Reduced Erythrocyte Deformability Research Articles

Reduced erythrocyte deformability alters pulmonary hemodynamics.

Isolated rat lungs were perfused with suspensions containing normal and stiffened erythrocytes (RBCs) to assess the effect of altered RBC deformability on pulmonary hemodynamics. RBC suspensions were prepared using cells previously incubated in isosmolar phosphate-buffered saline with or without 0.0125 or 0.01875% glutaraldehyde. Washed RBCs were resuspended in isosmolar 4% albumin saline solution. Isolated rat lungs were perfused with control and stiffened cells by the use of a perfusion system that allowed rapid switching between suspensions. Pressure-flow (P/Q) curves were constructed by measuring pulmonary arterial pressure (Ppa) over a range of flow rates. In a second set of experiments, P/Q curves were generated for perfusion with control and stiffened cells (0.0125% glutaraldehyde) before and after vasoconstriction with a synthetic prostaglandin analogue (U 46619). RBC deformability was quantified in all experiments by determination of filtration time of a dilute cell suspension through a 4.7 microns Nuclepore filter. Incubation with 0.0125 or 0.01875% glutaraldehyde produced a 6 or 21% decrease in RBC deformability, respectively. These decreases in deformability were associated with significant increases in Ppa at each flow rate. The increases in Ppa correlated significantly with the degree of RBC stiffening. With 0.0125% glutaraldehyde, the P/Q curve was shifted upward without a change in slope, whereas incubation with 0.01875% glutaraldehyde resulted in a significant increase in slope. Vasoconstriction and perfusion with stiffened RBCs had additive effects on Ppa. These findings suggest that decreases in RBC deformability cause physiologically significant elevations in hemodynamic resistance in the pulmonary circuit independent of vasoactivity.

The Effect of Improved Control on Blood Filtration Properties and Non‐enzymatic Glycosylation of Erythrocyte Proteins in Type 2 Diabetes

Abnormal blood flow in the microcirculation has been reported in diabetes and may be important in the pathogenesis of diabetic complications. The mechanism of this is not understood but non-enzymatic glycosylation of erythrocyte membrane or haemoglobin causing reduced erythrocyte deformability and a secondary change in filtration properties of blood have been suggested as possible factors. The relationship of non-enzymatic glycosylation of erythrocyte membrane and glycosylated haemoglobin to filtration time of blood was investigated during stabilization of diabetes with sulphonylurea therapy. Over an 8-month period, glycosylated haemoglobin, non-enzymatic glycosylation of erythrocyte membrane, and filtration time fell by 41%, 71%, and 53% of the initial value, respectively, but the rate of decline was slower for filtration time which did not change significantly until the last month. Due to the different time-course of improvement, no relationship was found between filtration time and glycosylated haemoglobin or non-enzymatic glycosylation of erythrocyte membrane whereas glycosylated haemoglobin and non-enzymatic glycosylation of erythrocyte membrane correlated significantly (r = 0.49, p less than 0.001). These results suggest that the abnormal filtration property of blood in diabetes is not a direct consequence of non-enzymatic glycosylation, and suggest that the erythrocytes made in the hyperglycaemic milieu are abnormally rigid. The filtration properties of blood are only improved when new generations of erythrocytes enter the circulation.

Inhibition of K+ efflux and dehydration of sickle cells by [(dihydroindenyl)oxy]alkanoic acid: an inhibitor of the K+ Cl- cotransport system.

[(Dihydroindenyl)oxy]alkanoic acid (DIOA) was recently introduced as a potent inhibitor of the K+Cl- cotransport system without side effects on other cation transport systems [Garay, R. P., Nazaret, C., Hannaert, P.A. & Cragoe, E. J., Jr. (1988) Mol. Pharmacol. 33, 696-701]. In sickle cells, an abnormal activation of this K+Cl- cotransport system was proposed to be involved in cell K+ loss and dehydration. We found that DIOA inhibited the abnormal sickle cell K+ loss and specifically reduced sickle cell density upon stimulation of the net outward K+Cl- cotransport--i.e., low pH, hypoosmolarity, and activation by N-ethylmaleimide. DIOA opens another therapeutic approach to sickle cell disease by inhibiting cell dehydration, which favors HbS polymerization and reduces erythrocyte deformability.

Effect of statil (ICI 128436) on erythrocyte viscosity in vitro.

