Reduced deformability of stored red blood cells is associated with generation of extracellular vesicles.

Throughout storage, red blood cells (RBCs) undergo detrimental changes in viability and their ability to effectively transport oxygen. RBC storage lesions are mediated, in part, by a progressive loss of cell deformability, and associated with the release of extracellular vesicles (EVs). Accumulation of EVs during the storage of RBCs correlates with a decrease in RBC surface area to volume ratio. Similarly, the loss of RBC-deformability is associated with loss of RBC surface area to volume ratio. In this study we thus tested whether loss of RBC-deformability is associated with increased RBC-EV production during blood storage. EVs obtained by differential centrifugation of stored RBCs (non-leukoreduced non-irradiated or leukoreduced γ-irradiated RBCs stored 35 or 28 days respectively) were enumerated by high-sensitivity flow cytometry. RBC deformability was quantified, using a cell-flow-properties-analyzer, by measuring the median cell elongation ratio (MER) and percentage of low and high deformable cells in the population (%, LDFC, and HDFC, respectively). The number of EVs was inversely correlated with the MER and positively correlated with the %LDFC with both measures showing highly significant logarithmic dependence with EV levels in stored RBCs. Considering how highly deformable cells did not correlate with EV formation as compared with low deformable RBCs we propose that the formation of EVs is a key factor leading to increased RBC-rigidity.

Hemolytic effect of polymeric nanoparticles: role of albumin.

Nanoparticles (NP) have drawn increasing interest from many fields in medicine and are a relatively new class of biomedical products. Because there are concerns about the health effects of nanoparticles, it is important to understand how nanoparticles interact specifically with red blood cells (RBC), a central object in the blood circulation. As numerous studies that have examined NP/RBC interaction concentrated on the hemolytic potential of nanoparticles, we describe an investigation of hemolytic activity of polystyrene nanoparticles (PS-NP) in protein free medium and its modulation by albumin. We found that treatment of RBCs with PS-NP induces hemolysis (dose and particle size dependent) in plasma free medium but not in full plasma or in buffer, which contain albumin. Critical albumin concentration is 0.05% wt. According to our results hemolytic effect of nanoparticles is strongly modulated by protein concentration in the medium.

Increased red blood cell aggregation in patients with Gaucher disease is non-inflammatory.

Red blood cell (RBC) aggregation is enhanced in the presence of ongoing inflammation, because of plasma protein effects, especially fibrinogen. Large RBC aggregates, in addition to being a marker of systemic inflammation, may hinder tissue perfusion and oxygenation. Gaucher disease, the most common lysosomal storage disorder, evinces many of the hallmarks of chronic inflammation. Manifestations of Gaucher disease which may be related to microvascular occlusion include avascular necrosis (AVN), bone crisis, and pulmonary hypertension. This study aims to determine whether increased RBC aggregation in non-splenectomized patients with Gaucher disease is due to Gaucher-related inflammation. The Cell Flow Properties Analyzer (CFA) monitors blood under conditions of different shear stress by creating varying pressure gradients. Blood from non-splenectomized patients with Gaucher disease showed only a slight correlation between aggregation parameters and fibrinogen levels, whereas blood from non-splenectomized patients treated with enzyme replacement therapy (ERT) showed marked correlation between aggregation parameters and fibrinogen, as in the control group. These results underscore the hypothesis that RBC aggregation in Gaucher disease is increased by (at least) two mechanisms: a fibrinogen-mediated inflammatory process and another non-inflammatory process that may be induced by elevated glucocerebroside levels in the RBC and/or inhibited by elevated plasma cerebroside levels.

Additive effect of red blood cell rigidity and adherence to endothelial cells in inducing vascular resistance.

