Spontaneous endocytosis in human neonatal and adult red blood cells: comparison to drug-induced endocytosis and to receptor-mediated endocytosis

Neonatal RBC contain many more spontaneous endocytic vacuoles than do adult RBC. It is not known if this difference is a result of an increase in production of vacuoles in the neonatal RBC (as is the case in drug-induced endocytosis), or is the result of a less effective neonatal macrophagic "pitting" process. Using an in vitro model of spontaneous endocytosis, we compared the rate and quantity of vacuoles and the shape of cord and adult RBC containing pits, visible by interference contrast microscopy (Nomarski method). The mechanism of the spontaneous endocytosis was explored using different inhibitors: sodium vanadate an inhibitor of ATPases, sodium fluoride which inhibits the generation of ATP and sodium cyanide a potent inhibitor of oxidative phosphorylation. We then compared spontaneous endocytosis with two other forms of RBC endocytosis: drug-induced endocytosis and receptor-mediated endocytosis. Spontaneous endocytosis is in fact increased in neonatal RBC initially but the increase in number of RBC containing pits after 144 hr of incubation is almost the same in adult RBC and neonatal RBC. Comparing spontaneous endocytosis with drug-induced endocytosis, it appears that their mechanisms are different in that spontaneous endocytosis is not preceded by stomatocytic shape change and is not inhibited by sodium vanadate or sodium fluoride as is the case for drug-induced endocytosis. Spontaneous endocytosis is different than transferrin receptor-mediated endocytosis because it occurs in many RBC, not only in the motile R1 reticulocytes and is not inhibited by sodium cyanide as is receptor-mediated endocytosis. Thus spontaneous endocytosis appears to be different than drug-induced endocytosis and transferrin receptor-mediated endocytosis. The increase in spontaneous endocytosis in cord RBC seen in vivo is probably a consequence of an immaturity of the neonatal macrophage pitting process.     

Directly observed reversible shape changes and hemoglobin stratification during centrifugation of human and Amphiuma red blood cells

This paper describes changes that occur in human and Amphiuma red blood cells observed during centrifugation with a special microscope. Dilute suspensions of cells were layered, in a centrifuge chamber, above an osmotically matched dense solution, containing Nycodenz, Ficoll, or Percoll (Pharmacia) that formed a density gradient that allowed the cells to slowly settle to an equilibrium position. Biconcave human red blood cells moved downward at low forces with minimum wobble. The cells oriented vertically when the force field was increased and Hb sedimented as the lower part of each cell became bulged and assumed a "bag-like" shape. The upper centripetal portion of the cell became thinner and remained biconcave. These changes occurred rapidly and were completely reversible upon lowering the centrifugal force. Bag-shaped cells, upon touching red cells in rouleau, immediately reverted to biconcave disks as they flipped onto a stack. Amphiuma red cells displayed a different type of reversible stratification and deformation at high force fields. Here the cells became stretched, with the nucleus now moving centrifugally, the Hb moving centripetally, and the bottom of the cells becoming thinner and clear. Nevertheless, the distribution of the marginal bands at the cells' rim was unchanged. We conclude that centrifugation, per se, while changing a red cell's shape and the distribution of its intracellular constituents, does so in a completely reversible manner. Centrifugation of red cells harboring altered or missing structural elements could provide information on shape determinants that are still unexplained.     

The behavior,as regards shape and volume,of human red cell ghosts in fresh and in stored blood

(1) When human red cells are hemolyzed in very hypotonic media (NaCl ofa tonicity of 0.167) and when the tonicity is restored, by adding appropriateamounts of NaCl, to tonicities such as 0.3, 0.5, 1.0, and 1.7, the mean volume ofthe ghosts appears to be linear with the reciprocal of the tonicity. This mightlead one to conclude that the ghosts are osmometers and that their volume isgoverned by simple osmotic considerations such as those expressed by amodified van’t Hoff-Mariotte law. Examination of the shapes and volumes of individual ghosts by a technic which combines phase optics, electronic flash (exposure time 0.001 second) and photography shows that three distinct populations of ghost coexist in any tonicity. These are spherical ghosts with a meanvolume of 150 µ³, discoidal biconcave ghosts with a mean volume of 85 µ³, andcrenated ghosts with a smaller volume which can be calculated. The most likelyreason for this complexity is that the shape and volume of the ghost dependspartly on its structure, and not altogether on the tonicity of the surroundingmedium. Simple osmotic laws have no real application to systems of this kind.

