVD that underlies blood flow regulation^{51, 60}. In fact, because of substrate (O₂) limitation, NO production by eNOS is most likely attenuated by hypoxia^61-63. Morover, NOS inhibitors do not block the acute change in blood flow that is coupled to Hb desaturation⁴⁸. Rather, it appears that NO is transported by RBCs to effect HVD at a time and place remote from the original site of NO synthesis (the NO groups deployed by RBCs do, however, originally arise from eNOS⁵⁴ and perhaps, other NOS isoforms⁶⁴ or nitrite⁶⁵). RBCs are therefore vascular control elements (FIGURE 2). Thus, it is increasingly appreciated that the transition in Hb conformation that occurs in the course of O₂ loading and delivery during arterio-venous (A-V) transit also governs transactions between RBCs and circulating NO groups in plasma: oxygenated (R-state) Hb sequesters, and deoxygenated (T-state) Hb releases, net NO bioactivity. As such, Hb R- to T-state cycling drives processing of circulating NO groups (through RBCs) to plasma or cellular thiols, to form vasoactive NO adducts known as S-nitrosothiols (SNOs). This system provides functional coupling between regional blood flow and biochemical cues of perfusion insufficiency, which may include hypoxia, hypercarbia and acidosis (FIGURE 3). As described below, the system is vulnerable to disruption during periods of oxidative stress and sustained alterations in PO₂.

FIGURE 2.

RBCs transduce regional O₂ gradients in tissue to control NO bioactivity in plasma by trapping or delivering NO groups as a function of 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 deoxyHbFeNO (A; NO sink), oxySNOHb (B; NO store), and acceptor thiols including the membrane protein SNO-AE1 (C; bioactive NO source). Direct SNO export from RBCs or S-transnitrosylation from RBCs to plasma thiols (D) 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 (panels 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.

FIGURE 3.

Hb O₂ saturation (Hb SO2%) exerts coordinated governance of RBC SNO content, RBC vasoactivity and human peripheral blood flow. A) Human blood gas measurements of SNO and O₂. RBCs with (black) or without (red) added extracellular glutathione (GSH) were deoxygenated under inert gas⁵⁰. The natural logarithm of SNO content in RBCs (SNORBC) is linearly related to Hb O₂ saturation. GSH accelerated SNORBC decay, consistent with O₂ -linked export of NO groups to extra-erythrocytic thiols. B) RBCs induce graded relaxation of systemic arteries (aortic ring bioassay) that is inversely related to Hb O₂ saturation across the physiological range, recapitulating hypoxic vasodilation⁵². Hb O₂ saturations spanned from red (oxy, > 90% Hb SO₂) to blue (deoxy, < 40% Hb SO₂). C) Leg vascular conductance increases as blood O₂ content falls (hypoxic vasodilation)⁵¹. Hb O₂ saturation and thus arterial blood O₂ content were manipulated by CO exposure ± varying FiO₂ (fraction of inspired O₂; hypoxia versus hyperoxia). Neither vascular conductance nor blood flow correlated with blood PO₂ per se.

Evidence for RBC Vasoactivity

Multiple lines of evidence indicate that NO is transported by RBCs to distal tissues. This RBC paracrine function is governed by O₂-linked transitions in Hb conformation, and Hb O₂ saturation thereby couples export of vasodilatory signals to tissue PO₂. Importantly, the only known endogenous NO compounds that retain bioactivity in the presence of Hb are S-nitrosothiols (SNOs). The data corroborating this biology is extensive. For example, Stamler et al.^{52, 55} and Datta et al.⁶⁶ demonstrated that RBCs dilate pre-constricted aortic rings at low PO₂ (1% O₂) but constrict at high PO₂ (95% O₂). More broadly, vasoconstriction and vasodilation by RBCs is graded across the physiological range of PO₂ encountered in the microcirculation⁵². In addition, Kubes’ group^67-69 has demonstrated bioactivity of inhaled NO in the mesenteric circulation that is commensurate with SNO production, and Cannon et al.⁷⁰ observed that NO gas inhalation lowers forearm vascular resistance in human volunteers who had undergone pharmacological NOS inhibition. McMahon and colleagues⁷¹ identified inhaled NO-based activity, at least in part, with SNO-Hb. Moreover, Reynolds and colleagues⁷² and Diesen and colleagues⁷³ have shown that NO added to RBCs potentiates vasodilatory activity to the extent that SNO-Hb is generated. Sonveaux et al.⁷⁴ have reported that the lifetime of infused SNO-Hb is increased by high PO₂ and destabilized at low PO₂, in direct contrast to NO itself. Additional studies support the conclusion that SNOs are impervious to inactivation by RBCs^{73, 75}. It therefore appears that NO bioactivity is preserved by RBCs in the form of stable SNOs that can be ‘exported’ to peripheral tissues on demand. Notably, hypoxic vasodilation by RBCs is entirely independent of endothelium^{54, 73, 76-78} and is facilitated by the presence of extra-erythrocytic low-molecular-weight thiols.

