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Competing Interests: None declared.

Seminars in Thrombosis and Hemostasis (STH) celebrates 50 years of publishing in 2024. To celebrate this landmark event, STH is republishing some archival material. This article represents the most highly cited paper ever published in STH. The original abstract follows.Blood is a two-phase suspension of formed elements (i.e., red blood cells [RBCs], white blood cells [WBCs], platelets) suspended in an aqueous solution of organic molecules, proteins, and salts called plasma. The apparent viscosity of blood depends on the existing shear forces (i.e., blood behaves as a non-Newtonian fluid) and is determined by hematocrit, plasma viscosity, RBC aggregation, and the mechanical properties of RBCs. RBCs are highly deformable, and this physical property significantly contributes to aiding blood flow both under bulk flow conditions and in the microcirculation. The tendency of RBCs to undergo reversible aggregation is an important determinant of apparent viscosity because the size of RBC aggregates is inversely proportional to the magnitude of shear forces; the aggregates are dispersed with increasing shear forces, then reform under low-flow or static conditions. RBC aggregation also affects the in vivo fluidity of blood, especially in the low-shear regions of the circulatory system. Blood rheology has been reported to be altered in various physiopathological processes: (1) Alterations of hematocrit significantly contribute to hemorheological variations in diseases and in certain extreme physiological conditions; (2) RBC deformability is sensitive to local and general homeostasis, with RBC deformability affected by alterations of the properties and associations of membrane skeletal proteins, the ratio of RBC membrane surface area to cell volume, cell morphology, and cytoplasmic viscosity. Such alterations may result from genetic disorders or may be induced by such factors as abnormal local tissue metabolism, oxidant stress, and activated leukocytes; and (3) RBC aggregation is mainly determined by plasma protein composition and surface properties of RBCs, with increased plasma concentrations of acute phase reactants in inflammatory disorders a common cause of increased RBC aggregation. In addition, RBC aggregation tendency can be modified by alterations of RBC surface properties because of RBC in vivo aging, oxygen-free radicals, or proteolytic enzymes. Impairment of blood fluidity may significantly affect tissue perfusion and result in functional deteriorations, especially if disease processes also disturb vascular properties., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: In the Fontan palliation for single ventricle heart disease (SVHD), pulmonary blood flow is non-pulsatile/passive, low velocity, and low shear, making viscous power loss a critical determinant of cardiac output. The rheologic properties of blood in SVHD patients are essential for understanding and modulating their limited cardiac output and they have not been systematically studied. We hypothesize that viscosity is decreased in single ventricle circulation., Methods: We evaluated whole blood viscosity, red blood cell (RBC) aggregation, and RBC deformability to evaluate changes in healthy children and SVHD patients. We altered suspending media to understand cellular and plasma differences contributing to rheologic differences., Results: Whole blood viscosity was similar between SVHD and healthy at their native hematocrits, while viscosity was lower at equivalent hematocrits for SVHD patients. RBC deformability is increased, and RBC aggregation is decreased in SVHD patients. Suspending SVHD RBCs in healthy plasma resulted in increased RBC aggregation and suspending healthy RBCs in SVHD plasma resulted in lower RBC aggregation., Conclusions: Hematocrit corrected blood viscosity is lower in SVHD vs. healthy due to decreased RBC aggregation and higher RBC deformability, a viscous adaptation of blood in patients whose cardiac output is dependent on minimizing viscous power loss., Impact: Patients with single ventricle circulation have decreased red blood cell aggregation and increased red blood cell deformability, both of which result in a decrease in blood viscosity across a large shear rate range. Since the unique Fontan circulation has very low-shear and low velocity flow in the pulmonary arteries, blood viscosity plays an increased role in vascular resistance, therefore this work is the first to describe a novel mechanism to target pulmonary vascular resistance as a modifiable risk factor. This is a novel, modifiable risk factor in this patient population., (© 2024. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Sickle cell anemia (SCA) is characterized by decreased red blood cell (RBC) deformability due to polymerization of deoxygenated hemoglobin, leading to abnormal mechanical properties of RBC, increased cellular adhesion, and microcirculatory obstruction. Prior work has demonstrated that NO• influences RBC hydration and deformability and is produced at a basal rate that increases under shear stress in normal RBC. Nevertheless, the origin and physiological relevance of nitric oxide (NO•) production and scavenging in RBC remains unclear. We aimed to assess the basal and shear-mediated production of NO• in RBC from SCA patients and control (CTRL) subjects. RBCs loaded with a fluorescent NO• detector, DAF-FM (4-Amino-5-methylamino- 2',7'-difluorofluorescein diacetate), were imaged in microflow channels over 30-min without shear stress, followed by a 30-min period under 0.5Pa shear stress. We utilized non-specific nitric oxide synthase (NOS) blockade and carbon monoxide (CO) saturation of hemoglobin to assess the contribution of NOS and hemoglobin, respectively, to NO• production. Quantification of DAF-FM fluorescence intensity in individual RBC showed an increase in NO• in SCA RBC at the start of the basal period; however, both SCA and CTRL RBC increased NO• by a similar quantity under shear. A subpopulation of sickle-shaped RBC exhibited lower basal NO• production compared to discoid RBC from SCA group, and under shear became more circular in the direction of shear when compared to discoid RBC from SCA and CTRL, which elongated. Both CO and NOS inhibition caused a decrease in basal NO• production. Shear-mediated NO• production was decreased by CO in all RBC, but was decreased by NOS blockade only in SCA. In conclusion, total NO• production is increased and shear-mediated NO• production is preserved in SCA RBC in a NOS-dependent manner. Sickle shaped RBC with inclusions have higher NO• production and they become more circular rather than elongated with shear., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Described to be antithrombotic and antihypertensive, nattokinase is consumed for putative cardiovascular benefit. However, no large-scale, long-term cardiovascular study has been conducted with nattokinase supplementation., Objective: To determine the effect of nattokinase on subclinical atherosclerosis progression and atherothrombotic biomarkers., Methods: In this double-blinded trial, 265 individuals of median age 65.3 years, without clinical evidence of cardiovascular disease (CVD) were randomized to oral nattokinase 2,000 fibrinolytic units or matching placebo. Primary outcome was rate of change in subclinical atherosclerosis measured by serial carotid ultrasound every 6 months as carotid artery intima-media thickness (CIMT) and carotid arterial stiffness (CAS). Additional outcomes determined at least every 6 months were clinical parameters including blood pressure and laboratory measures including metabolic factors, blood rheology parameters, blood coagulation and fibrinolysis factors, inflammatory markers and monocyte/macrophage cellular activation markers., Results: After median 3 years of randomized treatment, annualized rate of change in CIMT and CAS did not significantly differ between nattokinase supplementation and placebo. Additionally, there was no significant effect of nattokinase supplementation on blood pressure or any laboratory determination., Conclusions: Results of this trial show that nattokinase supplementation has a null effect on subclinical atherosclerosis progression in healthy individuals at low risk for CVD.

