Modifications in Erythrocyte Membrane Protein Content Are... : Shock (original) (raw)
INTRODUCTION
Alterations of the microcirculation (vessels with a diameter <100 μm) are common in critically ill patients, especially during sepsis (1–4), and persistence of these alterations during sepsis is associated with a poor outcome (2, 3). Red blood cells (RBCs) are now considered to have a central role in the microcirculation, not only because they transport oxygen to the cells but also because they can induce local vasodilatation in hypoxic zones by liberation of mediators such as nitric oxide and/or adenosine-5-triphosphate. The RBCs must exhibit a remarkable ability to undergo cellular deformation because their diameter (8 μm in humans) far exceeds that of the capillaries (2–3 μm). This deformability capacity is a complex phenomenon that depends on a number of different cell characteristics, including material properties of the cell membrane (lipids, proteins, and carbohydrates) and, to a lesser extent, cellular geometry and cytoplasmic viscosity (5).
The RBC membrane is composed of 52% in weight of proteins, 40% lipids, and 8% carbohydrates. Membrane proteins are divided into two classes, depending on their relation to the lipid bilayer. The first group consists of “integral” membrane spanning proteins, especially protein band 3, membrane channels or transporters, glycophorins and glycoproteins; and carbohydrate moieties coupled to “integral” proteins from an additional leaflet, also called “glycocalyx.” Sialic acids, in particular _N_-acetylneuraminic acid (SA), an acidic carbohydrate, are bound to glycophorin A and are responsible for 60% to 90% of the surface negative charge of the RBC membrane. The second group of membrane proteins is composed of “peripheral” proteins constituting the inner membrane skeleton. These include spectrin (α and β subunits), actin, protein 4.1, protein 4.2, tropomyosin, adducin, myosin, and ankyrin (6).
We have previously observed early alterations in RBC rheology (deformability, aggregation) in critically ill patients, especially in sepsis (7). We also found that RBC membrane SA content was decreased early in septic patients (8, 9). These modifications of the membrane were associated with a more spherical RBC shape, as assessed by a flow cytometry technique, and the altered RBCs were unable to modify their shape in hypo-osmolar solutions (8). Incubation of RBCs from healthy volunteers with neuraminidase—the enzyme that cleaves SA—was associated with the same alterations in shape as observed in septic patients (9).
These in vitro studies suggested a link between membrane SA content and RBC shape. Nevertheless, other compounds, such as RBC membrane proteins, may also be altered during sepsis and could influence RBC rheology. Indeed, in a mouse model of sepsis induced by cecal ligature and perforation, Spolarics et al. (10) investigated the effects of glucose-6-phosphate dehydrogenase deficiency on RBC membrane and rheology. These authors observed increased RBC rigidity, as assessed by an ektacytometry technique, and a tendency for hemolysis together with alterations in band 3/α-spectrin interactions. Interestingly, they also observed an increase in this ratio in septic wild-type mice compared with naive mice, suggesting a possible alteration in the RBC membrane integral/peripheral protein ratio, which may contribute to altered RBC deformability (10). As there are interspecies differences in membrane composition and rheology (11, 12), we investigated the RBC membrane protein content in healthy volunteers and in critically ill patients with and without sepsis. To exclude the effect of alterations occurring later during the course of sepsis, we also measured RBC membrane proteins at day 3 in the septic population.
SUBJECTS AND METHODS
After approval by the Erasme University Hospital Ethics Committee, this prospective study was performed in a 34-bed medicosurgical intensive care unit (ICU) from April 2007 to April 2009. We included 15 critically ill adult patients with severe sepsis or septic shock, nine critically ill patients without sepsis, and 10 healthy hospital employees of both sexes as volunteers. Written consent was obtained from all subjects or their next of kin. Severe sepsis and septic shock were defined according to the criteria established by the International Sepsis Definitions Conference (13). Exclusion criteria were age younger than 18 years, RBC transfusion in the previous 72 h or during the study period, acute bleeding, hematologic disorders including abnormal mean corpuscular volume (values <80 μm3 or >100 μm3), chemotherapy in the last 3 months, history of diabetes mellitus, burns, cardiogenic shock, chronic renal failure requiring hemodialysis, proven or suspected cirrhosis and pregnancy, or the administration of any medication known to influence RBC shape or rheologic properties (pentoxifylline, erythropoietin, cyclosporine, aspirin, nitrovasodilators).
The following data were recorded at ICU admission: age, sex, ICU admission diagnosis, presence or absence of sepsis, and outcome. Blood samples were taken once on the first day of ICU admission for all patients and also on day 3 for 12 of the septic patients (two patients had died, and one had been discharged from the ICU by this timepoint). We recorded RBC, leukocyte, and platelet counts; hematocrit; hemoglobin concentration; mean corpuscular volume; mean corpuscular hemoglobin; mean corpuscular hemoglobin concentration; and C-reactive protein (CRP) and lactate concentrations. In addition, the Sequential Organ Failure Assessment (SOFA) score (14) was calculated on the day of blood sampling.
