M. Piagnerelli rheology in K. Zouaoui Boudjeltia M. Vanhaeverbeek J.-L. Vincent

Abstract Changes in red blood cell membrane components such as sialic (RBC) function can contribute to acid, and an increase in others such alterations in microcirculatory blood as 2,3 diphosphoglycerate. Other flow and cellular dysoxia in sepsis. factors include interactions with Decreases in RBC and neutrophil white blood cells and their products deformability impair the passage of (reactive oxygen species), or the these cells through the microcircula- effects of temperature variations. tion. While the role of leukocytes Understanding the mechanisms of has been the focus of many studies altered RBC rheology in sepsis, and in sepsis, the role of erythrocyte the effects on blood flow and oxygen rheological alterations in this syn- transport, may lead to improved drome has only recently been inves- patient management and reductions tigated. RBC rheology can be influ- in morbidity and mortality. enced by many factors, including alterations in intracellular calcium Keywords Erythrocyte · and adenosine triphosphate (ATP) Deformability · Nitric oxide · concentrations, the effects of nitric Sialic acid · Multiple organ failure · oxide, a decrease in some RBC Oxygen transport

Introduction ogy of microcirculatory alterations and, perhaps, the treatment of sepsis. Severe sepsis and septic shock are the commonest causes This review evaluates alterations occurring in RBC of death in intensive care units (ICUs), with associated rheology during sepsis and possible underlying mecha- mortality rates of 30–50% [1]. Sepsis is a complex nisms. The potential implications of blood transfusion pathophysiological process that involves both alterations and erythropoietin administration in sepsis will not be in the microcirculation and changes in the biochemical discussed. and physiological characteristics of the blood constitu- ents. Microvascular damage plays a crucial role in the Major determinants of RBC rheology impairment of tissue oxygenation that can contribute to multiple organ failure and death [2]. Viscosity Microcirculatory alterations include slowing of capil- lary blood flow as a result of decreased perfusion pres- Haemorheology is the study of deformation and flow of sure and local arteriolar constriction [2, 3], viscosity al- blood and blood cells. The prime function of blood is terations [4, 5], and disturbances of red (RBC) and white transport by flow, and the most important rheological (WBC) blood cell rheology [6, 7]. property of blood is its resistance to flow, or viscosity. Some recent studies [8, 9, 10] have also defined the The definition of viscosity is explained in Fig. 1. Plasma RBC as a possible oxygen sensor and regulator of vascu- viscosity is about 1.6 times that of water (normal range lar tone, opening new perspectives into the pathophysiol- 1.15–1.35 mPa/s). Blood is a non-Newtonian fluid and 1053 230

viscometers. Serum viscosity (i.e., plasma viscosity less the effect of ) and whole blood viscosity can be measured.