Although enhanced Red Blood Cell (RBC) - Endothelial Cell (EC) interaction, as well as RBC induced EC activation, have been extensively studied in several RBC-linked pathologies, the specific individual effects of oxidatively modified RBC on EC activation has not yet been documented. However, increasing evidence in both experimental and clinical studies suggests that oxidatively modified RBC could be considered potential pathogenic determinants in several acute and chronic diseases displaying systemic oxidative stress. Therefore, the present study aimed to explore the specific effects of oxidized RBC interaction with endothelial cells on intracellular signaling pathways that promote EC activation. RBC were exposed to oxidative stress induced by phenazine methosulphate (PMS). It is shown that the interaction of oxidatively modified RBC with cultured human umbilical vein endothelial cells (HUVEC) results in: a) EC activation as indicated by the increased surface expression of intercellular adhesion molecule -1 (ICAM-1); b) the activation of transcription factor NF-κB, an indicator of cellular oxidant stress. These results emphasize the specific contribution of oxidatively modified RBC interaction to EC activation and their possible pathological role in vascular diseases and oxidative stress., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Abnormal adhesion of red blood cells (RBC) to the endothelium has been linked to the pathophysiology of several diseases associated with vascular disorders. Various biochemical changes on the outer membrane of RBC, as well as plasma protein levels, have been identified as possibly playing key roles, but the detailed interplay between plasma factors and cellular factors often remains unclear. In this work, we identified an alternative pathway by demonstrating that non-adsorbing macromolecules can also have a marked impact on the adhesion efficiency of RBC from patients with type 2 Diabetes (T2DM) to endothelial cells (EC). RBC isolated from blood samples of T2DM patients were suspended in isotonic solutions of dextran in order to mimic the impact of non-adsorbing macromolecules. Static and continuous flow adhesion assays were used to determine the adhesion behavior of T2DM RBC with EC and the results compared with those of normal controls. We found that the presence of non-adsorbing molecules promotes an increase in T2DM RBC - EC adhesion and that these RBC exhibit much greater adhesion than normal red cells. Our results thus suggest that the depletion mechanism might be an alternative phenomenon through which plasma proteins could cause enhanced RBC-EC adhesion in diabetes mellitus. These findings contribute towards the comprehensive understanding of pathophysiological mechanisms of vascular complications in diabetes and other diseases with similar vascular sequelae., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We have previously demonstrated that sickle cell disease (SCD) patients maintain normal global systemic and cerebral oxygen delivery by increasing cardiac output. However, ischemic end-organ injury remains common suggesting that tissue oxygen delivery may be impaired by microvascular dysregulation or damage. To test this hypothesis, we performed fingertip laser Doppler flowmetry measurements at the base of the nailbed and regional oxygen saturation (rSO 2 ) on the dorsal surface of the same hand. This was done during flow mediated dilation (FMD) studies in 26 chronically transfused SCD, 75 non-transfused SCD, and 18 control subjects. Chronically transfused SCD patients were studied prior to and following a single transfusion and there was no acute change in rSO 2 or perfusion. Laser Doppler estimates of resting perfusion were 76% higher in non-transfused and 110% higher in transfused SCD patients, compared to control subjects. In contrast, rSO 2 was 12 saturation points lower in non-transfused SCD patients, but normal in the transfused SCD patients. During cuff occlusion, rSO 2 declined at the same rate in all subjects suggesting similar intrinsic oxygen consumption rates. Upon cuff release, laser doppler post occlusive hyperemia was blunted in SCD patients in proportion to their resting perfusion values. Transfusion therapy did not improve the hyperemia response. FMD was impaired in SCD subjects but partially ameliorated in transfused SCD subjects. Taken together, non-transfused SCD subjects demonstrate impaired conduit artery FMD, impaired microcirculatory post-occlusive hyperemia, and resting hypoxia in the hand despite compensated oxygen delivery, suggesting impaired oxygen supply-demand matching. Transfusion improves FMD and oxygen supply-demand matching but not microcirculation hyperemic response., (© 2019 Wiley Periodicals, Inc.)

