1036 SECTION IX Pediatric Critical Care Hematology and Oncology RBCs in the spleen However, during critical illness, RBC endothelial interactions are altered by RBC injuries associated with sepsis22,2[.]
1036 S E C T I O N I X Pediatric Critical Care: Hematology and Oncology RBCs in the spleen However, during critical illness, RBCendothelial interactions are altered by RBC injuries associated with sepsis22,23,33,34 and/or oxidative stress.19 This is more prominent with activated endothelium, as frequently occurs in critical illness.34–36 Such RBC-endothelial aggregates create a physiologically significant increase in apparent blood viscosity.17 Moreover, RBC adhesion directly damages endothelium37–40 and augments leukocyte adhesion,41–44 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 to mm in thickness, which act like droplets that deform reversibly under the shear encountered during circulatory transit.17,45 Unique RBC geometry and deformability arises from cytoplasmic viscosity and specific interactions between the plasma membrane and underlying protein skeleton.28 Cytoplasmic viscosity is mainly determined by Hb concentration, which varies with intraerythrocytic hydration that is actively regulated by ATP-dependent cation pumps.46 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.47 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.46 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 periods48 and in RBCs subjected to significant metabolic stress during critical illness.49,50 Regulation of Blood Flow Distribution by Red Blood Cells Microcirculatory blood flow is physiologically regulated to instantaneously match O2 delivery to metabolic demand This extraordinarily sensitive programmed response to tissue hypoperfusion is termed hypoxic vasodilation (HVD).51 This process involves the detection of point-to-point variations in arteriolar O2 content52 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 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 supply.53 It was later demonstrated in vivo that HbSO2 was independent of plasma or tissue PO2 but was directly correlated with blood flow.54 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 utilization.55 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 ATP: (1) formation and export of S-nitrosothiols (SNOs), “catalyzed” by Hb (SNOHb hypothesis)56–58; (2) reduction of nitrite (NO22) to NO by deoxygenated Hb (nitrite hypothesis)59; and (3) hypoxia-responsive release of ATP (ATP hypothesis).55,60 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 1980,61 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.61,62 Identification of EDRF consequently led to a race to discover its chemical identity It was not until years later that two groups simultaneously published definitive studies characterizing and identifying EDRF as NO.63,64 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 39,59-monophosphate (cGMP) that appeared to clarify the means by which NO achieves its myriad effects.65 Over time, however, it is now appreciated that this pathway has little to 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.54,66 Because of O2 substrate limitation, NO production by endothelial nitric oxide synthase (eNOS) is most likely attenuated by hypoxia.67,68 In fact, NO derived from eNOS56 (and perhaps other NOS isoforms69 and/or nitrite70) 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.56,57 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.71 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 (Fe21) of sGC72 and (2) in the vessel lumen, with the heme group (Fe21) 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 CHAPTER 87 The Erythron autoxidation reaction),73 and/or O2-derived free radicals, including superoxide (O22), hydrogen peroxide (H2O2), or hydroxyl radicals (OH2) 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,74,75 and (3) an RBC-free zone adjacent to endothelium that results from laminar flow streaming.76 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.72 Metabolism