Aggregation

At low shear rates, blood evolves from a low viscosity emulsion to a high viscosity suspension. The electrostat- ic repulsion of RBC is overcome by the presence of mac- romolecules which aggregate the cells. In inflammatory states, acute phase proteins (especially fibrinogen and large serum proteins such as a2 macroglobulin) increase RBC aggregation. Rouleaux of cells bind together in a side-by-side fashion and, together with the continuous uptake of individual cells, networks of larger aggregates are formed. Fig. 1 Blood viscosity. Schematic representation of a vessel lu- RBC aggregation is reversible by shear forces. At men. The curved line represents the flow velocity profile in lami- -1 nar flow. The viscosity (unit: Poise) is expressed as a ratio of shear shear rates of 7–10 s , the aggregates in normal blood stress to shear rate, where the shear stress (unit: dynes/cm2) is the are dispersed, cells become orientated with flow stream- force (F) parallel to the direction of flow per unit area of fluid lines, and blood viscosity is reduced as the shear rates in- sheared (A), and the shear rate (unit: second-1) is the velocity gra- crease further. In vitro, this process of aggregation and dient between adjacent layers in laminar flow. [dv velocity differ- ence of adjacent fluid laminae, dx distance between the fluid lami- disaggregation can continue for hours. nae (adapted from [5], with permission)] In addition to the plasma protein pattern, RBC aggre- gation is primarily determined by cellular properties; re- duction in cell size increases aggregation as does RBC its viscosity is therefore variable at any given tempera- ageing. The haematocrit shows a biphasic effect on red ture, depending on the shear rate. Haematocrit has a cell aggregation, with a peak effect at around 40–45%. large effect on blood viscosity and blood flow. For ex- Conditions of low shear rate in vivo are found primari- ample, hyperviscosity syndromes, such as polycythae- ly in the post-capillary venules. An increase in RBC ag- mia, are associated with profound perfusion problems gregation would increase blood viscosity at this level [11]; in contrast, anaemia favours an increase in cardiac [12]. The increased viscosity may promote blood stasis, output. In physiological conditions, changes in blood which may induce local hypoxia and endothelial damage. viscosity do not have a pronounced effect on blood flow. There are several methods of estimating RBC aggre- However, in low flow states, a reduction in shear rate gation other than low shear rate viscometry, for example, will cause an increase in viscosity. Blood viscosity, erythrocyte sedimentation rate and direct microscopic therefore, has the potential to reduce flow under low observation of aggregation. Microscopic techniques with flow, low shear conditions [3, 4, 5]. image analysis have also been developed [13], but the When RBCs are added to plasma, blood viscosity in- most widely used technique is the light scattering analy- creases logarithmically with a linear increase in hae- sis of RBC suspensions that employs light transmission matocrit over the range 20–60%. At high shear rates, through a RBC suspension to obtain indices of RBC ag- bulk blood viscosity is low because RBC aggregates are gregation. This is expressed mainly as the average aggre- dispersed and deformed into ellipsoids, oriented in paral- gate size at a certain shear stress. lel with flow streamlines, and with the membrane sliding around its cytoplasm. As shear rates are reduced, blood viscosity rises exponentially. In low flow states, when Deformability the shear stress acting on the cell is reduced, the RBC is less deformed; furthermore, the RBCs aggregate to form ‘Cellular deformability’ is the term generally used to rouleaux, increasing blood viscosity. In the normal circu- characterize the RBC’s ability to undergo deformation lation the shear stress is generally sufficient to disperse during flow [14]. The deformation response of a RBC to the rouleaux, allowing RBC deformation and facilitating fluid forces is a complex phenomenon that depends on a blood flow [3, 4, 5]. Therefore, the major determinants number of different cell characteristics including mem- of whole blood viscosity are shear rate, plasma viscosity, brane material properties [15], cell geometry, and cyto- haematocrit, RBC deformability, and RBC aggregation. plasmic viscosity [16]. It is possible to measure plasma and whole blood vis- As measures of cellular deformability are dependent cosity over a wide range of shear rates in a variety of on the technique used, quoted values are not comparable. 1054 231