To date, the mechanisms behind red blood cell (RBC) adhesion remain unclear. However, polymer depletion at the red cell surface has been shown to play a significant role. Interestingly, most previous studies have focused on the adhesion-promoting effects of one type of large polymer or plasma protein. However, the situation in vivo is more complex in that one needs to consider a mixture of various bio-macromolecules. To explore this complexity, Interference Reflection Microscopy was used to investigate how mixtures of various polymers affect RBC adhesion. RBC adhesion to albumin-coated glass coverslips was studied in the presence of two pro-adhesion polymers [dextran70 kDa and 35 kDa poly(ethylene glycol) (PEG 35)] with and without three types of smaller polymers: dextran 10 kDa, PEG 10 kDa and Poloxamer 188. Our findings show that the presence of small polymers can inhibit the adhesion-promoting effects of dextran 70 and PEG 35, with a more pronounced reduction for heterogeneous mixtures. Interpretation of our results in terms of the depletion model appears appropriate, in that our findings are consistent with the assumption that this reduction occurs because of an increase of small molecules in the depletion region. This study thus suggests that depletion interaction can control cell-cell interactions in complex environments (e.g., in vivo), and indicates that considering the interplay of all plasma constituents is important in order to understand the pathophysiology of diseases associated with cell adhesion and vascular complications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Therapeutic plasma exchange has been tried as a treatment approach for systemic sclerosis since 1978 based on the rationale that some circulating factor is involved in disease pathogenesis, for example, autoantibodies or immune complexes, and that removing the potential pathogenic factors could lead to symptom improvement. Based on our impression that clinicians and researchers are largely unaware that a large volume of research has been published about the use of therapeutic plasma exchange as a treatment for systemic sclerosis, we conducted a comprehensive review and analysis of all published research on this topic., Results: We identified 46 relevant articles that met our search criteria, involving a total of 572 patients. Of these, 19 were case studies; the rest ranged from small observational studies to prospective randomized clinical trials. In all but two studies, most patients receiving therapeutic plasma exchange showed improvements in both clinical symptoms and laboratory markers, including significant improvement in Raynaud's symptoms and healing of digital ulceration after three to four weekly treatments. The beneficial effects from even a short course of therapeutic plasma exchange treatments were long-lasting, typically 6 months or longer. Therapeutic plasma exchange was very well tolerated. Adverse events were rare and, in almost all cases, mild and transitory., Conclusion: These results suggest that long-term therapeutic plasma exchange may offer a low-risk way to control and in some cases reverse systemic sclerosis symptoms. The mechanism for the clinical improvements seen from therapeutic plasma exchange in systemic sclerosis patients is unclear. Therefore, additional studies of therapeutic plasma exchange effects in systemic sclerosis appear to be highly desirable., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2018.)

Purpose: We sought a human blood T 2 -oximetery calibration curve over the wide range of hematocrits commonly found in anemic patients applicable with T 2 relaxation under spin tagging (TRUST)., Methods: Blood was drawn from five healthy control subjects. Ninety-three in vitro blood transverse relaxation (T 2b ) measurements were performed at 37°C over a broad range of hematocrits (10-55%) and oxygen saturations (14-100%) at 3 Tesla (T). In vivo TRUST was performed on 35 healthy African American control subjects and 11 patients with chronic anemia syndromes., Results: 1/T 2 rose linearly with hematocrit (r 2  = 0.96), for fully saturated blood. Upon desaturation, 1/T 2 rose linearly with the square of the oxygen extraction, (1-Y) 2 , and the slope was linearly proportional to hematocrit (r 2  = 0.88). The resulting bilinear model between 1/T 2 , (1-Y) 2 , and hematocrit had a combined r 2 of 0.96 and a coefficient of variation of 6.1%. Using the in vivo data, the bilinear model had significantly lower bias and variability than existing calibrations, particularly for low hematocrits. In vivo Bland Altman analysis demonstrated clinically relevant bias that was -6% (absolute saturation) for hematocrits near 30% and rose to + 6% for hematocrits near 45%., Conclusion: This work introduces a robust bilinear calibration model that should be used for MRI oximetry. Magn Reson Med 77:2364-2371, 2017. © 2016 International Society for Magnetic Resonance in Medicine., (© 2016 International Society for Magnetic Resonance in Medicine.)