(2) The changes in ghost volume and shape, as they depend on the durationof storage of the blood, at 4 C, from which the ghosts are prepared, and as theyreflect changes in ghost structure, can be expressed simply. Crenated and discoidal ghosts certainly have some of the elements of red cell structure; thespherical ghost, which soon fragments and gives rise to myelin forms, may alsoretain some of the original elements of structure, but the fragment and themyelin form have certainly lost them. The latter objects are so small and lightthat they are not thrown down into the ghost column in the hematocrit tube,and so, as ghost structure disappears with increasing time of storage of the redcells from which they are prepared, a discrepancy appears between the volumeof the ghost column as measured by the hematocrit and the volume which onewould expect. This discrepancy can be used as a measure of the extent to whichghost structure is lost, and there comes a time, as the duration of red cell storageis increased, when the ghosts prepared from these red cells begin to be replacedby breakdown products such as myelin forms, etc.

(3) The less the efficiency of the conditions of red cell preservation, the shorteris this time. In human blood rendered incoagulable with heparin, structuralbreakdown in the preceding sense and measured by a simple expression whichchanges sign when the loss of structure has reached a certain point, occurs afterabout 26 days. In human blood preserved in ACD, structural breakdown measured in an identical manner occurs after about 55 to 60 days. In human bloodpreserved in ACD-inosine, structural breakdown does not occur until about 75to 80 days.

These results are based on a large amount of preliminary work of an exploratory nature and then on three runs with heparin, five runs with ACD and fiveruns with ACD-Inosine.

Submitted on January 30, 1957 Accepted on April 1, 1957     

Echinocyte-stomatocyte transformation and shape control of human red blood cells: morphological aspects

Red cell morphology was studied after the induction of echinocytic transformation by metabolic depletion, Ca2+ loading, and salicylate and stomatocytic transformation with chlorpromazine. The results indicate that the red cell has an energy-dependent shape control mechanism that allows it to counteract shape-changing stimuli such as metabolic depletion. Albumin was found to induce stomatocytic transformation, whereas gamma-globulins induced echinocytic transformation. Loading of the red cell with calcium resulted in polymorphous membrane damages such as submembranous, "blister-like" lesions, and membrane disintegration; the red cell age had no influence on this process. Conversely, the stomatocyte-echinocyte transformation induced by chlorpromazine and salicylate was shifted towards echinocytes in density-separated old red cells. Sphero-stomatocytes were capable of echinocytic transformation with spicule formation within the red cell vacuoles, whereas sphero-echinocytes were unable to undergo stomatocytic transformation without hemolysis. These observations may help to unravel the complexity of echinocyte-stomatocyte transformation of red blood cells.     

Respiratory changes in human red cells

To investigate physiological respiratory changes in human red cells, we measured automated red cell parameters in samples from the pulmonary and radial arteries of 86 patients undergoing aorto-coronary bypass and from the pulmonary artery and the aorta in 23 patients. Our results showed higher mean corpuscular volume (88.53 +/- 5.06 fl vs. 88.12 +/- 4.94 fl, P < 0.000001), haematocrit (0.369 +/- 0.039 vs. 0.367 +/- 0.038, P < 0.0005), red cell distribution width (43.38 +/- 4.16 vs. 43.04 +/- 4.05 fl, P < 0.000001) and a lower mean corpuscular haemoglobin concentration (338.3 +/- 15.9 vs. 339.9 +/- 16.8 g/l, P < 0.005) in pulmonary arterial as compared to radial arterial blood. There were no differences with respect to haemoglobin concentration, red blood cell count, or mean corpuscular haemoglobin. Similar differences were observed between pulmonary arterial and aortic blood. Our results suggest cyclic respiratory modifications of red cell parameters attributable to the CO2 Jacobs-Stewart cycle.     