Cryohemolysis test
Cryohemolysis was performed according to Streichman and Gescheidt (15). Briefly, blood, anticoagulated with EDTA, was washed three times with cold saline. Buffered sucrose was added to packed RBCs, which were kept at 37°C for 10 min than transferred to an ice-cold bath for another 10 min. The samples were then vortexed and centrifuged. Absorbance of the supernatant aliquots was measured at 540 nm, and percent cryohemolysis was calculated by relating the absorbance of each sample to the absorbance of totally lysed packed cells and multiplied by 100. The normal value is less than 10% (15).
Membrane proteins by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
Blood samples were collected in heparin tubes. Erythrocyte membrane proteins were analyzed by a continuous buffer system (Fairbanks System) in a 4% to 12% acrylamide linear concentration gradient gel as described elsewhere (16). Proteins were stained with 0.1% Coomassie blue ethanol acetic acid solution. After destaining and drying of gels, the protein bands were quantified by densitometry (Hyrys CA 112 densitometer; Sebia, Evry, France). The amounts of the major membrane proteins are expressed as ratios.
Statistical Analysis
For the statistical analysis, the SigmaStat v 3.5 software package (Systat Software Inc, San Jose, Calif) was used. Data are presented as mean (SD) or median value with 25% to 75% interquartile ranges. Comparisons between groups were made using the t test or the Mann-Whitney U test with Dunn or Bonferroni post hoc adjustments. All statistics were two-tailed, and P < 0.05 was considered to be statistically significant.
RESULTS
The biological characteristics of the healthy volunteers and patients are shown in Tables 1 and 2. The causes of ICU admission for the septic patients were: pneumonia (n = 6), urinary infection (n = 4), peritonitis (n = 3), cholangitis (n = 1), and unknown origin (n = 1), and for nonseptic patients were neurologic pathologies (n = 7 subarachnoid hemorrhage, brain trauma, and seizure) and suicide attempts (n = 2). At admission, eight of the patients with sepsis were classified as having septic shock, and seven had severe sepsis. As expected, CRP concentrations were more elevated in the septic population on day 1 compared with nonseptic patients and volunteers and decreased by day 3 in the septic patients (Tables 1 and 2). The SOFA score was higher in septic compared with nonseptic patients on day 1 and also decreased significantly by day 3. Critically ill patients had a lower hemoglobin concentration than the healthy volunteers, but there were no differences in the hemogram between nonseptic and septic patients on day 1 or between day 1 and day 3 for the septic population (Tables 1 and 2).
Biological characteristics of healthy volunteers and patients on day 1
Biological characteristics of 12 of the septic patients on day 1 and on day 3
Cryohemolysis test
There were no significant differences in the cryohemolysis test among healthy volunteers and nonseptic and septic patients on day 1, or between days 1 and 3 for the septic group (Fig. 1). In general, values were within the reference range. Two healthy volunteers had a cryohemolysis test result above the normal value (14% and 16%), as did one patient with sepsis (16% on day 1, 21% on day 3).
Results of the cryohemolysis test for healthy volunteers, nonseptic patients, and septic patients on days 1 and 3 (for 12 of the septic patients). Results are expressed in median values with (25th–75th) interquartiles.
Membrane protein electrophoresis
There were several differences in the protein membrane content in critically ill patients compared with healthy volunteers (Table 3, Fig. 2). Protein content ratios were typically decreased in critically ill patients compared with healthy volunteers, but two ratios (band 3/spectrin and protein 4.2/band 3) were significantly increased (Table 3). There were no significant differences in any ratios between nonseptic and septic patients or during sepsis (Table 4).
Comparisons of RBC membrane proteins in healthy volunteers and nonseptic and septic patients on day 1
Comparisons of RBC membrane proteins on days 1 and 3 in 12 of the septic patients
Coomassie blue–stained RBC membrane proteins separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Example of a septic patient at day 1 (1) and day 3 (2), a nonseptic patient (3), and a healthy volunteer (4).
DISCUSSION
Red blood cell rheology is rapidly altered in sepsis, and this may contribute to the alterations in the microcirculation that are observed in these patients (5). The RBC membrane is the key element influencing RBC deformability, and alterations of the membrane have specific effects on RBC rheology (17–20). In this study, we investigated the membrane protein content of RBCs from healthy volunteers and from ICU patients with and without sepsis. There were differences in the RBC membrane protein content between critically ill patients within 24 h of ICU admission and healthy volunteers, but no differences in membrane protein content in septic patients compared with nonseptic patients, suggesting that sepsis per se does not alter the RBC membrane protein content.