Painful vaso-occlusive crisis (VOC), a complication of sickle cell disease (SCD), occurs when sickled red blood cells obstruct flow in the microvasculature. We postulated that exaggerated sympathetically mediated vasoconstriction, endothelial dysfunction and the synergistic interaction between these two factors act together to reduce microvascular flow, promoting regional vaso-occlusions, setting the stage for VOC. We previously found that SCD subjects had stronger vasoconstriction response to pulses of heat-induced pain compared to controls but the relative degrees to which autonomic dysregulation, peripheral vascular dysfunction and their interaction are present in SCD remain unknown. In the present study, we employed a mathematical model to decompose the total vasoconstriction response to pain into: 1) the neurogenic component, 2) the vascular response to blood pressure, 3) respiratory coupling and 4) neurogenic-vascular interaction. The model allowed us to quantify the contribution of each component to the total vasoconstriction response. The most salient features of the components were extracted to represent biophysical markers of autonomic and vascular impairment in SCD and controls. These markers provide a means of phenotyping severity of disease in sickle-cell anemia that is based more on underlying physiology than on genotype. The marker of the vascular component (BMv) showed stronger contribution to vasoconstriction in SCD than controls (p = 0.0409), suggesting a dominant myogenic response in the SCD subjects as a consequence of endothelial dysfunction. The marker of neurogenic-vascular interaction (BMn-v) revealed that the interaction reinforced vasoconstriction in SCD but produced vasodilatory response in controls (p = 0.0167). This marked difference in BMn-v suggests that it is the most sensitive marker for quantifying combined alterations in autonomic and vascular function in SCD in response to heat-induced pain.

While a number of studies have shown short-term beneficial effects of therapeutic plasma exchange (TPE) for treating systemic scleroderma (SSc), there have been no reports on the very long-term usage of TPE as the sole systemic treatment intervention. We report the case of a male patient, originally diagnosed with limited systemic scleroderma (lcSSc) in early 1990, who has been undergoing regular plasmapheresis treatments for more than 22 years, beginning in late 1993. Prior to commencing treatment, the patient exhibited symptoms including severe gastro esophageal reflux disease (GERD) with esophagitis, frequent Raynaud's attacks, reduced lung function, and chronic chilling. With the exception of mild residual Raynaud's, all of the patient's symptoms reversed after three years of regular TPE treatments and he remains in complete remission. While the typical explanation for the therapeutic benefits seen with TPE focuses on temporary reduction of circulating antibodies or other pathogenic factors, we propose instead an explanation based on abnormal blood rheology as a novel disease pathogenesis model for SSc.

After the Fontan procedure, patients with univentricular hearts can experience long-term complications due to chronic low-shear non-pulsatile pulmonary blood flow. We sought to evaluate hemorheology and its relationship to hemodynamics in children with univentricular hearts. We hypothesized that low-shear blood viscosity and red blood cell (RBC) aggregation would be associated with increased pulmonary vascular resistance (PVR) and decreased pulmonary blood flow (PBF). We performed a cross-sectional analysis of 62 children undergoing cardiac catheterization-20 with isolated atrial septal defect (ASD), 22 status post Glenn procedure (Glenn), and 20 status post Fontan procedure (Fontan). Shear-dependent blood viscosity, RBC aggregation and deformability, complete blood count, coagulation panel, metabolic panel, fibrinogen, and erythrocyte sedimentation rate were measured. PVR and PBF were calculated using the Fick equation. Group differences were analyzed by ANOVA and correlations by linear regression. Blood viscosity at all shear rates was higher in Glenn and Fontan, partially due to normocytic anemia in ASD. RBC aggregation and deformability were similar between all groups. Low-shear viscosity negatively correlated with PBF in Glenn and Fontan only (R (2) = 0.27, p < 0.001); it also negatively correlated with pulmonary artery pressure in Glenn (R (2) = 0.15, p = 0.01), and positively correlated with PVR in Fontan (R (2) = 0.28, p = 0.02). Our data demonstrate that elevated low-shear blood viscosity is associated with negative hemodynamic perturbations in a passive univentricular pulmonary circulation, but not in a pulsatile biventricular pulmonary circulation.