The hypothesis that sorbitol accumulation could contribute to a reduced erythrocyte deformability in diabetes was investigated. Erythrocyte sorbitol and erythrocyte viscosity at high and low shear rates were studied in 20 insulin-dependent diabetic (IDDM) and 20 matched control subjects. An increased erythrocyte sorbitol and an increased low-shear erythrocyte viscosity were found in the IDDM patients, but there was no significant correlation (r = .11, NS) between the parameters. Incubation (3 h, 37 degrees C) in a Krebs buffer containing 33.3 mM glucose resulted in a significant sorbitol accumulation, but erythrocyte viscosity was not affected. Despite this fact, addition of 1 mM statil (ICI 128436) in the 5.5- and 33.3-mM glucose media not only prevented erythrocyte sorbitol accumulation but also improved erythrocyte viscosity in diabetic and control subjects. The effect was more pronounced at the low (approximately 16%) than at the high (approximately 2%) shear rate. The effect on erythrocyte viscosity disappeared by washing the erythrocytes after incubation, although erythrocyte sorbitol remained different. Our results suggest that sorbitol accumulation does not contribute to an increased erythrocyte viscosity in diabetes, and statil shows a positive effect on erythrocyte viscosity independent of its aldose reductase-inhibiting property.

Interaction between erythrocytes and a perfluorochemical blood substitute.

Perfluorochemicals (PFCs) are being widely developed for use as erythrocyte substitutes to carry oxygen to tissue in acute crises. Oxypherol, a commercially available PFC preparation commonly used for animal tests, reduces erythrocyte deformability in the presence of plasma. This undesirable effect further complicates oxygen delivery by erythrocytes. Our experiments indicate that one or more plasma proteins must be present to observe Oxypherol-induced reduction in erythrocyte deformability, but the latter is not protein specific. Neither platelet activation nor enhanced protein adsorption could account for the mechanism of altered erythrocyte flexibility. Using fluorine-19 NMR, it was found that a small amount of Oxypherol droplets is adsorbed on the surface of the erythrocytes. The amount of adsorbed droplets increases when Oxypherol is incubated with the erythrocytes in the presence of plasma, in parallel with a decrease in erythrocyte deformability. Therefore, it seems likely that the loss of deformability of the erythrocytes is caused by the adsorption of small Oxypherol droplets on the erythrocytes.

Effects of insulin on physical factors: Atherosclerosis in diabetes mellitus

Newton's laws of motion play a major role in blood flow. Inertia and conservation of momentum cause flow to separate at branches and curves in large blood vessels. Areas of separated flow in the arterial system are sites of atherogenesis. The place at which the separation ends, called the stagnation point, is the focus for plaque development. Pulsation of the arterial circulation causes the stagnation point to move downstream with each systole and upstream with each diastole. This movement generates forward and backward shearing force in the stagnation region as the separated flow migrates back and forth. Angular momentum, introduced into flowing blood with each heart beat and further enhanced by the asymmetry of origin of vessels branching from the aorta, generates a sidewise force component that is preserved during migration of the stagnation point. The sidewise force, added to the forward and backward shear stresses, creates an area of multidirectional shear stress under the migrating stagnation point that increases the permeability of the local endothelium. Blood is a complex fluid; it can generate greater shear stresses near the stagnation point than the simple fluids normally studied by fluid mechanicists. Blood is capable of retaining shear stress for short periods after it ceases to flow and extra work is required to establish its flow. In diabetes, reduced erythrocyte deformability further burdens flow onset. We are not yet able to establish whether the increase is only a few percent, or whether the burden is larger. Whatever its magnitude, diabetic modifications of the flow properties of blood, directly affect the size, location, and rate of development of atherosclerotic plaques.

Reduced erythrocyte deformability and hyperfibrinogenemia in Nigerian diabetics with hemoglobin genotype HbAA.

Erythrocyte deformability (Vrbc) and plasma fibrinogen values were studied in 44 Nigerian diabetics and 41 age-matched controls, some of whom were hypertensives. All had normal hemoglobin genotype (HbAA). Vrbc was significantly reduced in the diabetic patients compared to the control group (p less than 0.005). Similarly, the Vrbc in hypertensive diabetics was significantly reduced below that in both non-hypertensive diabetics (p less than 0.01) and hypertensive controls (p less than 0.01). Plasma fibrinogen concentration was significantly raised in the diabetics compared to controls (p less than 0.01). Although the fibrinogen was higher in the hypertensive diabetics than in diabetics without hypertension the difference was not statistically significant. There was no significant difference between hypertensive controls and hypertensive diabetics even though the latter had a higher mean fibrinogen than controls without hypertension. We conclude that Nigerian diabetics with normal hemoglobin genotype, HbAA, have poorly deformable RBCs and a hyperfibrinogenemic plasma compared to a non-diabetic control group. These findings may have a considerable prognostic significance in terms of the patients' subsequent progress.