To explore the contribution of red blood cell (RBC) deformability and interaction with endothelial cells (ECs) to circulatory disorders, these RBC properties were modified by treatment with hydrogen peroxide (H(2)O(2)), and their effects on vascular resistance were monitored following their infusion into rat mesocecum vasculature. Treatment with 0.5 mM H(2)O(2) increased RBC/EC adherence without significant alteration of RBC deformability. At 5.0 mM H(2)O(2), RBC deformability was considerably reduced, inducing a threefold increase in the number of undeformable cells, whereas RBC/EC adherence was not further affected by the increased H(2)O(2) concentration. This enabled the selective manipulation of RBC adherence and deformability and the testing of their differential effect on vascular resistance. Perfusion of RBCs with enhanced adherence and unchanged deformability (treatment with 0.5 mM H(2)O(2)) increased vascular resistance by about 35% compared with untreated control RBCs. Perfusion of 5.0 mM H(2)O(2)-treated RBCs, with reduced deformability (without additional increase of adherence), further increased vascular resistance by about 60% compared with untreated control RBCs. These results demonstrate the specific effects of elevated adherence and reduced deformability of oxidized RBCs on vascular resistance. These effects can be additive, depending on the oxidation conditions. The oxidation-induced changes applied in this study are moderate compared with those observed in RBCs in pathological states. Yet, they caused a considerable increase in vascular resistance, thus demonstrating the potency of RBC/EC adherence and RBC deformability in determining resistance to blood flow in vivo.

Premature red blood cells have decreased aggregation and enhanced aggregability.

Preterm infants are highly susceptible to ischemic damage. This damage is most obvious in the brain, retina, and gastrointestinal tract. Studies focusing on the rheological properties of premature red blood cells (pRBCs) have consistently shown minimal or no RBC aggregation. Previously, measurements of pRBC aggregation kinetics indicated that specific plasma properties are responsible for the decreased RBC aggregation observed in the neonates, but that their specific RBC properties do not affect it. However, the strength of interaction in the pRBC aggregates as a function of medium composition has not been tested. In our previous research, we described clinically relevant parameters, that is, the aggregate resistance to disaggregation by flow. With the help of a cell flow property analyzer (CFA), we can monitor RBC aggregation by direct visualization of its dynamics during flow. We used the CFA to examine pRBC (from 9 premature babies) in the natural plasma and in PBS buffer supplemented with dextran (500 kDa) to distinguish between RBC intrinsic-cellular and plasma factors. pRBCs suspended in the native plasma showed minimal or no aggregation in comparison to normal adult RBC. When we transferred pRBCs from the same sample to the dextran solution, enhanced resistance to disaggregation by flow was apparent.

Flow-resistant red blood cell aggregation in morbid obesity.

OBJECTIVE: Enhanced red blood cell (RBC) aggregation has an adverse effect on microcirculatory blood flow and tissue oxygenation. It has been previously shown that obesity is associated with increased RBC aggregation. The objectives of the present study were to further characterize obesity-related RBC aggregation and to examine whether the enhanced aggregation is a plasma- or cellular-dependent process. METHODS: Obese (body mass index (BMI)=40+/-6.3 kg/m2, n=22) and nonobese (BMI=24+/-3.4 kg/m2, n=18) individuals were evaluated for inflammation markers and aggregation parameters. Aggregation parameters were derived from the distribution of RBC population into aggregate sizes, and from the variation of the distribution as a function of flow-derived shear stress, using a cell flow properties analyzer. To differentiate plasmatic from cellular factors, we determined the aggregation in the presence of autologous plasma or dextran-500 kDa and calculated the plasma factor (PF) in the obese group. PF ranges from 0 to 1. When the PF=1, the aggregation is all due to plasmatic factors, when PF=0, the altered aggregation depends entirely on cellular factors, whereas 0

Thrombolytic therapy reduces red blood cell aggregation in plasma without affecting intrinsic aggregability.

Red blood cell (RBC) aggregation may contribute to occlusion of the coronary microcirculation during myocardial infarction. We studied the effect of thrombolytic therapy on RBC aggregation in patients with acute myocardial infarction (AMI). Compared with patients with myocardial infarction who did not receive thrombolytic therapy, those treated with systemic thrombolysis exhibited significantly reduced RBC aggregation, reduced plasma fibrinogen levels and increased plasma D-dimer levels. Using measurement of RBC aggregation in a standardized dextran-500 solution, reduction in RBC aggregation after thrombolysis was shown to be plasma dependent. Thrombolytic therapy had no direct effect on intrinsic RBC aggregability in patients with AMI. We conclude that thrombolytic therapy has rheologic consequences that may contribute to its overall efficacy. Inhibition of RBC aggregation by thrombolytic therapy may result from the degradation of fibrinogen, a key factor in the formation of RBC aggregates, and from the generation of fibrinogen degradation products capable of disaggregating RBCs.