Background: Current generation mechanical circulatory assist devices are designed to minimize high shears to blood for prolonged durations to avoid hemolysis. However, red blood cells (RBC) demonstrate impaired capacity to deform when exposed to shear stress (SS) well below the "hemolytic threshold"., Objective: We endeavored to identify how changes in the magnitude and duration of SS exposure alter RBC deformability and subsequently develop a model to predict erythrocyte subhemolytic damage., Methods: RBC suspensions were exposed to discrete magnitudes of SS (1-64 Pa) for specific durations (1-64 s), immediately prior to RBC deformability being measured. Analyses included exploring the maximal RBC deformation (EImax) and SS required for half EImax (SS1/2). A surface-mesh was interpolated onto the raw data to predict impaired RBC deformability., Results: When SS was applied at <16Pa, limited changes were observed. When RBC were exposed to 32 Pa, mild impairments in EImax and SS1/2 occurred, although 64 Pa caused a dramatic impairment of RBC deformability. A clear relation between SS duration and magnitude was determined, which could predict impaired RBC deformability., Conclusion: The present results provide a model that may be used to predict whether RBC deformability is decreased following exposure to a given level and duration of SS, and may guide design of future generations of mechanical circulatory assist devices.

Tricuspid regurgitant (TR) jet velocity and its relationship to pulmonary hypertension has been controversial in sickle cell disease (SCD). Plasma free hemoglobin is elevated in SCD patients and acutely impairs systemic vascular reactivity. We postulated that plasma free hemoglobin would be negatively associated with both systemic and pulmonary endothelial function, assessed by flow-mediated dilation (FMD) of the brachial artery and TR jet velocity, respectively. Whole blood viscosity, plasma free hemoglobin, TR jet, and FMD were measured in chronically transfused SCD pre- and posttransfusion (N = 25), in nontransfused SCD (N = 26), and in ethnicity-matched control subjects (N = 10). We found increased TR jet velocity and decreased FMD in nontransfused SCD patients compared with the other 2 groups. TR jet velocity was inversely correlated with FMD. There was a striking nonlinear relationship between plasma free hemoglobin and both TR jet velocity and FMD. A single transfusion in the chronically transfused cohort improved FMD. In our patient sample, TR jet velocity and FMD were most strongly associated with plasma free hemoglobin and transfusion status (transfusions being protective), and thus consistent with the hypothesis that intravascular hemolysis and increased endogenous erythropoiesis damage vascular endothelia., (© 2015 by The American Society of Hematology.)

The glucose transporter GLUT1 at the blood-brain barrier (BBB) mediates glucose transport into the brain. Alzheimer's disease is characterized by early reductions in glucose transport associated with diminished GLUT1 expression at the BBB. Whether GLUT1 reduction influences disease pathogenesis remains, however, elusive. Here we show that GLUT1 deficiency in mice overexpressing amyloid β-peptide (Aβ) precursor protein leads to early cerebral microvascular degeneration, blood flow reductions and dysregulation and BBB breakdown, and to accelerated amyloid β-peptide (Aβ) pathology, reduced Aβ clearance, diminished neuronal activity, behavioral deficits, and progressive neuronal loss and neurodegeneration that develop after initial cerebrovascular degenerative changes. We also show that GLUT1 deficiency in endothelium, but not in astrocytes, initiates the vascular phenotype as shown by BBB breakdown. Thus, reduced BBB GLUT1 expression worsens Alzheimer's disease cerebrovascular degeneration, neuropathology and cognitive function, suggesting that GLUT1 may represent a therapeutic target for Alzheimer's disease vasculo-neuronal dysfunction and degeneration.

Objective: To test the hypothesis that abnormal hemorheology and chronic low-grade inflammation are more prevalent in Lewis negative individuals, possibly contributing to premature atherosclerosis., Methods and Results: We enrolled 223 healthy subjects (154 females, mean age: 64yrs). Conventional risk factors, markers of inflammation and hemorheological profiles were measured; Lewis blood group was determined by serology. Conventional risk factors (age, gender, BMI, blood pressure, lipid profile, smoking habit) did not differ among Lewis phenotypes. However, markers of inflammation (WBC, hs-CRP, ESR) were significantly elevated and rheological parameters (RBC aggregation, plasma viscosity) were abnormal in Lewis negative subjects, especially when compared to the Le(a-b+) group., Conclusions: With a prevalence of 33% in select populations, our data support the hypothesis that Le(a-b-) represents a pro-inflammatory phenotype that may contribute to the elevated cardiovascular risk in this group., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Cell-cell and cell-surface adhesion modulated by water-soluble polymers continues to be of current interest, especially since prior reports have indicated a role for depletion-mediated attractive forces., Objective: To determine the effects of concentration and molecular mass of the neutral polymer dextran (40 kDa to 28 MDa) on the adhesion of human red blood cells (RBC) to coated glass coverslips., Methods: Confocal-reflection interference contrast microscopy (C-IRM), in conjunction with phase contrast imaging, was utilized to measure the adhesion dynamics and contact mechanics of RBC during the initial stages of cell contact with several types of substrates., Results: Adhesion is markedly increased in the presence of dextran with a molecular mass ⩾ 70 kDa. This increased adhesiveness is attributed to reduced surface concentration of the large polymers and hence increased attractive forces due to depletion interaction. The equilibrium deformation of adhering RBC was modeled as a truncated sphere and the calculated adhesion energies were in close agreement with theoretical results., Conclusions: These results clearly demonstrate that polymer depletion can promote RBC adhesion to artificial surfaces and suggest that this phenomenon may play a role in other specific and non-specific cell-cell interactions, such as rouleau formation and RBC-endothelial cell adhesion.