Methods to measure cellular deformability have been de- scribed in detail elsewhere [17, 18]. Briefly, micropipette aspiration provides the most detailed characterization of membrane properties. Single RBCs are aspirated into mi- cropipettes with diameters in the range 1–2 µm; the rela- tionship between the applied negative pressure and the membrane tongue extension is then quantitated. Using ektacytometry, RBCs are subjected to a laminar shear stress field in a cuvette viscometer; the resultant change in cell shape is continuously monitored by laser dif- fractometry. Unstressed discoid cells generate a circular diffraction pattern. By measuring optical densities at two points along the major and minor axes of the elliptical diffraction pattern, a parameter termed “deformability Fig. 2 RBC membrane. Schematic representation of protein orien- tations in the human RBC. The membrane is composed of a phos- index” is generated which is a direct measure of cell pholipid membrane bilayer and transmembrane proteins including ellipticity. A numerical value of zero corresponds to non- glycophorin A and Band 3 proteins. Glycophorin A is the major deformable cells, while increasingly positive values cor- sialoglycoprotein of the RBC. SA bound to glycophorin A is re- respond to increasing cellular deformability. The mem- sponsible for the negative charge of the RBC membrane. The in- tracellular compartment (IC) is constituted by spectrin ( and brane fragmentation assay using the ektacytometer is subunits), actin, protein 4.1, protein 4.2, and ankyrin particularly useful for documenting decreased mechani- cal integrity of the membrane due to protein defects. Micropore filtration is limited by the possible occlu- Reversible deformation of the RBC membrane occurs sion of the filter pores by WBCs [6]. This technique with a change in geometric shape without any change in has been replaced by the cell transit analyzer (CTA) as it surface area. With increased deformation, some of the is insensitive to the presence of WBCs while the passage spectrin molecules can attain their maximal linear exten- of individual RBCs are monitored by a computerized sion, reaching the limit of reversible deformability [14]. system. The most abundant and best-studied integral protein Flow cytometry techniques can also be used to appre- of the RBC membrane is glycophorin A. This protein is ciate RBC shape, and to study the effect of modifications highly glycosylated, with approximately 60% of its of osmolality on shape in critically ill patients [19, 20]. weight being carbohydrate, mostly in the form of 15 The advantage of this technique is that it can provide an O-glycosidically linked tetrasaccharides. The two sialic easier and more rapid estimation of erythrocyte shape. acid residues (N-acetyl-neuraminic acid; SA) account for the negative electrostatic force on the RBC membrane [14, 15, 16], a necessary feature of the cell’s repellent RBC Membrane physiology properties. The importance of SA to RBC shape is dem- onstrated by the observation that neuraminidase-treated To undertake oxygen delivery, the RBC must be able to cells, which release their membrane SA content, undergo undergo considerable cellular deformation since its di- increased aggregation and have a reduced mean curva- ameter (8 µm in humans) far exceeds that of the capillar- ture [21]. ies (2–3 µm) through which it must pass [14]. The RBC membrane is composed of proteins (52% in weight), lip- ids (40%), and carbohydrates (8%). Membrane elasticity Alterations in microcirculation and blood rheology depends on the structural interactions between the outer in sepsis plasma membrane and the underlying protein skeleton. The proteins of the RBC membrane are divided into two Sepsis induces profound changes in the microcirculation groups: integral and peripheral (Fig. 2). Integral proteins [2] with loss of capillary density [22], maldistribution of (glycophorin and Band 3 proteins) are tightly bound to blood flow, increased flow heterogeneity [23], changes the membrane through hydrophobic interactions lipids in in microvascular reactivity [24], and WBC-endothelial the bilayer [14, 15, 16]. A filamentous network of pro- cell adhesion and vascular leakage [2]. teins is anchored to the bilayer by the integral proteins. Microcirculatory dysfunction may be further aggra- This network has three principal components: spectrin, vated by alterations in blood rheology resulting from de- actin, and protein 4.1. The peripheral membrane proteins creased RBC [2, 6, 12, 25, 26, 27] and WBC deformabil- are located on the cytoplasmic surface of the lipid bilay- ity [2, 7], RBC aggregation [28, 29] and coagulation dis- er and can be readily released from the membrane by turbances [30]. Interactions with WBCs cause release of simple manipulation of the ionic strength of the milieu or oxygen free radicals, stimulating RBC intracellular pro- variation in the concentrations of other proteins [14]. teolysis, membrane lipid peroxidation, and nitric oxide 1055 232

(NO) production. Alterations in RBC membrane (de- creased carbohydrate content, alterations of membrane pumps) with increased free calcium concentrations, de- creased ATP reserve, and modifications of 2,3 diphos- phoglycerate (DPG) concentrations have also been de- scribed. Simchon et al. [31] noted that a reduction in RBC deformability in rats led to RBC entrapment in the microcirculation of specific regions (spleen, lung, liver, and femur). This resulted in a reduction in regional blood flow proportional to the number of trapped RBCs. The role of RBC rheological alterations in sepsis has been investigated relatively recently. Several studies in animals and patients with sepsis have demonstrated de- creased RBC deformability, increased aggregation and Fig. 3 Role of 2,3 diphosphoglycerate. Diagrammatic representa- adhesiveness between WBCs, platelets, and endothelial tion of the subunit interaction in haemoglobin as O2 is added. De- cells [6, 25, 27, 29, 32]. Moreover, alterations in RBC oxyhaemoglobin (right), with low O2 affinity, is in the T-state, constrained by salt bridges (black crosses) and 2,3 DPG. As O2 is deformability have been described as an early indicator added in haem (black double triangles), the salt bridges are broken of in trauma patients [33], and as a prognostic (black dots), and the 2,3 DPG molecule is expelled, resulting in factor in canine septic shock [34]. the R configuration with higher O2 affinity. As 2,3 DPG lowers the affinity of haemoglobin toward O2, O2 transfer from blood to tissues in the microcirculation is increased Mechanisms underlying alterations in RBC deformability during sepsis Nitric oxide (NO)