Phospholipid patterns of erythrocytes in schizophrenia: relationships to symptomatology.

The phospholipid composition of red blood cells (RBC) from 32 haloperidol-treated schizophrenic patients, classified according to the positive and negative syndrome scale (PANSS) as showing either predominantly positive or predominantly negative symptoms, was determined and compared with that of normal controls. While the levels of phosphatidylcholine and phosphatidylserine were similar in all three groups, sphingomyelin (SM) and phosphatidylethanolamine (PE) were, respectively, increased and decreased in RBCs of schizophrenic patients. In both patient groups, the SM/PE ratios correlated directly with the PANSS negative symptom scale scores and inversely with the positive symptom scale scores. However, the inverse changes in the contents of SM and PE were much more expressed in the negative group. It is suggested that a main source of that difference is a higher activity of the polyunsaturated acid-selective phospholipase A(2) in the negative syndrome patients than in the positive syndrome and control groups.

Parameters of red blood cell aggregation as correlates of the inflammatory state.

To identify clinically relevant parameters of red blood cell (RBC) aggregation, we examined correlations of aggregation parameters with C-reactive protein and fibrinogen in unstable angina (UA), acute myocardial infarction (AMI), and bacterial infection (BI). Aggregation parameters were derived from the distribution of RBC population into aggregate sizes (cells per aggregate) and changing of the distribution by flow-derived shear stress. Increased aggregation was observed in the following order: UA, AMI, and BI. The best correlation was obtained by integration of large aggregate fraction as a function of shear stress. To differentiate plasmatic from cellular factors in RBC aggregation, we determined the aggregation in the presence and absence of plasma and formulated a "plasma factor" (PF) ranging from 0 to 1. In AMI the enhanced aggregation was entirely due to PF (PF = 1), whereas in UA and BI it was due to both plasmatic and cellular factors (0 < or = PF < or = 1). It is proposed that clinically relevant parameters of RBC aggregation should express both RBC aggregate size distribution and aggregate resistance to disaggregation and distinguish between plasmatic and cellular factors.

Kinetics of linear rouleaux formation studied by visual monitoring of red cell dynamic organization.

Red blood cells (RBCs) in the presence of plasma proteins or other macromolecules may form aggregates, normally in rouleaux formations, which are dispersed with increasing blood flow. Experimental observations have suggested that the spontaneous aggregation process involves the formation of linear rouleaux (FLR) followed by formation of branched rouleaux networks. Theoretical models for the spontaneous rouleaux formation were formulated, taking into consideration that FLR may involve both "polymerization," i.e., interaction between two single RBCs (e + e) and the addition of a single RBC to the end of an existing rouleau (e + r), as well as "condensation" between two rouleaux by end-to-end addition (r + r). The present study was undertaken to experimentally examine the theoretical models and their assumptions, by visual monitoring of the spontaneous FLR (from singly dispersed RBC) in plasma, in a narrow gap flow chamber. The results validate the theoretical model, showing that FLR involves both polymerization and condensation, and that the kinetic constants for the above three types of intercellular interactions are the same, i.e., k(ee) = k(er) = k(rr) = k, and for all tested hematocrits (0.625-6%) k < 0.13 +/- 0.03 s(-1).

Enhanced adherence of beta-thalassaemic erythrocytes to endothelial cells.

The adherence of red blood cells (RBC) to endothelial cells (EC), shown to correlate with microvascular occlusion in sickle cell disease and malaria, is considered a major contributor to microcirculatory disorders. In the present study the adherence to EC was markedly enhanced with RBC from beta-thalassaemia major (TM) patients, and even more so with RBC from beta-thalassaemia intermedia (TI) patients (10-fold and 25-fold higher than normal, respectively). It is proposed that enhanced RBC/EC adherence may contribute to the microcirculatory disorders observed in thalassaemia, especially in TI patients who are particularly known to suffer from leg ulcers.

Alteration of red cell aggregability and shape during blood storage.