A theoretical framework based on macromolecular depletion has been utilized in order to examine the energetics of red blood cell interactions. Three different glycocalyx structures are considered and cell-cell affinities are calculated by superposition of depletion, steric and electrostatic interactions. The theoretical model predicts a non-monotonic dependence of the interaction energies on polymer size. Further, our results indicate that the glycocalyx segment distribution has a large impact on adhesion energies between cells: a linear segment distribution induces the strongest adhesion between cells followed by pseudo-tail and uniform distributions. Our approach confirms the concept of a depletion mechanism for RBC aggregation, and also provides new insights that may eventually help to understand and quantify cellular factors that control red blood cell interactions in health and disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Sickle cell disease (SCD) is characterized by decreased erythrocyte deformability, microvessel occlusion and severe painful infarctions of different organs. Ektacytometry of SCD red blood cells (RBC) is made difficult by the presence of rigid, poorly-deformable irreversibly sickled cells (ISC) that do not align with the fluid shear field and distort the elliptical diffraction pattern seen with normal RBC. In operation, the computer software fits an outline to the diffraction pattern, then reports an elongation index (EI) at each shear stress based on the length and width of the fitted ellipse: EI=(length-width)/(length+width). Using a commercial ektacytometer (LORCA, Mechatronics Instruments, The Netherlands) we have approached the problem of ellipse fitting in two ways: (1) altering the height of the diffraction image on a computer monitor using an aperture within the camera lens; (2) altering the light intensity level (gray level) used by the software to fit the image to an elliptical shape. Neither of these methods affected deformability results (elongation index-shear stress relations) for normal RBC but did markedly affect results for SCD erythrocytes: (1) decreasing image height by 15% and 30% increased EI at moderate to high stresses; (2) progressively increasing the light level increased EI over a wide range of stresses. Fitting data obtained at different image heights using the Lineweaver-Burke routine yielded percentage ISC results in good agreement with microscopic cell counting. We suggest that these two relatively simple approaches allow minimizing artifacts due to the presence of rigid discs or ISC and also suggest the need for additional studies to evaluate the physiological relevance of deformability data obtained via these methods.

Consumption of red wine is associated with a decreased risk of several cardiovascular diseases (e.g., coronary artery disease, stroke), but unfortunately literature reports regarding ethanol's effects on hemorheological parameters are not concordant. In the present study, red blood cell (RBC) deformability was tested via laser ektacytometry (LORCA, 0.3-30 Pa) using two approaches: 1) addition of ethanol to whole blood at 0.25%-2% followed by incubation and testing in ethanol-free LORCA medium; 2) addition of ethanol to the LORCA medium at 0.25%-6% then testing untreated native RBC in these media. The effects of ethanol on deformability for oxidatively stressed RBC were investigated as were changes of RBC aggregation (Myrenne Aggregometer) for cells in autologous plasma or 3% 70 kDa dextran. Significant dose-related increases of RBC deformability were observed at 0.25% (p < 0.05) and higher concentrations only if ethanol was in the LORCA medium; no changes occurred for cells previously incubated with ethanol then tested in ethanol-free medium. The impaired deformability of cells pre-exposed to oxidative stress was improved only if ethanol was in the LORCA medium. RBC aggregation decreased with concentration at 0.25% and higher for cells in both autologous plasma and dextran 70. Our results indicate that ethanol reversibly improves erythrocyte deformability and irreversibly decreases erythrocyte aggregation; the relevance of these results to the health benefits of moderate wine consumption require further investigation.

Background: Abnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium., Methods: RBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500kDa dextran with a flow chamber assay., Results: Our results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction., Conclusion: These results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids., General Significance: Our findings should therefore be of great value in understanding abnormal RBC-EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions., (© 2013.)

Background: Previous studies have demonstrated that red blood cells (RBC) either lyse or at least experience mechanical damage following prolonged exposure to high shear stress (≥100 Pa). Conversely, prolonged shear stress exposure within the physiological range (5-20 Pa, 300 s) was recently reported to improve RBC deformability. This study investigated the relationships between shear stress and RBC deformability to determine the breakpoint between beneficial vs. detrimental exposure to shear stress (i.e., "subhemolytic threshold"). A second aim of the study was to determine whether the frequency of intermittent application of shear stress influenced the subhemolytic threshold., Methods: RBC were exposed to various levels of shear stress (0-100 Pa) in a Couette type shearing system for 300 s. RBC deformability was then immediately measured via ektacytometry. Parallel experiments were conducted at the same shear stresses, except the application time differed while keeping constant the total exposure time: shear stress was applied either for 30 s and repeated 10 times (10×30 s) or applied for 15 s and repeated 20 times (20×15 s)., Results: For a range of donors, the subhemolytic threshold with constant shear stress application was between 30-40 Pa. When physiological shear stress was applied in an intermittent manner, more frequent applications tended to improve (i.e., increase) RBC deformability. However, when supra-physiological shear stress was applied, both continuous and intermittent protocols damaged RBC. Changes of RBC mechanical behavior occurred without increases of hemoglobin in the suspending media, thus attesting to the absence of hemolysis., Conclusion: Shear stress has a biphasic effect on the mechanical properties of RBC, with the duration and rate of exposure appearing to have minimal impact on the subhemolytic threshold when compared with the magnitude of applied shear stress.

