To assess the influence of intracellular hemoglobin concentration on red cell viscoelasticity and to better understand changes related to in vivo aging, membrane shear elastic moduli (mu) and time constants for cell shape recovery (tc) were measured for age-fractionated human erythrocytes and derived ghosts. Time constants were also measured for osmotically shrunk cell fractions. Young and old cells had equal mu, but tc was longer for older cells. When young cells were shrunk to equal the volume (and hence hemoglobin concentration and internal viscosity) of old cells, tc increased only slightly. Thus membrane viscosity (eta = mu . tc) increases during aging, regardless of increased internal viscosity. However, further shrinkage of young cells, or slight shrinkage of old cells, caused a sharp increase in tc. Because this increased tc is not explainable by elevated internal viscosity, eta increased, possibly due to a concentration-dependent hemoglobin-membrane interaction. Ghosts had a greater mu than intact cells, with proportionally faster tc; their membrane viscosity was therefore similar to intact cells. However, the ratio of old/young membrane viscosity was less for ghosts than for intact cells, indicating that differences between young and old cell eta may be partly explained by altered hemoglobin-membrane interaction during aging. It is postulated that these changes in viscoelastic behavior influence in vivo survival of senescent cells.

Little data exist for the mechanical properties of individual irreversible or reversible sickle cells (ISC and RSC, respectively), nor is the process of ISC formation well understood. For oxygenated ISC and density-fractionated RSC, we have used micropipette techniques to measure cell surface area (SA) and volume (V), membrane shear elastic modulus (mu), time constant for viscoelastic shape recovery (tc), and hence to calculate membrane surface viscosity (eta = mu X tc). Volume loss associated with increasing cell density was accompanied by a proportionately smaller surface area decrease; SA/V ratio thus increased for denser cells, with ISC having the highest values. Membrane area loss by fragmentation must thus be accompanied by an accelerated decrease in cell volume. ISC had relatively rigid membranes (mu 130% above normal controls) and tc close to normal values, so that their effective membrane viscosity was more than double control. RSC had viscoelastic properties close to control, but showed wider variation between sickle cell donors and within samples. Measurements on density-separated RSC showed that, on average, mu was nearly constant, but that tc was longer for the densest cells, with their eta approaching ISC levels. A small subpopulation of RSC were found that had mu close to ISC values. Hypotonically swollen ISC (with internal hemoglobin concentration decreased to normal levels) retained their increased membrane stiffness but had markedly decreased tc, so that their eta approached normal values. The results show that elevated hemoglobin concentration influences the viscoelastic behavior of ISC and RSC, but that an irreversible change in membrane elasticity also occurs for ISC. These data suggest that ISC formation occurs via a two- stage process: (1) accelerated volume loss leading to increased cytoplasmic and effective membrane viscosity; (2) a sharp rise in membrane rigidity, presumably linked to membrane structural alteration.

Although there is evidence that the deformability of the entire red blood cell (RBC) decreases during aging, reports on changes in relevant specific properties associated with the aging process are limited and not in total agreement. The purpose of this study was to evaluate some of the factors that might contribute to this decreased deformability. Geometric, osmotic, and membrane mechanical properties of unfractionated, top (“young”) and bottom (“old”) RBC from 5 healthy adult donors were measured using micropipette techniques. Surface area, volume, and diameter of RBC were measured at osmolalities of 297, 254, 202, and 153 mosm/kg. Two membrane mechanical properties, surface shear modulus of elasticity (mu) and time constant (tc) of viscoelastic recovery, were studied only in isotonic media. At each of the osmolalities, volume and surface area of the bottom cells were about 25% lower than those of the top cells. Bottom cells showed smaller increases in volume with decreasing osmolality than top cells; the surface area remained constant with changing osmolality for all three groups. The surface area-to-volume ratio and the minimum cylindrical diameter of the bottom cells were essentially identical to the top cells. However, both the surface area index (actual are of RBC divided by area of a sphere of same volume) and the swelling index (maximal volume divided by actual volume) of the bottom cells were significantly lower than top RBC. The shear modules of elasticity (mu) was about 0.006 dyne/cm in all 3 RBC populations, indicating that the forces necessary to deform a portion of the membrane did not change with RBC aging. The viscoelastic time constant (tc) was 0.148 +/- 0.020 (SD) sec for the bottom RBC and 0.099 +/- 0.017 sec for the top cells. This difference indicates that shape recovery following membrane deformation is delayed in old RBC. The membrane surface viscosity (eta), calculated as the product of tc times mu was 0.95 +/- 0.22 x 10(-3) dyne-sec/cm for the bottom cells and 0.54 +/- 0.15 x 10(-3) for the top RBC. These data indicate that the relative deficit in membrane surface area and the increased membrane viscosity of old RBC may be important determinants for their decreased deformability and their eventual removal from the circulation.