2,3 diphosphoglycerate (2,3 DPG) RBC and modulation of vascular tone via NO

2,3 DPG is one of the most important organic phos- NO is produced by endothelial cells from L-arginine by phates in the RBC (Fig. 3) as it increases oxygen deliv- constitutive (cNOS) or inducible (iNOS) synthase. In ery to the tissues by decreasing the interaction between physiological conditions, cNOS plays an essential role in oxygen and haemoglobin. Hypoxaemia stimulates 2,3 producing small amounts of NO, thus maintaining capil- DPG production. Han and colleagues [35] reported an lary patency. In sepsis, lipopolysaccharide (LPS) and increase in 2,3 DPG in critically ill children, even in the cytokines such as tumour necrosis factor (TNF) and in- absence of hypoxaemia. This may represent a possible terferon- (INF- ) induce iNOS, resulting in the produc- response to illness with increased oxygen unloading to tion of much greater quantities of NO. By its action on the tissues. However, increased 2,3 DPG can also de- vascular smooth muscle, NO causes vasodilatation and crease RBC deformability. Suzuki et al. [36] reported increased tissue blood flow, but may also lead to arterial that increasing the 2,3-DPG concentration (by incubat- hypotension [37]. In the lung, NO rapidly binds to hae- ing human RBCs with inosine and pyruvate) increased moglobin (T-state, ‘tense’, low oxygen affinity state, par- intracellular haemoglobin concentrations (MCHC) and tially nitrosylated), forming a relatively stable haemoglo- ATP content, and decreased intracellular pH. The de- bin-NO complex (R-state, ‘relaxed’, high oxygen affinity formability of 2,3-DPG-enriched RBC was greatly im- state, ligand-bound). Oxyhaemoglobin (R-state) is thus proved when the MCHC (and thus the internal viscosi- converted to methaemoglobin and nitrate. NO is released ty) was normalized by suspension in a hypotonic solu- by haemoglobin (R-state) in the peripheral circulation, tion, but not when the intracellular pH was altered from resulting in opening of the microvasculature [38, 39]. 6.5 to 7.5, or when the ATP concentration was adjusted Allosteric transition from the R- to the T-state is trig- by incubation with various concentrations of adenine, gered by oxygen release in the pre-capillary resistance inosine, and glucose (0.6–2.1 mM/cells). Hence, de- vessels, which is then followed by release of NO. In the + creased RBC deformability is due in part to the increase lungs, after eliminating carbon dioxide (CO2) and H , in internal viscosity, and in part to the increase in mem- haemoglobin (in the T-state) re-captures oxygen and NO brane viscoelasticity [36]. and switches back to the R-state [38, 39]. Sprague and colleagues [40, 41] demonstrated this es- sential role of RBCs on NO release in isolated perfused rabbit lungs. Based on several observations, they showed that ATP is a key mediator for NO release as RBCs con- tain large amounts of ATP [42] that are released in re- sponse to physiological stimuli [8]. Moreover, ATP stim- 1056 233 ulates endothelial NO synthesis [43], thereby reducing brane fragility. L-NAME significantly decreased RBC vascular resistance in isolated lungs; this is prevented by deformability but did not influence fragility. However, the NO synthase inhibitor NG-nitro-L-arginine methyl L-NAME administration 10 min prior to endotoxin ad- ester (L-NAME) [40]. ministration also improved endotoxin-induced fragility. In dogs, ATP is also a mediator of NO release. How- This indicates that NO influences RBC deformability ever, in response to mechanical deformation, dog RBCs and membrane fragility in the first stages of sepsis, prob- release much less ATP than rabbit or human RBCs [41]. ably by stimulation of cNOS [50]. This may be related to the much lower ATP concentra- Bateman et al. [51] recently demonstrated that amino- tions found in dog RBCs compared to rabbits and hu- guanidine, an inhibitor of iNOS, prevented the accumu- mans [44]. lation of NO within the RBC in a rat model of peritoni- NO is also a modulator of the membrane properties of tis, and also prevented the decrease in RBC deformabili- RBC although the exact mechanism involved is as yet ty. Thus, NO may play a role in modulating the mechani- unclear. By an effect on the membrane RBC Ca2+- cal properties of the RBC in vivo. Whether this is a ATPase channel, NO may increase free intracellular Ca2+ direct action, with NO interfering with cytoskeletal ele- and thus decrease RBC deformability. In blood samples ments, or indirect, via some intermediate such as peroxy- from healthy volunteers, endotoxin induced a significant nitrite oxidizing cellular proteins [52], is unknown. increase in both intracellular Ca2+ concentration (evalu- ated by a fluorescent membrane probe) and membrane viscosity (anisotropy, evaluated by fluorescence spectro- RBC calcium (Ca2+) photometry) [45]. Addition of the NO synthase inhibitor N- monomethyl-arginine (NMA) had no effect in control RBC membrane fluidity is dependent upon the mainte- conditions but prevented the changes induced by endo- nance of a normal intracellular Ca2+ concentration [53]. toxin, suggesting that NO plays a role in intracellular This is kept within a narrow range (20–30 nM), some Ca2+ homeostasis and erythrocyte membrane deformabil- 50,000-fold lower than the external free Ca2+ concentra- ity in sepsis. tion, by a membrane-associated ATPase pump mecha- nism. In human RBCs, as in most cell types, a rise in cytosolic free Ca2+ induces a rapid increase in the potas- RBCs as producers of NO sium permeability by activation of a Ca2+-activated K+ channel, resulting in membrane hyperpolarization [54]. Several studies have indicated that RBCs have the capac- Increased intracellular Ca2+ levels in aged RBC may ity to synthesize NO under certain conditions [46, 47]. precipitate their removal from the circulation [53]. Sep- Jubelin et al. [48] suggested that the RBC possesses all sis-associated changes in the RBC membrane may alter the cellular machinery necessary to synthesize its own pump binding sites, inhibit their function, and disrupt in- NO. tracellular Ca2+ homeostasis [55]. Alterations in the RBC Plasmodium falciparum-infected human RBCs can Ca2+-ATPase pump in sepsis remain controversial. Todd also produce NO, probably by the activation of iNOS and Mollitt [56] reported that intracellular (free cytosol- [47]. By this means, RBCs may modulate their mem- ic) RBC Ca2+ concentrations (determined by fluorescent brane deformability and oxygen affinity, and perhaps spectroscopy) are increased in septic surgical patients as also contribute to the inflammatory host response by re- in experimental endotoxemia. RBCs incubated with en- acting with reactive oxygen species (ROS). dotoxin had increased intracellular Ca2+ concentrations, but these did not correlate with extracellular Ca2+ levels. This phenomenon was not prevented by dantrolene, an Effect of NO on RBC deformability inhibitor of intracellular Ca2+ release, and only partially prevented by calcium-channel blockade, but it was mini- Korbut and Gryglewski [49] noted that alterations in de- mized by adenosine or ATP. It was also partially reversed formability of isolated rabbit RBCs depended on the by post-treatment with ATP, but not with adenosine [56]. WBC concentration; WBCs decreased RBC deformabili- These same authors [57] noted in an in vitro study that ty when the WBC count was below 1.2 106 cells/ml, endotoxin increased intracellular free Ca2+ within RBCs while higher WBC counts abolished this effect. In the but only in the presence of WBCs. Pretreatment of these presence of a low WBC count, RBC deformability was RBCs with allopurinol (xanthine oxidase inhibitor), su- increased by NO donors, such as sydnonimine (a metab- peroxide dismutase (free radical scavenger), or pentoxi- olite of molsidomine; SIN-1) and sodium nitroprusside, fylline (WBC modulator) significantly limited the rise in but was reduced by the NO synthase inhibitor L-NAME. intracellular Ca2+ concentrations induced by endotoxin. In endotoxaemic rats, these same authors [50] noted al- Ca2+ channel blockers are unable to influence the de- tered RBC deformability (as measured by shear stress la- formability of normal RBCs [58, 59]. In diabetic pa- ser diffractometry) associated with increased RBC mem- tients, where intracellular Ca2+ are increased, some stud- 1057 234