BACKGROUND: Storage of blood units (for 35-42 days, depending on the preservative solution) has been reported to induce changes (e.g., reduction of sialic acid level) in red cells that are expected to alter their aggregability. STUDY DESIGN AND METHODS: The aggregability of stored red cells was monitored in their autologous plasma and compared to that obtained with washed cells in dextran-containing buffer throughout the storage period. Red cell aggregability was determined by using a computerized image analyzer of cell flow properties. RESULTS: Blood storage induced changes in red cells that are associated with continuous increase of their aggregability. At the same time, blood storage was associated with a reduction in the level of plasma fibrinogen, the major aggregating agent in plasma. Accordingly, the increased red cell aggregability was observed in red cells stored in dextran-containing buffer, but not in red cells stored in autologous plasma. CONCLUSION: Because blood transfusion is routinely given to patients with normal or high fibrinogen level, the transfusion of stored red cells has the potential to induce increased aggregation in vivo, depending on the storage period. This should be taken into account when blood transfusion is considered, particularly for patients with microcirculatory disorders.

Red blood cell intercellular interactions in oxidative stress states.

Red blood cell (RBC) intercellular interactions, i.e., self-aggregation and adherence to endothelial cells (EC), play important roles in microcirculation. These RBC flow properties are determined by cell membrane components, which are prone to damaging reactive oxygen species (ROS) produced in oxidative stress (OS) states. Alterations in RBC aggregability and adherence have been linked to the pathophysiology in numerous diseases associated with OS. We investigated RBC intercellular interactions in four OS states--thalassemia, treatment of RBC with phenyl-hydrazine or H2O2, and photodynamic virus inactivation of blood units. All these OS states increased RBC adherence to EC, but only part of them elevated their aggregability, while others abolished it. It is proposed that (1) different OS states might induce disparate effects on RBC intercellular interactions; (2) RBC aggregability and adherence to EC, although both intercellular interactions, are controlled by different cell surface factors.

Enhanced aggregability of human red blood cells by diving.

In vitro studies have shown that mild pressure increases red blood cell (RBC) aggregation. Enhanced RBC aggregation in pathologic states can drive the circulation into stasis. This investigation examined the effects of pressure on RBC aggregation in human volunteers. The hypothesis tested is that RBC aggregation is increased during hyperbaric exposure. Eleven subjects participated in dives to 300 feet of seawater (fsw) in a man-rated chamber complex. Blood samples were taken at the surface, at 66 fsw, and at 300 fsw. Data were analyzed with a repeated measures one-way analysis of variance for a complete randomized design. Statistical significance was achieved at P < 0.05. Data are expressed as mean +/- SEM. The median aggregate size (number of RBC/aggregate) of RBCs was significantly increased at depth. At a shear stress of 0.1 dyne/cm2, median aggregate size was 12.0 +/- 2.1, 33.0 +/- 7.3, and 48.8 +/- 10.8 at the surface, at 66 fsw, and at 300 fsw, respectively. These results show that mild pressure increases RBC aggregation in the human circulation.

Red blood cell rouleaux formation in dextran solution: dependence on polymer conformation.

The velocity of rouleaux formation (RF), as previously shown, increases with increasing dextran concentration up to a critical concentration (Ca), beyond which the addition of dextran reduces the RF velocity (RFV). de Gennes' model for polymer solutions suggests that dextrans exist in two conformations: a coil structure at low concentrations, which changes to a network beyond a critical concentration (C*). In the present study we examined the relation between Ca and C* for dextrans of different molecular weight, and found that they coincide. This suggests that the change in dextran behavior, from increasing to decreasing RFV, occurs when their conformation changes from coil to network. In addition, it has been reported that in dilute dextran solutions the intercellular distance (D) between RBC in rouleaux increases with the molecular weight of the dextran. We found that D correlates with Rf, the end-to-end distance of the polymer molecule, and for all dextrans D < or = 1.5 Rf. In accord with de Gennes' Model for polymers between surfaces, this corresponds to intercellular interaction with two overlapping surface-associated polymer layers, which may extend "tails" to interact with the opposing cells.

Reduction of protein volume and compressibility by macromolecular cosolvents: dependence on the cosolvent molecular weight.

The partial specific volume (V) and adiabatic compressibility (beta) of myoglobin have been shown to be reduced by small cosolvents such as glycerol (A. Priev, A. Almagor, S. Yedgar, B. Gavish, Biochemistry 35 (1996) 2061-2066). To elucidate the effect of the cosolvent size on these protein properties, in the present study we determined V and beta of myoglobin in solutions containing a homologous cosolvent series from sucrose to dextran--500 (M.W. 500,000). It was found that in addition to the expected effect of the cosolvent concentration, V and beta decrease with increasing cosolvent M.W. This suggests that structural properties of the cosolvent contribute to its effect on the protein interior.