If a surface is in contact with a solution containing macromolecules or proteins, and the loss of configurational entropy of these molecules at the surface is not balanced by adsorption energy, a polymer-poor layer will develop near the surface. If two such layers overlap, an attractive force develops due to the osmotic pressure difference between these depletion zones and the bulk phase. Recent studies have shown that depletion interaction plays a major role in red blood cell (RBC) aggregation and hence it is a major determinate of blood flow stability; depletion interaction also markedly affects RBC adhesion to vascular endothelial cells. Understanding and quantitating factors that regulate depletion in vivo are thus of importance, yet made difficult since only very small changes of the cell surface (e.g., glycocalyx thickness) such as seen during RBC aging can lead to massive changes of depletion interaction and hence cell-cell adhesion. It is suggested that insight into the in vivo relevance of depletion mechanisms may lead to an improved understanding of how and why blood flow is altered in many diseases, and may also provide new biomarkers (e.g., surface properties) that will aid in the development of novel or improved diagnostic and therapeutic tools.

Cardiovascular diseases (CVD) are the most frequent cause of death throughout the world. The coronary vessel system is a special part of the circulation since there is a continuous change in blood flow, perfusion pressure and shear rate during each cardiac cycle. It is also the place of the narrowest capillaries in the human body, therefore the role of rheological alterations may be of greater importance than in the other parts of the circulatory system. During the past decades, our group has investigated hemorheological parameters (HP) in over 1,000 patients diagnosed with various forms of ischemic heart disease (IHD). In one prospective study, we measured the HP of patients with acute coronary syndrome (ACS). On admission, all examined variables were significantly worse than those of control subjects. During the hospital phase, some of the HP showed further deterioration, and HP remained in the pathologic range during the follow-up period. In another study, we showed that HP are in close correlation with the severity of coronary artery disease. In patients treated with percutaneous coronary intervention, changes in HP were very similar to those observed in subjects with ACS. In a recent study, we analyzed HP in patients undergoing CABG surgery. Our data suggest a hemorheological advantage of off-pump surgery. In another study low Hct/WBV ratio can be regarded as a risk factor of cardiac death in IHD. Our data indicate that rheological parameters are significantly altered in patients with IHD: the extent of the alterations is in excellent correlation with the clinical severity of the disease. Our findings prove that HP play a critical role in the pathogenesis of myocardial ischemia. In recent in vitro and in vivo studies we have investigated the effects of red wine on hemorheological parameters. Our results show that moderate red wine consumption has beneficial effects on hemorheological parameters which may contribute to the French paradox.

The role of membrane fluidity in determining red blood cell (RBC) deformability has been suggested by a number of studies. The present investigation evaluated alterations of RBC membrane fluidity, deformability and stability in the presence of four linear alcohols (methanol, ethanol, propanol and butanol) using ektacytometry and electron paramagnetic resonance (EPR) spectroscopy. All alcohols had a biphasic effect on deformability such that it increased then decreased with increasing concentration; the critical concentration for reversal was an inverse function of molecular size. EPR results showed biphasic changes of near-surface fluidity (i.e., increase then decrease) and a decreased fluidity of the lipid core; rank order of effectiveness was butanol > propanol > ethanol > methanol, with a significant correlation between near-surface fluidity and deformability (r = 0.697; p<0.01). The presence of alcohol enhanced the impairment of RBC deformability caused by subjecting cells to 100 Pa shear stress for 300 s, with significant differences from control being observed at higher concentrations of all four alcohols. The level of hemolysis was dependent on molecular size and concentration, whereas echinocytic shape transformation (i.e., biconcave disc to crenated morphology) was observed only for ethanol and propanol. These results are in accordance with available data obtained on model membranes. They document the presence of mechanical links between RBC deformability and near-surface membrane fluidity, chain length-dependence of the ability of alcohols to alter RBC mechanical behavior, and the biphasic response of RBC deformability and near-surface membrane fluidity to increasing alcohol concentrations.

The flow properties of blood play significant roles in tissue perfusion by contributing to hydrodynamic resistance in blood vessels. These properties are influenced by pathophysiological processes, thereby increasing the clinical relevance of blood rheology information. There is well-established clinical evidence for impaired blood fluidity in humans of advanced age, including enhanced plasma and whole blood viscosity, impaired red blood cell (RBC) deformability and enhanced RBC aggregation. Increased plasma fibrinogen concentration is a common finding in many studies owing to the pro-inflammatory condition of aged individuals; this finding of increased fibrinogen concentration explains the higher plasma viscosity and RBC aggregation in elderly subjects. Enhanced oxidant stress in advanced age is also known to contribute to altered blood fluidity, with RBC deformability being an important determinant of blood viscosity. Several studies have shown that physical activity may improve the hemorheological picture in elderly subjects, yet well-designed observational and mechanistic studies are required to determine the specific effects of regular exercise on hemorheological parameters in healthy and older individuals.

Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-βAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-βAS3-FB LV transduced BM CD34+ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total β-globin-like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34+ cells were transplanted into immunodeficient mice, and the human cells recovered after 2-3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34+ cells that were directly differentiated in vitro. These results demonstrate that the CCL-βAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling β-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells.

Background: Chronic transfusion therapy (CTT) is a mainstay for stroke prophylaxis in sickle cell anemia, but its effects on hemodynamics are poorly characterized. Transfusion improves oxygen-carrying capacity, reducing demands for high cardiac output, while decreasing hemoglobin (Hb)S%, reticulocyte count, and hemolysis. We hypothesized that transfusion would improve oxygen-carrying capacity, but that would be counteracted by a decrease in cardiac output due to increased hematocrit (Hct) and vascular resistance, leaving oxygen delivery unchanged., Study Design and Methods: To test this hypothesis, we examined patients on CTT immediately before transfusion and again 12 to 120 hours after transfusion, using echocardiography and near infrared spectroscopy., Results: Comparable increases in Hb and Hct and decreases in reticulocyte count and HbS with transfusion were observed in all patients, but males had a larger rebound of HbS%, reticulocyte count, and free Hb levels between transfusions. In males, transfusion decreased heart rate by 12%, stroke volume by 15%, and cardiac index by 24% while estimates for pulmonary and systemic vascular resistance increased, culminating in 6% decrease in oxygen delivery. In contrast, stroke volume and cardiac index and systemic and pulmonary vascular resistance did not change in women after transfusion, such that oxygen delivery improved 17%., Conclusion: In our sample population, males exhibit a paradoxical reduction in oxygen delivery in response to transfusion because the increase in vascular resistance is larger than the increase in oxygen capacity. This may result from an inability to adequately suppress their HbS% between transfusion cycles., (© 2012 American Association of Blood Banks.)

Elevated red blood cell (RBC) aggregation increases low-shear blood viscosity and is closely related to several pathophysiological diseases such as atherosclerosis, thrombosis, diabetes, hypertension, cancer, and hereditary chronic hemolytic conditions. Non-ionic linear polymers such as poly(ethylene glycol) (PEG) and Pluronic F68 have shown inhibitory effects against RBC aggregation. However, hypersensitivity reactions in some individuals, attributed to a diblock component of Pluronic F68, have been reported. Therefore, we investigated the use of an amphiphilic star-shaped PEG polymer based on a cholic acid core as a substitute for Pluronics to reduce RBC aggregation. Cholic acid is a natural bile acid produced in the human liver and therefore should assure biocompatibility. Cholic acid based PEG polymers, termed CA(PEG)(4), were synthesized by anionic polymerization. Size exclusion chromatography indicated narrow mass distributions and hydrodynamic radii less than 2 nm were calculated. The effects of CA(PEG)(4) on human RBC aggregation and blood viscosity were investigated and compared to linear PEGs by light transmission aggregometry. Results showed optimal reduction of RBC aggregation for molar masses between 10 and 16 kDa of star-shaped CA(PEG)(4) polymers. Cholic acid based PEG polymers affect the rheology of erythrocytes and may find applications as alternatives to linear PEG or Pluronics to improve blood fluidity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Laser-diffraction ektacytometry is a generally accepted technique for measuring RBC deformability induced by fluid shear stress (SS) and yields paired elongation index-SS data at several levels of stress. Unfortunately, comparison of results is hindered by the lack of simple indices that accurately characterize these data. Several mathematical models have been proposed, including those developed for analysis of enzyme kinetics (Lineweaver-Burk, Eadie-Hofstee) and curve fitting (Streekstra-Bronkhorst). All of these analytical approaches provide a value for cell deformation at infinite stress (EImax) and the shear stress required to achieve one-half of this deformation (SS1/2); the use of non-linear regression is essential when calculating these parameters. While the current models provide equivalent results for normal RBC if used with non-linear regression, EImax and SS1/2 are not always concordant for cells with abnormal mechanical behavior. This technical note examines such differences for three conditions: glutaraldehyde treatment, mechanical stress and non-isotonic media. It was found that none of the models yield completely satisfactory values for EImax and SS1/2, especially if there are large changes of EImax. However, the ratio of SS1/2 to EImax (SS1/2/EImax) is much less affected by these problems, has similar power (i.e., standardized difference) as SS1/2 and EImax and is more robust in reflecting alterations of deformability. We thus conclude that the SS1/2/EImax ratio can be used when reporting and comparing various populations of RBC or cells obtained from subjects having different clinical states.