In mice with septic shock induced by peritonitis, Spolarics et al. (10) observed an increase in the band 3/α-spectrin ratio, suggesting possible alterations in the integral/peripheral protein ratio of the RBC membrane. This modification was associated with alterations in RBC deformability as assessed by ektacytometry, especially at shear stresses ranging between 1.0 and 3.0 Pa (10). In the same model, these authors observed increased band 3 tyrosine-phosphorylation and altered band 3 organization without grossly altered RBC anion exchange activity (21). In a model of trauma/hemorrhagic shock, Caprio et al. (22) reported no significant quantitative differences in RBC membrane proteins between rats with trauma/hemorrhagic shock and those with trauma/sham shock. These authors investigated the levels of spectrin oxidation, phosphorylation, and ubiquitination and found no differences between groups for the first two but a decreased level of α-spectrin ubiquitination in the trauma/hemorrhagic shock rats, which may contribute to the RBC rigidity observed in trauma/hemorrhagic shock (22). However, observations on RBC rheology from animal models must be interpreted with caution. Indeed, RBC biochemistry, membrane, shape, and thus rheology (deformability and aggregation) are completely different among different species (11, 12, 23). Miseta et al. (23) observed large differences in the ionic content of RBCs among species. Baskurt et al. (11) compared RBCs from humans to those of rats and horses and observed lack of protein 4.2 in the RBCs from horses and complete modifications in the distributions of the carbohydrate groups on the RBC membrane surface. These modifications were associated with modifications of RBC rheology in these animals, with a higher aggregation tendency for horse RBCs and smaller deformability changes for the same shear stress in rat RBCs compared with humans and horses (11).
Few studies have measured the RBC membrane protein content in critically ill patients (8, 24). During the first 36 h of ICU admission, Nieuwland et al. (24) found no difference in the glycophorin A plasma content in septic meningococcal patients compared with healthy volunteers. In a previous work evaluating the RBC membrane SA content, we also measured glycophorin A content by flow cytometry at ICU admission (8). We did not observe any difference in the RBC membrane glycophorin A content at ICU admission between septic and nonseptic patients. However, glycophorin A content was increased in septic RBCs compared with those from healthy volunteers. This may be due to desialylation of RBC membrane glycoconjugates caused by cleavage of terminal SA residues.
Other components of the RBC membrane are also altered during sepsis. We reported an early decrease in the RBC membrane SA content at ICU admission, especially in septic patients (8, 9). We also observed an increased percentage of desialylated transferrin isoforms in the sera of septic compared with nonseptic patients and healthy volunteers (25). These results suggest an increased concentration and/or activity of the SA degrading enzyme, neuraminidase. Indeed, we recently found an increase in the serum activity of neuraminidase in septic patients (9). Modifications of the lipid bilayer, especially the exposure of phosphatidylserine, have been reported in RBCs from septic patients. Kempe et al. (26) observed that incubation of RBCs from healthy volunteers in plasma from septic patients triggered phosphatidylserine exposure highlighted by increased fixation of annexin V on the surface of these RBCs. The same effect was also observed when RBCs from healthy volunteers were incubated with the supernatant of pathogens. The effect of patient plasma on RBC annexin V was paralleled by the formation of ceramide and a significant increase in intraerythrocytic calcium. This, in turn, could activate the calcium-sensitive K+ channel, which, together with chloride channels, can allow KCl to leave the cell, thus resulting in cell shrinkage (26).
In the present study, we observed some alterations in the protein membrane content in critically ill patients compared with healthy volunteers, but these were not different in septic compared with nonseptic patients. Our results showed increased band 3/spectrin and protein 4.2/band 3 ratios but a significant decrease in other protein ratios (spectrin/actin, ankyrin/actin, band 3/actin, protein 4.1/actin, ankyrin/protein 4.1). These findings could be explained by the interactions between band 3 and other membrane proteins to maintain membrane skeletal cohesion and resistance to rupture. Protein 4.2 binds to both band 3 and ankyrin and can regulate the avidity of the interactions between band 3 and ankyrin (6). This hypothesis is in agreement with the fact that there were no differences in the cryohemolysis results among groups.
Interestingly, there was no significant difference between nonseptic and septic patients despite higher levels of inflammatory markers (white blood cells and CRP) in the septic patients. This observation suggests that the alterations in RBC rheology (shape, deformability, and aggregability) observed in septic patients already at ICU admission (7–9) are the result not only of changes in the protein membrane content but also of changes in other compounds, such as sialic acid and/or lipids. Better preservation of the protein part of the RBC membrane, compared with the lipid or carbohydrate portions, may be important for the biochemistry of the RBC. Indeed, the majority of the glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase) have been shown to organize into multienzyme complexes on the band 3 protein. These complexes are regulated by oxygenation and phosphorylation (27, 28). Damage to the RBC membrane proteins could alter the biochemistry of the RBCs and their capacity to synthesize and liberate adenosine-5-triphosphate as a vasodilator agent to improve the microcirculation in sepsis.
In conclusion, RBC membrane skeletal protein content was modified in critically ill patients compared with healthy volunteers but was not influenced by the presence of sepsis. Therefore, the alterations in RBC rheology observed during sepsis are mainly the result of alterations in other membrane components, like carbohydrates, such as SA and/or lipids. A better understanding of the mechanisms of RBC rheologic alterations in septic patients and the effects of these alterations on blood flow and on oxygen transport is important and may help in the development of strategies to improve outcomes.
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Keywords:
Red blood cell; microcirculation; sepsis; spectrin; cryohemolysis; rheology
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