ies have demonstrated a beneficial effect of Ca2+ channel White blood cells (WBC) blockers on RBC deformability, but at concentrations up to ten times higher than those clinically attainable Various factors can alter RBC membrane properties in- (10-8 mol/l) [59]. Further investigations are necessary to cluding direct contact between RBCs and WBCs [29, 68, fully define the effects of Ca2+-channel blockers on RBC 69] or ROS that stimulate intracellular proteolysis and deformability in sepsis. membrane lipid peroxidation [70, 71]. Several studies have reported that WBCs in sepsis have increased rigidity and enhanced aggregation with ATP platelets and RBCs [7, 72, 73]. During , large numbers of WBCs adhere to or roll along the mi- RBCs contain millimolar quantities of ATP [44]. These crovascular endothelium (margination), primarily in the are produced within the cell by membrane-bound glyco- postcapillary venules and only occasionally in the arteri- lytic pathways, and are used to maintain intracellular oles [13, 29, 74]. In the capillaries, the WBCs usually hydration and electrolyte composition [42]. ATP content appear to flow smoothly without rotation. Occasionally, is decreased in old RBCs [42], and this is accompanied WBCs may impede RBC flow [74]. Berliner et al. [13], by a loss of surface membrane SA, which might be a pri- using a method involving a slide test and image analysis, mary factor in the removal of old RBCs by the reticulo- demonstrated that both WBC and RBC adhesiveness/ag- endothelial system. gregation were increased in the peripheral venous blood Bergfeld and Forrester [60] documented that human of septic patients. This phenomenon may impair micro- RBC can release ATP in response to a hypoxic chal- vascular flow in sepsis. Moreover, activation of WBCs lenge. As ATP binds to receptors on the vascular endo- stimulates multiple mediator networks including the thelium, vessel calibre increases and regional blood flow complement, kinin, coagulation and fibrinolytic cas- improves [8, 43]. RBCs may act not only as oxygen cades, along with the release of chemokines, cytokines, transporters but also as oxygen sensors able to modify soluble receptors, lipid mediators, reactive oxygen spe- oxygen delivery. cies (ROS) and numerous enzymes, including elastase, Intracellular RBC ATP levels are decreased in sepsis myeloperoxidase and many proteases [75]. Claster and [56], causing a decrease in energy for the Ca2+ RBC mem- colleagues [76] demonstrated that ROS released by these brane pump, thereby increasing RBC intracellular Ca2+ activated WBC can attack RBC membranes, causing al- and resulting in a decrease in cell deformability. Pretreat- terations in lipid and protein structure that may decrease ment with pentoxifylline may improve RBC deformability RBC deformability and, ultimately, result in hemolysis. through a direct increase in intracellular ATP content [57]. These authors and others also demonstrated a dose- response curve for WBC-mediated lipid peroxidation in RBCs [76, 77]. Sialic acid content of the RBC membrane