Effects of age-dependent membrane transport changes on the homeostasis of senescent human red blood cells

Little is known about age-related changes in red blood cell (RBC) membrane transport and homeostasis. We investigated first whether the known large variation in plasma membrane Ca(2+) (PMCA) pump activity was correlated with RBC age. Glycated hemoglobin, Hb A1c, was used as a reliable age marker for normal RBCs. We found an inverse correlation between PMCA strength and Hb A1c content, indicating that PMCA activity declines monotonically with RBC age. The previously described subpopulation of high-Na(+), low-density RBCs had the highest Hb A1c levels, suggesting it represents a late homeostatic condition of senescent RBCs. Thus, the normal densification process of RBCs with age must undergo late reversal, requiring a membrane permeability increase with net NaCl gain exceeding KCl loss. Activation of a nonselective cation channel, Pcat, was considered the key link in this density reversal. Investigation of Pcat properties showed that its most powerful activator was increased intracellular Ca(2+). Pcat was comparably selective to Na(+), K(+), choline, and N-methyl-D-glucamine, indicating a fairly large, poorly selective cation permeability pathway. Based on these observations, a working hypothesis is proposed to explain the mechanism of progressive RBC densification with age and of the late reversal to a low-density condition with altered ionic gradients.     

Start-up shape dynamics of red blood cells in microcapillary flow

Red blood cell (RBC) deformability plays a key role in oxygen exchange between blood and tissues in microcirculation by allowing RBCs to flow in vessels of diameter even smaller than cell size. Hence, RBC flow in microcapillaries has been widely studied in vitro, mostly under steady-state conditions. Here, we provide the first quantitative investigation of the transient behavior of RBC shape in confined Poiseuille flow in vitro. Our approach is based on high-speed video microscopy imaging of RBCs flowing in silica microcapillaries and quantitative data processing by image analysis techniques. In start-up flow, RBCs undergo a complex transition from the biconcave shape to a parachute-like configuration through membrane folding and cytoplasm reorganization. The time scale of this transient process is independent on the applied pressure drop and the measured value for healthy cells (around 0.1 s) is in agreement with previous micropipette data from the literature. Glutaraldehyde (GA)-hardened RBCs exhibit a faster shape evolution at higher GA concentration, thus showing that the corresponding time scale becomes shorter at increasing cytoskeleton elasticity. Our results provide a novel microfluidics methodology to measure the RBC characteristic time which is a potential diagnostic parameter of altered cell deformability.     

Insulin effects on human red blood cells

The effect of insulin on human red blood cells was investigated, both on intact cells and on isolated plasma membranes, testing the responsiveness of membrane-bound enzymes--such as (Na+-K+)-ATPase and 5'-nucleotidase--as well as the ouabain binding and ionic fluxes. It appears that insulin stimulates Na-pumping mechanisms increasing (Na+-K+)-ATPase activity through an enhanced availability of pumping sites, as can be inferred from the increased ouabain binding. The apparent unresponsiveness of fluorescence polarization parameters, following insulin treatment of isolated plasma membranes and intact cells, rules out--at present--an involvement of membrane lipid fluidity in the mechanism of action of insulin on human erythrocytes.     