of Endothelium-Derived Nitric Oxide by Red Blood Cells: Contemporary View A much broader biological chemistry of endothelial NO has been elucidated.77–79 Most notable is the covalent binding of NO1 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 (Snitrosoalbumin80), 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.81 The SNO hypothesis was extended to include a reactive thiol of Hb (Cysb93) that was demonstrated to undergo S-nitrosylation and sustain bioactivity under oxygenated conditions and NO release under low O2 conditions (see HbSNO hypothesis).56 In this SNO paradigm, the NO radical must be oxidized to an NO1 (nitrosonium) equivalent, which can then be passed between thiols in peptides and proteins, preserving NO bioactivity.77,78 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.79,82 Processing and Export of S-Nitrosothiols by Red Blood Cells Hb S-nitrosylation (HbSNO), which has been characterized by both mass spectrometry83 and X-ray crystallography,84 provides an explanation as to how NO circumvents terminal reactions with Hb, enabling RBCs to conserve NO bioactivity and transport it throughout the circulation56,57 (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 O2 loading/unloading during arteriovenous (AV) transit This is due to conformationaldependent change in reactivity of the Cysb93 residue toward NO, which is higher in the R (oxygenated) Hb state and lower in the T (deoxygenated) Hb state.56,57 In a tightly regulated fashion, Hb captures and binds NO at its b-hemes and then passes the NO group from the heme to a thiol (Cys-b93-SNO).70,85 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.86 Once in R state, the Cys-b93-SNO is protected through confinement 1037 to a hydrophobic pocket.84 NO group export from Cys-b93SNO occurs when steep O2 gradients are encountered in the periphery (HVD) The R- to T-state conformational transition that occurs on Cys-b93-SNO deoxygenation (or oxidation) results in a shift in the location of the b-chain from its hydrophobic niche toward the aqueous cytoplasmic solvent.84 This allows the Cysb93-SNO to be “chemically available” for transfer to target thiolcontaining proteins, including those associated with the RBC membrane protein AE-1 (Band 3)87 and extra-erythrocytic thiols.88,89 Resultant plasma or other cellular SNOs then become vasoactive at low nM concentrations.56,57 Importantly, all NO transfers in this process involve NO1,56,58 which protects bioactivity from Fe21 heme recapture and/or inactivation S-nitrosothiols are the only known endogenous NO compounds that retain bioactivity in the presence of Hb.56,89,90 Extensive evidence supports SNO-Hb biology, whereby RBCs exert graded vasodilator and vasoconstrictor responses across the physiologic microcirculatory O2 gradient RBCs dilate preconstricted aortic rings at low PO2 (1% O2), while constricting at high PO2 (95% O2).57,90–92 The vasodilatory response at low O2 is enhanced following the addition of NO (or SNO) to RBCs commensurate with SNO-Hb formation.56,87,90,93 Additionally, the vasodilatory response is enhanced in the presence of extracellular free thiol,90 occurs in the absence of endothelium58,90 (which is consistent with in vivo observation that HVD is endothelium independent94), and transpires in the time frame of circulatory transit, as confirmed by measurements of AV gradients in SNOHb.56,88,91,92 In addition to these ex vivo experiments, numerous groups have also demonstrated bioactivity of inhaled NO commensurate with SNO-Hb formation.95–99 Metabolism of Nitrite by Red Blood Cells NO22, 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.100 Some have suggested that the reduction of nitrite by deoxyHb may serve as the RBC-derived signaling mechanism regulating HVD.101 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 NO22 must escape RBCs at low O2 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 (107 M-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 (HbFe21NO) being further converted into SNO-Hb,70,85 as HbFe21NO itself