Expanding the NO Paradigm: RBCs Control NO Bioactivity in Blood

Originally, the “classic” NO paradigm was based on several tenets: 1) NO^• is the primary bioactive product of NOS; 2) NO life-time and fate is explained by the diffusion of NO^• “gas” in solution and by its terminal reactions with Hb (and superoxide); and 3) the heme center of guanyl cyclase is the primary NO^• receptor. More recent evidence suggests that NO-based signaling is significantly more complex. In particular, the landscape has been fundamentally altered by appreciation that NO acts principally through covalent binding to cysteine thiols to form S-nitrosothiols (SNOs), rather than by signaling exclusively via sGC. Approximately one thousand SNO-proteins have now been identified in cells and tissues, indicating the central role of SNOs in NO-based signaling ^79-81.

It is now understood that through context-responsive reactions between NO and Hb (that is, reactions coupled to PO₂ and influenced by CO₂ and pH), RBCs exert functional control over NO group bioavailability in blood, and thereby perform crucial roles as both sensors of O₂ supply and effectors in vascular signaling at local, organ, and system levels ^{54-56, 82}. Thus, RBCs shuttle 3 gases in the respiratory cycle (NO/O₂/CO₂) to control delivery of O₂. Both the complexity of the biological chemistry of NO and the specificity of NO-group reactions in signaling are well exemplified in the case of Hb. Through tightly regulated reactions at its heme center and a reactive cysteine thiol (βcys⁹³), in the course of circulatory transit, Hb captures (binds NO at β-hemes), activates (converts β-heme-NO into βcys⁹³-SNO) and deploys NO groups (transnitrosylates receptor thiols, see details below). Heme-redox coupled activation of the NO group, a requirement for the oxidative chemistry that subserves conversion of heme-NO into βcys⁹³-SNO, is governed by O₂-coupled allosteric transitions in Hb^{54, 82-84}. The O₂-linked change in Hb conformation also initiates export of SNO from RBCs by promoting NO transfer to receptor thiols, including those associated with the erythrocytic membrane protein AE-1 (Band 3)⁸⁵ and thence to extra-erythrocytic thiols^{50, 75} to form plasma or other cellular SNOs, which are vasoactive at concentrations as low as 1 to 5 nM^{54, 55}. By governing the bioavailability of NO in the microcirculation through regulated sequestration, transport and delivery of NO groups, RBCs maintain appropriate coupling between regional blood flow and both O₂ availability in the lung and O₂ need in the periphery.

S-nitrosylation (SNO synthesis) in RBCs

The chemical basis of S-nitrosylation reactions and the molecular determinants of targeting specificity are reviewed in detail elsewhere^{56, 80, 86}. In brief, hemoglobin processes NO^•, low-molecular-weight SNO or nitrite (NO ^-₂) into S-nitroso-Hb (SNO-Hb), and thus has been analogized to a SNO reactor^87-92. As described below, the locus of NO binding to the Hb globin chain is the highly conserved βcys⁹³, which conforms to both acid-base and hydrophobic motifs for S-nitrosylation^{87, 89, 92-95}. Importantly, as a result of conformation-dependent variation in the βcys⁹³ microenvironment, the likelihood of NO ligation to βcys⁹³ thiol is increased in R-structure (oxygenated Hb) and diminished in T-structure (deoxygenated Hb)^{54, 55, 82}. Therefore, S-nitrosylation is favored upon Hb oxygenation, whereas NO group (NO/NO⁺) release is promoted upon Hb deoxygenation⁸⁹. By contrast, NO binding to heme (at physiological ratios of NO/Hb) is favored in T state and disfavored in R. Subsequent binding of O₂ to heme as RBCs transit the lung, which promotes Hb R structure thus facilitates NO group transfer from heme to thiol (auto-S-nitrosylation).