Studies on oral contraceptive-induced changes in blood coagulation and fibrinolysis and the estrogen effect on endothelial cells

Summary Blood coagulation (fibrinogen, thrombinantithrombin III complexes, TAT, and prothrombin fragment F₁₊₂) and fibrinolytic parameters [fibrin split-product D-dimer, tissue plasminogen activator (t-PA) activity, plasminogen activator inhibitor-1 activity (PAI-1), and plasmin-antiplasmin-complexes (PAP)] were evaluated in 16 women on low estrogen (EE) oral contraceptive (OC) therapy. Blood samples were taken before and between days 18 and 22 of the first, third, and sixth treatment cycle. Fibrinogen levels were found significantly elevated during OC treatment compared with pretreatment values, while TAT and also F₁₊₂ levels remained unchanged. Treatment-induced activation of fibrinolysis was documented by elevated D-dimer [pretreatment (pt): 172 ng/ml (range: 65–640 ng/ml), cycle 6 (c.6): 351 ng/ml (range: 93–960 ng/ ml),p<0.05)] and PAP [(pt: 46.6 ng/ml (13–220 ng/ml), c.6: 66.4 ng/ml (21–200 ng/ml),p<0.05] plasma levels. Among the fibrinolytic components a decrease in PAI-1 [pt: 10.8 ng/ml (2–56 ng/ml), c.6: 5.3 ng/ml (2.2–14.4 ng/ml),p<0.05] and an increase in t-PA activity [pt: 0.23 U/ml (0.17–0.45 U/ml), c.6: 0.33 U/ml (0.2–0.9 U/ml),p<0.05] were detected. Experiments with cultured human endothelial cells (EC) showed that EE influenced neither EC hemostatic regulatory activities (tissue factor, thrombomodulin) nor the secretion of the fibrinolytic components t-PA and PAI-1.     

Studies on the aggregation behaviour of pegylated human red blood cells with the Zeta sedimentation technique

Covalent binding of poly(ethylene glycol), abbreviated as PEG, to red blood cells (RBC) surface leads to masking of the RBC blood group determinants and the PEG layer on the cell surface sterically hinders RBC-RBC and RBC-plasma protein interactions. We cross-linked linear mPEG-SPA of various molecular mass (2000, 5000, 20000) to washed human RBC under varying incubation ratios polymer to RBC. The electrophoretic mobility (EM) of the modified RBC decreases with increasing of chain length and concentration of PEG up to 50%. It may reflect the alteration in the surface layer thickness and friction. The aggregation behaviour of the pegylated RBC was studied with the Zeta sedimentation technique modifying the cell-cell interactions pressing them toward each other under centrifugal forces of various magnitudes. As a rule at low centrifugation forces the increase in chain length and concentration of PEG linked to RBC surface reduces the dextran-induced aggregation probably via elevation of the steric repulsion, which counteracts the depletion force generated by the free polymer. This effect was reversed to some extent by elevation of free dextran concentration and centrifugation forces. If cell-cell polymer bridging starts playing a role under these conditions requires further experimental and theoretical investigations.     

The effect of red blood cells on thrombin generation

We have developed an assay of thrombin generation in clotting whole blood. Because our goal was to study patients with red blood cell diseases that are associated with thrombosis, the initial evaluation of this assay analysed the effect of the haematocrit and haemolysate on the total amount of thrombin activity generated and the maximal concentration of thrombin achieved. Both of these parameters were proportional to the haematocrit throughout a wide range of clinically relevant cell concentrations. Red cell lysate also augmented thrombin generation. Because this effect was removed by filtration of the lysate, we propose that it was related to red cell microparticles.     