acts as a vasoconstrictor rather than vasodilator.102 The second shortcoming relates to the fact that the NO22 reductase activity of deoxyHb is purportedly symmetric across the physiologic O2 gradient,103,104 with maximal activity occurring at the P50 of Hb (27 mm Hg).103,105 This reaction profile does not match the HVD response, which increases in a steadily graded fashion as PO2 falls in the physiologic range from 100 mm Hg down to approximately mm Hg (HbSO2 1%–2%).51,54 If RBC-based vasoactivity were maximal at Hb’s P50, then blood flow would be diverted away from regions with PO2 below 27 mm Hg, where it would be needed most Additionally, based on the symmetry of Hb nitrite reductase activity at the P50, RBCs traversing vascular beds with PO2 at 25 or 75 would 1038 S E C T I O N I X Pediatric Critical Care: Hematology and Oncology generate equal NO-based activity,101 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,55 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,55,106 with release stimulated by conditions associated with diminished O2 supply relative to demand, hypoxia, hypercapnia, and low pH.55,107 O2 offloading from membrane-associated Hb is thought to initiate RBC ATP release,106 stimulating heterotrimeric G protein,108 as a result of membrane deformation This leads to activation of adenylyl cyclase and an increase in cyclic adenosine monophosphate (cAMP),109 which activates protein kinase A (PKA).109 PKA stimulates cystic fibrosis transmembrane conductance regulator,110 which activates release of ATP from the RBC via pannexin 1.111 Release of ATP via this pathway requires an increase in intracellular cAMP, which is controlled by the relative activities of adenylyl cyclase and phosphodiesterase 3.60 Despite potential as an HVD mediator, RBC-derived ATP falls short on two fronts First, HVD is unaltered by both endothelial denudation and eNOS deletion58; 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.58 Red Blood Cell Energetics and Consequences of Antioxidant System Failure RBCs produce ATP by glycolysis only, with two branches112: the Embden Meyerhof Pathway (EMP) and the Hexose Monophosphate Pathway (HMP).113 Importantly, the HMP is the sole means for recycling NADPH,114 which powers the thiol-based antioxidant system.114 HMP flux is gated by protein complex assembly upon the cytoplasmic domain of the Band membrane protein (cdB3 “metabolon”).115–122 HMP flux oscillates with PO2, as a function of Hb conformation and cdB3 phosphorylation.123–129 Of note, RBC antioxidant systems fail when HMP flux is blunted by altered cdB3 protein assembly/phosphorylation caused by aberrant Hbs or hypoxia.130,131 Strikingly similar perturbations to cdB3 are reported in sepsis,132,133 possibly arising from caspase activation134–136 and/or direct endotoxin or complement membrane binding21,137–143 (altering metabolon assembly, glycolysis, and ROS clearance; see Fig 87.1C).144–146 As such, it appears that sepsis (particularly in the setting of hypoxic and/or uremic/oxidative147–154 environments) disturbs cdB3-based metabolic control, leading to (1) EMP activation, (2) limited glucose6-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 O2 delivery homeostasis (SiRD) This full pattern has been reported in other settings impacting protein assembly at cdB3130,131; further, such HMP constraint has functional similarity to glucose-6-phosphate dehydrogenase (G6PD) deficiency,130 which amplifies vulnerability to sepsis.155–157 Moreover, hypoxia critically limits RBC energetics and depowers RBC antioxidant systems.158,159 In health, O2• abundance is tightly regulated by the superoxide dismutase (SOD) family.160 However, overwhelming O2• genesis161 is implicated in sepsis-associated injury cascades.162,163 Of note, sepsis-associated O2• excess injures RBCs, impairing O2 delivery by altering control of O2 affinity,5–8 NO processing,164–166 rheology,21,24,25,28,140,167,168 and adhesion.33,169 O2• excess also disrupts vasoregulation via NO consumption and catecholamine inactivation in