Control of NO Entry into RBCs

Although free Hb readily inactivates NO^•, Hb packaging in RBCs limits NO^• consumption by a factor of ~1,000^96-99. Specifically, four successive barriers separate eNOS-generated NO^• from intra-erythrocytic Hb: 1) the endothelial cell membrane, 2) an abluminal RBC-free zone, created by velocity gradients at the vascular edge of the migrating RBC column^99-101, 3) an unstirred layer of plasma surrounding moving RBCs¹⁰², and 4) the RBC membrane and cytoskeleton – which together provide a dynamically responsive diffusion barrier to NO^{• 97, 103, 104}. It is generally accepted that, due to its solubility properties, the neutral and (relatively) nonpolar NO^• radical freely permeates membranes. However, it has been recently reported that dynamic chemical and physical variation of the RBC membrane alters, and in fact may control, the rate of NO^• uptake by RBCs^{97, 104}. This variation appears to arise from conformation- dependent interactions between Hb and key RBC membrane proteins that modulate NO^• uptake (FIGURE 4), rather than from an O₂-linked change in the physical nature of the lipid bilayer itself.

FIGURE 4.

Conformation-specific binding between Hb and the RBC membrane protein AE-1 affords O₂ responsive control of NO trapping¹⁰³. A) Under oxygenated conditions (normoxic RBC, at left), the RBC membrane constitutes a significant barrier to NO^• entry via tight association between the sub-membrane cytoskeleton and the cytoplasmic domain of the Band 3 (AE-1) membrane protein (ankyrin successfully competes with oxyHb for binding to AE-1). Upon RBC deoxygenation (hypoxic RBC), oxyHb R-state is converted to deoxyHb T-state, which now successfully competes with ankyrin for the AE-1 cytoplasmic domain (affinity for AE-1 ranks as follows: deoxyHb > ankryn > oxyHb). Proximal apposition of heme to the membrane and diminished cytoskeleton-membrane interaction allows increased NO entry and affords intraerythrocytic Hb greater access to extraerythrocytic NO. Moreover, if deoxyHb encounters high concentrations of NO^•, “super-T” αFe(II)NOHb may form (in which NO bound to the α subunit disrupts normal heme-globin linkage, locking Hb in the deoxy or T conformation; see text for details). B) Increasing the proportion of intra-erythrocytic T-state Hb (by forming either deoxyHb or αFe(II)NO, both of which are T-state tetramers and bind avidly to AE-1) increases NO consumption by intact RBCs as measured by a competition assay. In this plot, increased consumption in treated vs control RBCs appears as an increased KRBC/KRBCcontrol ratio (y-axis). C) Increased NO uptake by NOpretreated hypoxic RBCs correlates with the formation of αFe(II)NO (i.e. “super-T” Hb).

NO Group Processing in RBCs

As described above, after formation of HbFeNO, the O₂-linked transition from T- to R- structure promotes intra-molecular transfer of the NO group from β-heme to β-thiol (Cysβ⁹³), thereby forming HbCysβ⁹³SNO (SNO-Hb)^{65, 84}. Once formed in R-state Hb, Cysβ⁹³SNO is stable and protected from solvent through confinement in a hydrophobic pocket^{54, 55, 105-107}. Additionally cycling of NO between Cysβ⁹³ and heme in vivo is supported by measurements of FeNO and SNO as a function of PO₂ (FIGURE 5)^{50, 52, 55}.

FIGURE 5.