Cobalt uptake and binding in human red blood cells

The basal uptake and cytoplasmic binding of cobalt was studied in human red cells using (57)Co as tracer. The basal uptake is linear with time, at a rate of about 10 μmol (l cells)(-1) h(-1) at 100 μM [Co(2+)](o), and is almost irreversible, as there is hardly any efflux into excess EDTA. Ionophore A23187 mediates a rapid equilibration of Co(2+) across the cell membrane leading to a marked accumulation, reflecting effective cytoplasmic buffering. The fraction (α(Co)) of total cell cobalt being present as free, ionized Co(2+) is estimated at α(Co)=0.01 from the equilibrium distribution of cobalt, and also from the initial slope of the cobalt buffering curve. The cobalt accumulation is similar in fed and ATP-depleted cells. The buffering curve for [Co(T)](c) can be fitted by a Michaelis type function with B(max)=24 mmol (l cells)(-1) and half-saturation at 240 μM [Co(2+)](c). The tracer influx curves are adequately fitted by single exponentials, whereas the net influx curves all require at least double exponential fits, probably due to non-stationary A23187 kinetics. The rate of tracer influx decreases with increasing cobalt concentration, and increases with delayed addition of (57)Co tracer during net uptake. This might be explained by an 'auto-inhibition' by cobalt. The kinetics for A23187-mediated net and tracer influx of (54)Mn is very similar to that of (57)Co, whereas the net influx of (65)Zn can be fitted by single exponentials. In cobalt-loaded cells the cobalt is partly reversibly bound, being releasable by excess extracellular EGTA in the presence of A23187, and partly tightly bound, remaining in the cells even at high ionophore concentrations. The tightly bound fraction builds up over time, and is larger and develops earlier in fed cells compared to ATP-depleted cells. However, all cell cobalt appears to exchange with (57)Co during tracer influx. It is speculated that oxidation of Co(2+) to Co(3+) could lead to the high affinity binding. Tight binding is not observed in the case of (54)Mn. Tightly bound and the major part of reversibly bound (57)Co co-migrate with hemoglobin in Sephadex column chromatography of a lysate of (57)Co-loaded cells. (57)Co also co-migrates with hemoglobin when added to a lysate of unlabeled cells or to a solution of purified hemoglobin, in both cases with a time-dependent development of tight binding. Cobalt is known to bind to the globin moiety of hemoglobin. The results imply that during long-term cobalt exposure in vivo cobalt will be taken up practically irreversibly in the red cells during their 120 days life span. Thus, for biomonitoring of cobalt exposure, it could be appropriate to measure the cobalt content in red cells to give, compared with timed or in-competition whole-blood and serum analysis, an average value for the exposure over the last couple of months.     

Extreme-value statistics of human red blood cells

Paper is devoted to answer the question: “How large is the largest red blood cell in a blood sample?” The red cell diameter was measured by photographing the cell in an interferometric microscope and processing the hologram by a numerical procedure of superresolution, which yields an improved resolution of 0.02 μm. Data on the largest cells in samples of specific size were collected and analyzed according to the theory of extreme-value statistics. It was found that the statistical distribution of the largest cells follows the asymptote derived for initial distribution of the exponential type. When the parameters of the asymptotic extreme distribution are determined from the observed data, the expected largest red cell in a sample of arbitrary size n can be easily determined as a function of n. For example, for the group of young adult male students examined the predicted largest diameter in a population of 10⁸ red blood cells lies between 15.66 and 17.06 μm.     

Abnormal permeability pathways in human red blood cells

A number of situations that result in abnormal permeability pathways in human red blood cells (RBCs) have been investigated. In sickle cell disease (SCD), RBCs contain HbS, rather than the normal HbA. When deoxygenated, an abnormal conductance pathway, termed P(sickle), is activated, which contributes to cell dehydration, largely through allowing Ca(2+) entry and subsequent activation of the Gardos channel. Whole-cell patch-clamp recordings from sickle RBCs show a deoxygenated-induced conductance, absent from normal RBCs, which shares some of the properties of P(sickle): equivalent Na(+) and K(+) permeability, significant Ca(2+) conductance, partial inhibition by DIDS and also Zn(2+). Gd(3+) markedly attenuates conductance in both normal and sickle RBCs. In addition, deoxygenated sickle cells, but not oxygenated ones or normal RBCs regardless of the oxygen tension, undergo haemolysis in isosmotic non-electrolyte solutions. Non-electrolyte entry was confirmed radioisotopically whilst haemolysis was inhibited by DIDS. These findings suggest that under certain circumstances P(sickle) may also be permeable to non-electrolytes. Finally, RBCs from certain patients with hereditary stomatocytosis have a mutated band 3, which appears able to act as a conductance pathway for univalent cations. These results extend our understanding of the abnormal permeability pathways of RBCs.