Decreased RBC SA may be an important mechanism of Reactive oxygen species (ROS) senescent RBC destruction by the reticulo-endothelial system [61]. Eichelbronner et al. [32] demonstrated that Sepsis is characterized by increased production of ROS - - endotoxin promotes adhesion of human RBCs to endo- [superoxide anion (O2 ), hydroxyl radical (OH ), and thelial cells in vitro, probably by decreasing SA on the hydrogen peroxide H2O2)] as well as a decrease in anti- RBC membrane, and thereby decreasing the repulsive oxidant defences. Damage occurs when ROS production force between RBCs and the endothelium. exceeds the tissues’ antioxidant defences [71, 78]. ROS While the effects of SA on RBC aggregation have produced by WBCs can also damage haemoglobin and been well described, the effects of SA on RBC shape induce haemolysis [76, 77]. Uyesaka et al. [79] demon- - remain controversial. We have recently demonstrated a strated that RBCs exposed to O2 displayed pronounced decreased RBC membrane SA content in patients with degradation of membrane proteins (band 3 and spectrin) sepsis that was associated with a modification of RBC with formation of new protein bands that can decrease shape [62]. Hence, there is a possible link between RBC RBC deformability. In sepsis induced by caecal ligation membrane SA content and RBC shape in sepsis, as is de- and puncture in rats, Powell et al. [80, 81] demonstrated scribed in other diseases such as diabetes mellitus [63, that the loss of RBC deformability—with increased sur- 64]. Several mechanisms may account for the decrease vival—could be prevented by pre-treatment with the an- in SA. There may be increased activity of the SA de- tioxidant a-tocopherol. grading enzyme, sialidase, either by WBC as has been described in diabetic patients [65], or by the RBC mem- brane sialidases themselves [66]. Another mechanism could be a direct effect of bacteria upon the RBC [67]. 1058 235