plasma.170–176 Specifically, ROS sourced directly to RBCs38,39,177,178 injure vessels.179,180 Such reciprocal injuries mutually escalate; as such, the dysoxia characteristic of septic shock (ischemia despite adequate blood O2 content and cardiac output)181–184 may arise from SiRD-vascular interactions.22,23 Notably, ROS excess is also a common consequence of uremia/kidney injury,147–154 particularly during sepsis.185–191 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.192 To date, clearance of normal senescent RBCs has not been clearly understood Two mechanisms have been proposed: (1) clustering of the band (B3) membrane protein193–196 and (2) externalization of membrane phosphatidylserine (PS).197–200 Both of these processes may be accelerated in the setting of critical illness, impairing O2 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.201 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,197–200 signaling RBC removal by the reticuloendothelial system.200 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 Ca21 entry through Ca21-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, CK1a, 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 disease.200,202,203 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,204 as well as RBC aggregation and deformability of RBCs, which also CHAPTER 87 The Erythron support clot assembly/retraction.205 In addition, RBCs interact directly and indirectly with ECs and platelets during thrombosis.206 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.207,208 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.209 Additionally, RBCs directly interact with fibrin(ogen) and affect the structure, mechanical properties, and lytic resistance of clots and thrombi.210 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.211 Summary Evidence is mounting in support of a causal relationship between acquired RBC dysfunction and a host of perfusion-related morbidities that complicate critical illness.20,92,178,212–225 Recently, it has been observed that levels of SNO-Hb are altered in several disease states characterized by disordered tissue oxygenat ion.92,93,164,165,226–231 In addition, where examined, RBCs from such patients exhibit impaired vasodilatory capacity.88,92,93,228,230–232 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,92 diabetes,93,227 pulmonary hypertension91,226 and sickle cell disease,228,233 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 1039 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 Key References Aird WC The hematologic system as a marker of organ dysfunction in sepsis Mayo Clin Proc 2003;78(7):869-881 Barvitenko NN, Adragna NC, Weber RE Erythrocyte signal transduction pathways, their oxygenation dependence and functional significance Cell Physiol Biochem 2005;15(1-4):1-18 Baskurt OK, Meiselman HJ Blood rheology and hemodynamics Semin Thromb Hemost 2003;29(5):435-450 Bennett-Guerrero E, Veldman TH, Doctor A, et al Evolution of adverse changes in stored RBCs Proc Natl Acad Sci U S A 2007;104(43):17063-17068 Doctor A, Stamler JS Nitric oxide transport in blood: a third gas in the respiratory cycle Compr Physiol 2011;1(1):541-568 Du VX, Huskens D, Maas C, Al Dieri R, de Groot PG, de Laat B New insights into the role of erythrocytes in thrombus formation Semin Thromb Hemost 2014;40(1):72-80 Foller M, Huber SM, Lang F Erythrocyte programmed cell death IUBMB Life 2008;60(10):661-668 Furchgott RF, Zawadzki JV The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine Nature 1980;288(5789):373-376 Hsia CCW Coordinated adaptation of oxygen transport in cardiopulmonary disease Circulation 2001;104(8):963-969 Hsia CCW Respiratory function of hemoglobin N Engl J Med 1998; 338(4):239-247 Ross JM, Fairchild HM, Weldy J, Guyton AC Autoregulation of blood flow by oxygen lack Am J Physiol 1962;202:21-24 Singel DJ, Stamler JS Chemical physiology of blood flow regulation by red blood cells: the role of nitric oxide and S-nitrosohemoglobin Annu Rev Physiol 2005;67:99-145 The full reference list for this chapter