Summary. The anti-neoplastic agent 5-fluorouracil (5-FU) in high therapeutic doses can induce angina pectoris and myocardial infarction. The pathophysiological mechanism of this side-effect has not yet been elucidated. We analysed the influence of 5-FU on blood rheology in vitro. Whole blood, blood cell suspensions and plasma were incubated with increasing concentrations of 5-FU (final concentrations 0, 0 . 08, 0 . 4, 2, 10 and 25 mg/ml 5-FU) at 37ЊC. Erythrocyte morphology was analysed after fixation with glutaraldehyde. Viscosity was measured at high and low shear rates (94 and 0 . 1 s -1 ). Erythrocyte aggregation and the cell transit times of erythrocytes through 5 mm pores and polymorphonuclear leucocytes through 8 mm pores were determined. 5-FU induced a dose-dependent formation of echinocytes within minutes and was reversible upon removal of 5-FU, which reflected a preferential intercalation of the drug in the outer hemileaflet of the cell membrane. High shear blood viscosity was increased at the highest 5-FU concentration (148 Ϯ 12%), and at low shear rate a dose-dependent decrease was found (0 mg/ml: 100%, 0 . 08 mg/ml: 87 Ϯ 10%, 0 . 4 mg/ml: 80 Ϯ 19%, 2 mg/ml: 70 Ϯ 15%, 10 mg/ml: 40 Ϯ 19%, 25 mg/ml: 33 Ϯ 5%). Erythrocyte aggregation was decreased by the 5-FU-induced echinocytosis. The transit time of erythrocytes through narrow pores was increased in a dose-dependent manner by 5-FU, whereas the transit time of polymorphonuclear leucocytes was initially decreased at 10 mg/ml and returned to control after 60 min incubation. We conclude that 5-FU interacts with the cell membrane, induces echinocytosis and vesiculation and affects blood rheology in several ways which may contribute to cardiovascular complications.

We investigated several rheologic variables in 17 patients (11 men, six women, mean age = 52. 1 + 9.8 years) with chronic stable angina. None took any medication except for sublingual nitroglycerin for 2 weeks before the study, and all had angiographically proven coronary artery disease with no history of myocardial infarction. Rheologic measurements included hematocrit, whole blood and plasma viscosity (750 and 1500 sec-'), degree of red cell aggregation via the zeta sedimentation ratio, and the extent and rate of red cell aggregation after stasis (Myrenne aggregometer). Compared with normal control donors, salient observations in the patients as a group included: (1) a small (6%) but significant increase in hematocrit, (2) a significant elevation in plasma viscosity (9%), (3) significant increases in whole blood viscosity at both shear rates (14% and 16%), (4) significant increases in the degree (12%), the extent (41 %), and the rate (28% faster time constant) of red cell aggregation, (5) an elevated a2 level (15% increase) and a significantly increased fibrinogen concentration (25% increase), both of which correlated with the enhanced red cell aggregation. Rheologic abnormalities were evident when patients with disease in either one vessel or two to three vessels were compared with controls, but differences between these subgroups of patients were not significant. We conclude that patients with angina have rheologic abnormalities that are compatible with disturbed blood flow and an enhanced tendency for coronary arterial thrombosis.