Photodynamic treatment of red blood cell concentrates for virus inactivation enhances red blood cell aggregation: protection with antioxidants.

Photodynamic treatment (PDT) using phthalocyanines and red light appears to be a promising procedure for decontamination of red blood cell (RBC) concentrates for transfusion. A possible complication of this treatment may be induced aggregation of RBC. The production of RBC aggregates was measured with a novel computerized cell flow properties analyzer (CFA). The PDT of RBC concentrates with sulfonated aluminum phthalocyanine (AIPcS4) and the silicon phthalocyanine Pc 4 under virucidal conditions markedly enhanced RBC aggregation and higher shear stress was required to disperse these aggregates. The clusters of cells were huge and abnormally shaped, unlike the rouleaux formed by untreated RBC. This aggregation was prevented when a mixture of antioxidants was included during PDT. Addition of the antioxidants after PDT reduced aggregation only partially. It is concluded that inclusion of antioxidants during PDT of RBC concentrates prior to transfusion may reduce or eliminate the hemodynamic risk that the virucidal treatment may present to the recipient.

Membrane lipid order of human red blood cells is altered by physiological levels of hydrostatic pressure.

The effect of hydrostatic pressure at levels applied in diving or hyperbaric treatment (thus considered "physiological") on the order of lipid domains in human red blood cell (RBC) membrane was studied. Membrane order was determined by measuring 1) the fluorescence anisotropy (FAn) of lipid probes, 2) the resonance energy transfer from tryptophan to lipid probes, and 3) spectral shifts in Laurdan fluorescence emission. It was found that the application of mild pressure (< 15 atm) 1) increased, selectively, the FAn of lipid probes that monitor the membrane lipid core, 2) increased the tryptophan FAn, 3) increased the resonance energy transfer from tryptophan to lipid probes residing in the lipid core, and 4) induced changes in the Laurdan fluorescence spectrum, which corresponded to reduced membrane hydration. It is proposed that the application of pressure of several atmospheres increases the phase order of membrane lipid domains, particularly in the proximity of proteins. Because the membrane lipid order ("fluidity") of RBCs plays an important role in their cellular and rheological functions, the pressure-induced alterations of the RBC membrane might be pertinent to microcirculatory disorders observed in humans subjected to elevated pressure.

Enhanced aggregability of red blood cells of beta-thalassemia major patients.

beta-Thalassemia major (TM), a congenital hemoglobinopathy, is associated with hemodynamic disorders and with structural red blood cell (RBC) anomalies that may indicate impairment of RBC rheological properties. To gain insight into the possible contribution of RBC to the hemodynamic disorders, we studied RBC aggregability, which plays a central role in blood flow, particularly in the microcirculation. RBC aggregate size distribution and morphology of TM RBC were determined using a novel system for image analysis of blood cells in a flow chamber. It was found that the aggregability of RBC of TM patients is markedly enhanced. These cells form large clusters, as opposed to normal rouleaux, and higher shear stress is required to disperse them. The aggregate size of TM RBC is reduced to the normal range after the patients have received a blood transfusion. This study suggests that the hemodynamic disorders observed in TM may be linked to the enhanced RBC aggregability and that improvement of RBC rheological properties may be considered in the treatment of thalassemia.

Human red blood cell shape and volume are changed by physiological levels of hydrostatic pressure.

Application of hydrostatic pressure of several atmospheres (atm), such as that applied in diving or hyperbaric treatment, has been previously shown to induce the release of membrane components into the extracellular medium. As the shape of red blood cells (RBC) is sensitive to membrane composition, this might imply a subsequent change in RBC shape and volume. The present study demonstrates that application of hydrostatic pressure of up to 15 atm changes the shape of RBC from the normal discoids to stomatocytes (cup-shaped) and accordingly increases their volume. Changes in RBC shape and volume are known to impair physiological and cellular function. Thus, these changes might be pertinent to hemodynamic and physiological disorders observed in humans subjected to elevated pressure.