is available at ExpertConsult.com e1 References Kaushansky K, Lichtman MA, Kipps TJ, Seligsohn U, Prchal JT, eds Williams Hematology 8e ed New York: McGraw-Hill; 2010 Doctor A, Stamler JS Nitric oxide transport in blood: a third gas in the respiratory cycle Compr Physiol 2011;1(1):541-568 Hsia CCW Respiratory function of hemoglobin N Engl J Med 1998;338(4):239-247 Hsia CCW Coordinated adaptation of oxygen transport in cardiopulmonary disease Circulation 2001;104(8):963-969 Leon K, Pichavant-Rafini K, Quemener E, et al Oxygen blood transport during experimental sepsis: effect of hypothermia Crit Care Med 2012;40(3):912-918 Ibrahim Eel D, McLellan SA, Walsh TS Red blood cell 2,3-diphosphoglycerate concentration and in vivo P50 during early critical illness Crit Care Med 2005;33(10):2247-2252 Tuynman HA, Thijs LG, Straub JP, Koopman PA, Bezemer PD, Bronsveld W Effects of glucose-insulin-potassium (GIK) on the position of the oxyhemoglobin dissociation curve, 2.3-diphosphoglycerate, and oxygen consumption in canine endotoxin shock J Surg Res 1983;34(3):246-253 Johnson Jr G, McDevitt NB, Proctor HJ Erythrocyte 2,3-diphosphoglycerate in endotoxic shock in the subhuman primate: response to fluid and-or methylprednisolone succinate Ann Surg 1974;180(5): 783-786 Geers C, Gros G Carbon dioxide transport and carbonic anhydrase in blood and muscle Physiol Rev 2000;80(2):681-715 10 Klocke RA Velocity of CO2 exchange in blood Annu Rev Physiol 1988;50:625-37 11 Robin Fahraeus TL The viscosity of the blood in narrow capillary tubes Am J Physiol 1931;96:562-568 12 Copley AL Fluid mechanics and biorheology Thromb Res 1990; 57(3):315-331 13 Tek I, Toprak SK, Hasdemir E, Rahatli S, Yesilkaya A Influence of febrile neutropenia period on plasma viscosity at malignancy ScientificWorldJournal 2013;2013:507270 14 Kwaan HC Role of plasma proteins in whole blood viscosity: a brief clinical review Clin Hemorheol Microcirc 2010;44(3):167-176 15 Kesmarky G, Kenyeres P, Rabai M, Toth K Plasma viscosity: a forgotten variable Clin Hemorheol Microcirc 2008;39(1-4):243-246 16 McHedlishvili G, Varazashvili M, Gobejishvili L Local RBC aggregation disturbing blood fluidity and causing stasis in microvessels Clin Hemorheol Microcirc 2002;26(2):99-106 17 Baskurt OK, Meiselman HJ Blood rheology and hemodynamics Semin Thromb Hemost 2003;29(5):435-450 18 Wang X, Wu Z, Song G, Wang H, Long M, Cai S Effects of oxidative damage of membrane protein thiol groups on erythrocyte membrane viscoelasticities Clin Hemorheol Microcirc 1999;21(2):137-146 19 Baskurt OK, Temiz A, Meiselman HJ Effect of superoxide anions on red blood cell rheologic properties Free Radic Biol Med 1998;24(1):102-110 20 Reggiori G, Occhipinti G, De Gasperi A, Vincent JL, Piagnerelli M Early alterations of red blood cell rheology in critically ill patients Crit Care Med 2009;37(12):3041-3046 21 Poschl JM, Leray C, Ruef P, Cazenave JP, Linderkamp O Endotoxin binding to erythrocyte membrane and erythrocyte deformability in human sepsis and in vitro Crit Care Med 2003;31(3):924-928 22 Aird WC The hematologic system as a marker of organ dysfunction in sepsis Mayo Clin Proc 2003;78(7):869-881 23 Goyette RE, Key NS, Ely EW Hematologic changes in sepsis and their therapeutic implications Semin Respir Crit Care Med 2004; 25(6):645-659 24 Condon MR, Kim JE, Deitch EA, Machiedo GW, Spolarics Z Appearance of an erythrocyte population with decreased deformability and hemoglobin content following sepsis Am J Physiol Heart Circ Physiol 2003;284(6):H2177-H184 25 Piagnerelli M, Boudjeltia KZ, Vanhaeverbeek M, Vincent JL Red blood cell rheology in sepsis Intensive Care Med 2003;29(7):1052-1061 26 Suzuki Y, Nakajima T, Shiga T, Maeda N Influence of 2,3diphosphoglycerate on the deformability of human erythrocytes Biochim Biophys Acta 1990;1029(1):85-90 27 Todd JC III, Mollitt DL Effect of sepsis on erythrocyte intracellular calcium homeostasis Crit Care Med 1995;23(3):459-465 28 Piagnerelli M, Boudjeltia KZ, Brohee D, et al Alterations of red blood cell shape and sialic acid