O₂-dependent variation in SNO–Hb (■) and Hb[FeNO] (□) demonstrate the association between Hb conformation and intramolecular heme → thiol migration of NO groups in RBCs. A - D)⁵², Moles NO per mole Hb tetramer in arterial and mixed venous blood from humans breathing either 21% O₂ at 1 ATA (absolute atmospheres) (A and D), 21% O₂ at 0.56 ATA (equivalent to ~ 12% O₂ at 1 ATA) (B) or 100% O₂ at 3 ATA (D). Total Hb-bound NO equals the sum of the 2 bars for each condition. These data demonstrate O₂ - dependent shuttling of Hb-bound NO groups between heme and Cys-ß93. (E and F), SNO content of blood Hb, presented as the fraction of Hb-NO (% SNO), correlates with Hb O₂ saturation (E), but not with pO₂ (F).

NO Group Delivery by RBCs

The described arterio-venous gradient of SNO-Hb^{50, 52, 54, 66, 108-110} is consistent with dynamic processing of NO by circulating RBCs, in which O₂ loading in the lung is accompanied by Hb S-nitrosylation and NO-group export is facilitated by O₂ delivery when steep O₂ gradients are encountered in the periphery (hypoxic vasodilation)⁵⁴. The R → T transition in Hb conformation that occurs with O₂ delivery shifts Cysβ⁹³ SNO from its sequestered hydrophobic niche to exposure in aqueous solvent^{54, 55, 106}, making the NO group available for transnitrosylation of target protein or small-molecular-weight thiol^{54, 75, 85, 111}. Importantly, NO is transferred in this reaction as a nitrosonium ion (NO⁺)^{54, 56} and is therefore protected from (Fe(II)) heme recapture and/or inactivation (as well as from pharmacological NO^• chelators)¹¹².

Hb deoxygenation (and/or heme oxidation) enables release of NO bioactivity by RBCs, which results in vasodilation ^{50, 54, 55, 66, 85, 113}. Export of NO bioactivity from RBCs occurs, at least in part, via low-mass thiols in plasma^{50, 52, 73, 75} (FIGURE 6), although a role for direct cell-cell contact in the microcirculation may be envisaged. In either case, signaling is believed to proceed via a transnitrosylation cascade; the RBC membrane protein AE-1, with which Hb is known to undergo conformation-dependent docking^{114, 115}, appears to initiate this cascade. SNO-Hb docking on AE-1 is followed by NO-group transfer from SNO-Cysβ⁹³ to cysteine thiol(s) of the cytoplasmic domain of AE-1; moreover, this transfer is required for RBC-induced vasodilation⁸⁵. The identity of the acceptor thiols that ferry the NO signal in vivo from AE-1 to the vascular wall is unknown.

FIGURE 6.

PO₂ - regulated export of NO groups from RBCs can occur via NO group transfer from SNO-Hb to an extra-erythrocytic thiol reactant⁷⁵. Circulatory transit was simulated for human whole blood in a thin-film tonometer (under 5% CO₂, balance N₂, pH 7.4) after spiking the sample with the non-native, low-mass thiol, N-acetyl cysteine (NAC, 100 M in plasma). The concentration of S-nitroso-N-acetyl cysteine (SNOAC) that formed in plasma was measured by mass spectrometry, and confirmed by mass labeling with ¹⁵N. The conversion of extra-erythrocytic NAC to SNOAC correlated with RBC O₂ and SNO content. Serum SNOAC formed as a function of Hb SO₂. A) Liquid chromatogram demonstrating co-elution of RBC-generated SNOAC with the ¹⁵N-labeled SNOAC standard. B) Mass spectrum demonstrating paired signals from RBC-generated SNOAC and the ¹⁵N-labeled SNOAC standard (m/z 194). C) Extra-erythrocytic SNOAC concentration follows oxyHb desaturation (by co-oximetry). D) RBC SNO content (black) decreased in tandem with HbO₂ desaturation (dashed blue). Note that, as SNO content in RBCs fell, extra-erythrocytic SNOAC (yellow symbols) accumulated. Note also that SNOAC levels were below the limits of detection when the HbO₂ saturation was above 80%.