Hemorheological parameters in some vascular diseases.

The origin of arterial diseases (acrosyndromes or peripheral vascular diseases is undoubtedly more generally attributed to parietal and vasomotor anomalies than to hemorheological disorders. However, in the light of recent investigations it would seem that increased erythrocyte aggregation is generally observed during arterial disease and is sometimes accompanied by reduced red cell deformability resulting in the appearance of a hyperviscosity syndrome. These rheological disturbances may have very significant bearing on the complications observed via three primary mechanisms: hemolysis (resulting in ADP release and promoting platelet aggregation), erythrocyte aggregation (resulting in a decreased blood flow (sludge)), and reduced erythrocyte deformability accompanied by reduced oxygen availability in the small blood vessels.

The effect of diabetes on blood flow properties.

Blood flow is a complex process combining fluid shearing in both plasma and the interior of red blood cells with elastic deformation of blood's solid elements. The red cell membrane is the major solid in blood, but platelets and white cells contribute solid behavior as well. Changes in blood's flow properties are often hidden by blood's ability to change its pattern of response. Capillary viscometry can be used to examine serum, plasma, and hemoglobin solutions directly, but rotational viscometry, where regularity of fluid shearing can be controlled to a narrow shear rate range, is required to study blood flow effectively. The most striking diabetic blood flow abnormality is best seen in time-based rotational viscometer studies that demonstrate the pattern of development of shear stress as flow becomes established. In such studies blood demonstrates both viscoelastic and thixotropic behavior; in diabetes blood's thixotropy is substantially increased. The diabetic pattern appears to be produced by a combination of reduced erythrocyte deformability and increased erythrocyte aggregation due to plasma protein changes. The plasma protein changes observed in diabetes are linked to the development of glucose intolerance but they are not specific to diabetes. The combined increase in aggregation and resistance to deformation of red blood cells produces blood flow abnormalities that can be detected primarily at low shear rate. Plasma protein changes and alteration in blood viscosity are absent from children with diabetes, while such changes tend to be associated with diabetic microangiopathy in adults. In some in vivo conditions, the effects on blood flow that can be linked to erythrocyte deformability are magnified out of proportion to measurements made in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Cigarette smoking in pregnancy and fetal hyperviscosity.

The effects of maternal cigarette smoking on fetal blood viscosity and its major determinants were investigated in an effort to determine if there might be a relation between the intrauterine growth retardation associated with cigarette smoking and fetal hyperviscosity. Blood was collected from the umbilical vein at birth in 40 infants born to mothers who had smoked more than 20 cigarettes a day throughout pregnancy and from 40 matched nonsmoking controls. Blood and plasma viscosity packed cell volume plasma fibrinogen concentration and erythrocyte deformability were measured by standard techniques and statistical comparison of the differences in means between the 2 groups made using Students t test. Cigarette smoking was associated with a 30% increase in fetal blood viscosity this rise being compounded of a 12% increase in packed cell volume and an 18% fall in erythrocyte deformability. Neither plasma viscosity nor plasma fibrinogen values were altered. The mean birth weight was 318 gm lower in infants born to smoking mothers. When adult smokers become pregnant the large physiological variation in blood viscosity factors obscure the effects of smoking in maternal blood. This is not the case in the fetus. In the extensive capillary microcirculation of the fetal placental villi raised blood viscosity and reduced erythrocyte deformability would be apt to reduce blood flow significantly in accordance with Poiseuilles law. Reduced intravillus perfusion would combine with reduced intervillus blood flow on the maternal side of the placenta owing to the effects of nicotine and with the already reduced oxygen availability in both maternal and fetal blood to cause fetal hypoxia stimulating fetal erythropoiesis with a further increase in blood viscosity leading to a vicious circle of decreasing flow and further hypoxia. The study indicates a new pathogenic pathway for the deleterious effects of maternal cigarette smoking on fetal growth and development in pregnancy.

Reduced erythrocyte deformability associated with calcium accumulation

Erythrocytes exposed to subhemolytic shear stress in vitro exhibit decreased deformability as determined by a filtration method. Intracellular calcium content of these cells has been measured by atomic absorption spectroscopy and found to be 35 and 55% higher than controls (0.0157 μmol/ml packed red blood cells) after shear stress levels of 100 and 130 N/cm 2, respectively. These alterations occur without significant changes in ATP level, intracellular magnesium content, cell volume, or morphology, and without large associated sodium and potassium fluxes. Results indicate that calcium may be responsible for or associated with changes in the viscoelastic properties of the red cell membrane caused by sublytic mechanical trauma.