Fig. 4 Schematic relationship between RBCs, WBCs and en- dothelium. Left panel: low PO2, acidic pH, and decreased RBC deformability lead to ATP liberation from RBCs. These ATP molecules stimulate the endothelial cells to release NO, promoting vasodilatation. Right panel: septic conditions with decreased RBC deformability and increased RBC volume with RBC haemolysis. ROS were liberated by the WBC and attack the RBC membrane. The increase in NO induced by sep- sis raised the intracellular Ca++. This elevated Ca++ impairs the RBC membrane skeleton caus- ing a decline in RBC deform- ability

Table 1 Some factors influencing RBC deformability

Factor Potential effects References

2,3 diphosphoglycerate Increases tissue O2 delivery [35] Decreases RBC deformability by increased internal viscosity [36] Nitric oxide Bound by haemoglobin [37, 38, 39, 40, 41, 42, 43] Modulates vascular tone [39, 40, 41, 43] Synthesized by RBC? [46, 47, 48] Modulates RBC membrane properties [45, 49, 50, 51, 52] Intracellular calcium Increased intracellular concentrations by decreased activity [53, 54, 55, 56, 57] of Ca++-ATPase pump Adenosine triphosphate Mediates NO release [8, 40, 41, 43] Maintains intracellular hydration and activity of ionic pumps [42, 56, 57, 60] Released by RBC to improve blood flow [8, 43, 60] Sialic acid Signal recognition for capture by the reticulo-endothelial system. [61, 64] Modifies RBC shape [62, 63, 64] Increases RBC aggregation [32, 61, 63] White blood cells Increase aggregation with RBCs [7, 13, 29, 68, 69, 72, 73, 74] Produce ROS [70, 71, 75, 76, 77, 79, 80, 81] Reactive oxygen species (ROS) Induce degradation of RBC membrane proteins [71, 76, 77, 78] Decrease RBC deformability [79, 80, 81] Temperature Influences in vitro results of RBC deformability [82, 83, 84, 85]

Effects of temperature blocking to passing through 1.3±0.2 µm micropipettes at a critical temperature of 36.4±0.3 °C. The authors attrib- RBC membrane mechanical properties are known to be uted these findings to an elastomeric transition of hae- temperature sensitive. Temperature can have significant moglobin from gel-like to fluid, and to an elastomeric effects on RBC deformability. In RBCs from healthy transition of membrane proteins such as spectrin [83]. subjects, the elongation index (representing deformabili- In septic and non-septic rats, Baskurt and Mat [84] ty) decreased significantly with a fall in temperature noted differences in RBC elongation index only at 37 °C from 37 °C to 5 °C [82]. Artmann and colleagues [83] using ektacytometry, while in RBCs from rats incubated noted that human RBCs undergo a sudden change from with endotoxin (E. coli; 75 µg/ml), Jagger et al. [85] not- 1059 236 ed alterations in RBC deformability measured using the in sepsis. Multiple factors may be involved including micropipette aspiration technique at 25 °C but not at NO and ROS, altered calcium homeostasis, decrease 37 °C. These authors underline the effect of room tem- in ATP reserves, increase in intracellular 2,3 DPG, perature measurement on physical membrane properties, membrane components (sialic acid), and WBC interac- which may perhaps exaggerate the differences between tions. normal and perturbed RBCs. The effects of temperature Importantly, the RBC is more than an oxygen trans- on RBC deformability in sepsis and, in particular, the in porter but also an oxygen sensor and may itself augment vivo relevance of these data clearly require further study. blood flow by liberation of ATP and O2 delivery wherev- The various factors involved in RBC deformability alter- er and whenever the need might arise. New thinking re- ations are summarized in Table 1 and Fig. 4. garding this well-studied cell will lead to a better under- standing of the mechanisms of RBC rheological altera- tions in sepsis and their effect on blood flow and O2 Conclusion transport. This may be important in the development of new therapeutic strategies to improve cellular oxygen Alterations in RBC rheology may contribute to the availability, and thereby reduce organ failure in severe microvascular injury and impaired oxygen supply seen sepsis and septic shock.

References

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