Vasodilation by SNO-Hb is readily detected at < 10^–8 M, which suggests that the endogenous SNO-Hb pool (10^–7 – 10^–5.5 M) dispenses NO bioactivity in a tightly regulated fashion and with significant potency. By exploiting the Hb conformation-based NO delivery system, RBCs actuate graded dilation of blood vessels over rapid time scales - that differ greatly from the slow relaxation of native vascular smooth muscle under hypoxia (a response that occurs over minutes, and which may set background vascular tone). Thus, baseline tone appears to be regulated further by RBC-based HVD (acute changes, over seconds)^{51, 60, 66, 73}. Hypercarbia and acidosis are additional effectors of the T-conformation in Hb, and thereby also promote vasodilation by RBCs¹¹⁶. Furthermore, oxidation of hemes can elicit a T-like conformation in SNO-Hb to promote NO release^{54, 113}. Thus, ischemia and oxidative stress may provide multiple biochemical cues that can be employed by RBCs to correct perfusion insufficiency.

Dysregulated RBC Vasoactivity Arising from the Storage Lesion

Strong evidence is mounting in support of a causal relationship between acquired, NO-related RBC dysfunction and a host of morbidities that complicate critical illness^117-124. Recently, it has been observed that levels of SNO-Hb are altered in several disease states characterized by disordered tissue oxygenation^{66, 71, 74, 77, 78, 125-127}. In addition, where examined, RBCs from such patients exhibit impaired vasodilatory capacity^{50, 71, 78, 127}. These data suggest that altered RBC-derived vasoactivity contributes to human pathophysiology. Specifically, alterations in NO metabolism by RBCs have been reported in congestive heart failure⁶⁶, diabetes^{78, 126}, pulmonary hypertension^{52, 71} and sickle cell disease^{127, 128}, all of which are conditions characterized by inflammation, oxidative stress and dysfunctional vascular control.

RBCs are progressively altered by processing and storage for transfusion (referred to as the ‘‘storage lesion’’, as described above)⁷, and the interval between RBC donation and administration appears to be an independent risk factor for transfusion-associated morbidity and mortality^{3, 129-131}. Altered O₂ loading/unloading by stored RBCs is well documented^132-134. It has been shown more recently that, in blood that has been collected, processed, and stored using blood-banking industry standard operating procedures, RBC SNO-Hb levels and RBC-dependent vasodilatory activity are profoundly depressed, and that these defects can be reversed by repletion of RBC SNO^{8, 72} (FIGURE 7). These observations provide mechanistic insight into clinical data that suggest impaired vasoregulation by administration of banked blood^{4, 132, 135-137}.

FIGURE 7.

Impact of processing and storage upon RBC NO content and vasoactivity (both in isolated vascular rings⁸ and in a whole-animal cardiac preparation⁷²). In these projects, blood from healthy volunteers was leukofiltered, processed into standard additive solution and stored according to AABB standards. (A) RBC NO content was significantly depressed, (B) as was vasoactivity in a rabbit vascular ring preparation. (C) Representative tracings from a similar project demonstrate the degree of vasorelaxation as percent change in tension in rabbit aortic rings produced by fresh, stored (expired and day 1), or renitrosylated (day 1) RBCs. (D) Hypoxic vasodilation by stored and renitrosylated RBCs in vivo. Shown are changes in canine coronary artery bloodflow(mean ± SD; n7) produced by infusion of SNO-depleted or renitrosylated RBCs. Increases in flow elicited by renitrosylated RBCs were significantly greater than those produced by SNO-depleted RBCs, and the degree of change was greater under hypoxic (5% FiO2) than normoxic (21% FiO2) conditions.

Summary

Trapping, sequestration, processing and delivery of NO groups by RBCs, which are coupled to Hb allostery, links regional blood flow to biochemical cues of perfusion insufficiency (metabolic demand). The respiratory cycle may thus be broadly viewed as comprised of three gases, NO, O₂ and CO₂, which are coordinately or covalently bound to Hb. RBCs therefore actively regulate regional blood flow (and thus, O₂ delivery) by coupling O₂ sensing by Hb to the formation and release of vasodilator SNO, conveying a PO₂-linked signal in the setting of perfusion lack. In addition to the multiple components of the ‘stroage lesaion’, which impact every aspect of RBC physiology, malfunction of this precise RBC vascular signaling system may undermine transfusion efficacy. Understanding of such should inform improvements in blood processing and storage, which are necessary to ameliorate the ‘storage lesion’.