Erythrocyte Spectrin Glucosylation in Diabetes

Because reduced erythrocyte deformability in diabetes might be mediated by increased noneniymatic glucosylation of membrane proteins, we have isolated and studied the degree of glucosylation of spectrin, the major protein of the inner membrane surface. Spectrin was recovered from the erythrocytes of seven age-and sex-matched diabetic and nondiabetic individuals. The thiobarbituric acid (TBA) method was used to measure the glucosylation of spectrin and of 10 mg of hemoglobin from the same ceils. Increased glucosylation of both spectrin and hemoglobin was found in diabetes. Glucosylation, expressed as a ratio of TBA absorbance to protein weight was nearly three times as high for spectrin as for hemoglobin. The ratio of TBA absorbance to hemoglobin content was examined and found to be curvilinear. A reaction model with both first and second order components was fitted to the data to obtain a reaction constant. When this model and constant were used to adjust TBA ab-sorbances, spectrin's glucosylation became less than double that of hemoglobin. Spectrin forms an inner membrane network responsible for returning deformed erythrocytes to their original shape. The reported normal response to stretching of diabetic erythrocytes suggests that increased glucosylation of spectrin fails to stiffen the membrane. Some other mechanism, such as reduced phosphorylation of spectrin and other membrane proteins, may be responsible for reduced deformability of diabetic erythrocytes. But spectrin's increased glucosylation might be responsible for the increased number of poorly deformable erythrocytes found among aged diabetic erythrocytes. It may produce this effect by inhibiting the membrane protein transglutamidation responsible for removal of aged erythrocytes.

Red cell deformability is not an independent risk factor in stroke.

Reduced erythrocyte deformability has been implicated in the causation of stroke. We have therefore tested this red cell property by filtration through polycarbonate membranes in 42 patients with either transient ischaemic attacks or completed ischaemic stroke together with age and sex paired controls. We have failed to demonstrate any difference between patients and controls suggesting that reduced red cell deformability is unlikely to be an important independent factor in the production of stroke.

Blood viscosity and haemostatic factors in late pregnancy, pre-eclampsia and fetal growth retardation.

Summary. Blood viscosity and several of its determinants (packed cell volume, plasma viscosity, plasma fibrinogen and erythrocyte deformability) and several haemostatic variables (platelet count, serum fibrin degradation products and plasma soluble fibrin) were measured in 106 women during normal third‐trimester pregnancy, 12 patients with moderate pre‐eclampsia, nine patients with severe pre‐eclampsia and 16 patients with confirmed fetal growth retardation. Blood viscosity was measured at high and low shear rates (94.5 and 0.94 s−1), with and without correction to a standard packed cell volume of 0.45. In normal pregnancy low‐shear blood viscosity increased significantly towards term, associated with significant increases in packed cell volume and plasma viscosity (but not fibrinogen); there was no change in high‐shear blood viscosity at native packed cell volume and a decrease in viscosity after correction for packed cell volume, associated with a significant increase in erythrocyte deformability. Significant decrease in the platelet count and significant increases in serum fibrin degradation products and plasma soluble fibrin suggested a degree of haemostatic activation in normal third‐trimester pregnancy. In moderate pre‐eclampsia blood viscosity was significantly lower at low shear rate, due to a significantly lower packed cell volume and despite a significantly elevated plasma viscosity. In severe pre‐eclampsia high‐shear blood viscosity was significantly elevated after correction for packed cell volume, associated with a significantly reduced erythrocyte deformability; plasma fibrinogen levels were reduced, but other variables were not significantly abnormal. In fetal growth retardation blood viscosity was significantly increased at both shear rates, despite a reduced packed cell volume: this was also associated with a significant reduction in erythrocyte deformability. In general there was little correlation between blood viscosity factors and haemostatic variables, but in normal‐term pregnancy soluble fibrin levels correlated with blood viscosity at both shear rates. We suggest that increased blood viscosity and decreased erythrocyte deformability may play a role in the placental insufficiency of severe pre‐eclampsia and fetal growth retardation.

Impaired erythrocyte doublet formation in diabetes.