membrane content in septic patients Crit Care Med 2003;31(8):2156-2162 29 Betticher DC, Keller H, Maly FE, Reinhart WH The effect of endotoxin and tumour necrosis factor on erythrocyte and leucocyte deformability in vitro Br J Haematol 1993;83(1):130-137 30 Machiedo GW, Powell RJ, Rush Jr BF, Swislocki NI, Dikdan G The incidence of decreased red blood cell deformability in sepsis and the association with oxygen free radical damage and multiple-system organ failure Arch Surg 1989;124(12):1386-1389 31 Somer T, Meiselman HJ Disorders of blood viscosity Ann Med 1993;25(1):31-39 32 Baskurt OK, Meiselman HJ Activated polymorphonuclear leukocytes affect red blood cell aggregability J Leukoc Biol 1998;63(1):89-93 33 Eichelbronner O, Sibbald WJ, Chin-Yee IH Intermittent flow increases endotoxin-induced adhesion of human erythrocytes to vascular endothelial cells Intensive Care Med 2003;29(5):709-714 34 Tissot Van Patot MC, MacKenzie S, Tucker A, Voelkel NF Endotoxininduced adhesion of red blood cells to pulmonary artery endothelial cells Am J Physiol 1996;270(1 Pt 1):L28-L36 35 Anniss AM, Sparrow RL Variable adhesion of different red blood cell products to activated vascular endothelium under flow conditions Am J Hematol 2007;82(6):439-445 36 Chappey O, Wautier MP, Boval B, Wautier JL Endothelial cells in culture: an experimental model for the study of vascular dysfunctions Cell Biol Toxicol 1996;12(4-6):199-205 37 Zoukourian C, Wautier MP, Chappey O, et al Endothelial cell dysfunction secondary to the adhesion of diabetic erythrocytes Modulation by iloprost Int Angiol 1996;15(3):195-200 38 Rattan V, Shen Y, Sultana C, Kumar D, Kalra VK Diabetic RBCinduced oxidant stress leads to transendothelial migration of monocyte-like HL-60 cells Am J Physiol 1997;273(2 Pt 1):E369-E375 39 Sultana C, Shen Y, Rattan V, Johnson C, Kalra VK Interaction of sickle erythrocytes with endothelial cells in the presence of endothelial cell conditioned medium induces oxidant stress leading to transendothelial migration of monocytes Blood 1998;92(10): 3924-3935 40 Sirois E, Charara J, Ruel J, Dussault JC, Gagnon P, Doillon CJ Endothelial cells exposed to erythrocytes under shear stress: an in vitro study Biomaterials 1998;19(21):1925-1934 41 Munn LL, Melder RJ, Jain RK Role of erythrocytes in leukocyteendothelial interactions: mathematical model and experimental validation Biophys J 1996;71(1):466-478 42 Sun C, Migliorini C, Munn LL Red blood cells initiate leukocyte rolling in postcapillary expansions: a lattice Boltzmann analysis Biophys J 2003;85(1):208-222 43 Migliorini C, Qian Y, Chen H, Brown EB, Jain RK, Munn LL Red blood cells augment leukocyte rolling in a virtual blood vessel Biophys J 2002;83(4):1834-1841 44 Zennadi R, Chien A, Xu K, Batchvarova M, Telen MJ Sickle red cells induce adhesion of lymphocytes and monocytes to endothelium Blood 2008;112(8):3474-3483 45 Evans EA, La Celle PL Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation Blood 1975;45(1):29-43 46 Mohandas N, Shohet SB The role of membrane-associated enzymes in regulation of erythrocyte shape and deformability Clin Haematol 1981;10(1):223-237 47 Mohandas N The red blood cell membrane In: Hoffman R BE, Shattil SJ, Furie B, Cohen HJ, eds Hematology: Basis, Principles and Practice New York: Churchill-Livingstone; 1991:264-269 48 Rendell M, Luu T, Quinlan E, et al Red cell filterability determined using the cell transit time analyzer (CTTA): effects of ATP depletion ... NO has been elucidated.77–79 Most notable is the covalent binding of NO1 to cysteine thiols, forming SNOs This paradigm developed following the discovery that endogenously produced NO circulated... thiol of Hb (Cysb93) that was demonstrated to undergo S-nitrosylation and sustain bioactivity under oxygenated conditions and NO release under low O2 conditions (see HbSNO hypothesis).56 In this... NO at its b-hemes and then passes the NO group from the heme to a thiol (Cys-b93-SNO).70,85 Transfer of NO between heme and thiol requires heme-redox coupled activation of the NO group, which