A new means by which to examine erythrocyte deformability, the rate at which erythrocytes in dilute suspension form doublets after settling to the surface of a microscope slide, has been developed and tested. Doublet formation consists of the evaluation and subsequent apposition of one cell over another, a process limited by the ability of each red cell membrane to bend. The cell-cell attraction that promotes doublet formation is controlled by adding an appropriate amount of dextran to the artificial suspending medium. Videotaping permits careful analysis of doublet formation rate and maintains a permanent record. When erythrocytes from 20 diabetic and 20 non-diabetic subjects were studied, doublet formation rates were found to be strikingly different. Cells from half of the diabetics studied formed less than three doublets in 20 min while the non-diabetic mean for the same period was 15 doublets. No overall correlation between doublet formation and fasting glucose could be found. No relation between doublet formation rate and type of diabetes, treatment, or microvascular complications was observed. Doublet formation rate is a simple and rapid means of detecting and studying reduced erythrocyte deformability in diabetes.

Physical factors important in the development of atherosclerosis in diabetes.

Circulation of the blood is a process controlled by physical laws. The same physical laws also determine the sites of atherosclerotic plaques. Our understanding of their role in atherogenesis is aided by a review of (1) the difference between a solid and fluid, (2) strain and shear or strain rate and shear rate as describers of motion, (3) the distinction between force as pressure and as shear stress, (4) the effects of viscosity and inertia on fluid motion, and (5) the nonlinear responses of blood and blood vessels. Moving blood affects the vessel wall principally through shear stress, the force due to its viscosity applied parallel to the surface of the vessel. Excessive shear stress occurs near the orifices of branches of the aorta, the arterial areas where atherosclerotic plaques develop. It is produced by a change in blood flow pattern at these sites. Arterial stiffening in diabetes reduces the ability of the branches of the aorta to dilate during systole, a motion that helps to control high wall shear stress. At wall shear stress levels under 400 dynes/cm2, a value only modestly higher than that normally achieved near the orifices of the major arterial branches, the endothelial lining of the arterial wall becomes disrupted, allowing entry of plasma proteins and lipids. Blood viscosity elevation in diabetes, linked to enhanced erythrocyte aggregation and reduced erythrocyte deformability, adds to the shear stress at the vessel wall, favoring increased lipid entry. Exposed to a high lipid influx secondary to excessive wall shear stress, the arterial wall must rely on physical as well as chemical means to remove the lipids. Subendothelial motion generated by diastolic arterial contraction and systolic expansion appears to cause plasmalemmal vesicle formation and dissolution, an event probably important in the removal process. The combination of arterial stiffening and hemorheologic disturbance adds to the burden of increased plasma lipids to favor the development of atherosclerosis in diabetes.

Higher levels of erythrocyte membrane microviscosity in diabetes.

Significantly higher levels of erythrocyte membrane microviscosity (MV) [n-: 5.22 +/- 0.17 (4.70--5.92), mean +/- SD (range), poise, N = 67, P less than 0.005] measured by fluorescence depolarization using 1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe were found in diabetic patients when compared with normal controls [5.05 +/- 0.15 (4.70--5.29), N = 22]. No significant differences in MV existed between males and females, nor was MV significantly correlated with diabetic age, duration of diabetes, plasma cholesterol, cholesterol/phsopholipid ratios, and plasma lecithin:cholesterol acyltransferase activities. No significant difference in MV was observed between groups with or without diabetic retinopathy. There was, however, significantly higher MV [5.29 +/- 0.19 (5.00--5.92), N = 20, P less than 0.05] in the group with fasting blood glucose (FBG) greater than or equal to 140 mg/dl than that [5.19 +/- 0.15 (4.70--5.46), N = 47] in the group with FBG less than 140 mg/dl. The changes in erythrocyte membrane MV presented in this study appear to be related to the current metabolic control of diabetic patients and are considered to be one of the factors responsible for the reduced erythrocyte deformability in diabetes.

IS HYPERVISCOSITY A TREATABLE COMPONENT OF DIABETIC MICROCIRCULATORY DISEASE?

Blood viscosity at low shear-rates was significantly higher in sixty-four patients with longstanding diabetes than in sixty-one matched non-diabetic controls. This increase was most striking in patients with either proliferative retinopathy or nephropathy, although it was present to a lesser extent in diabetic patients with evidence of myocardial or peripheral ischæmia. Erythrocyte deformability was lower in the fourteen diabetic patients with the most extensive microangiopathy than in twenty-two diabetics with slight or no complications or in controls. Hyperviscosity and reduced erythrocyte deformability may well be important and potentially treatable factors in the ætiology or progression of microcirculatory disease in diabetes.