Erythrocyte aggregation: experimental approaches and clinical implications

From a clinical point of view, quantitative studies of erythrocyte aggregation or rouleau formation is of great interest. However, in order to approach the phenomenon, structural parameters (rouleau shape), kinetic parameters (rouleau formation and dissociation velocity) and rheological parameters (dissociation thresholds) must be determined. At present, optical dynamic methods are available for use in clinical hemorheology. They involve optical measurements in a viscometer. The first method consists of a study of the light intensity transmitted through the measuring chamber of a cone plan viscometer. A device calculates and index I, which provides a whole approach to the aggregation kinetics. On the other hand, a systemic which also involves a cone-plain viscometer and an inverted microscope (Rheoscope) allows to collect data by means of microphotographs, microcinematography or numerical image processing. In that case, an adimensional K index typical of the morphology of the aggregates can be determined. The second method involves the analysis of the light intensity backscattered by the red blood cells sheared in a Couette flow. An automatic system has been developed and aggregation times, orientation and structure index, total and partial disaggregation thresholds can be determined on the basis of the curves of variations of backscattered light.     

Aggregation and sedimentation of mixtures of erythrocytes with different properties

The rouleau formation of erythrocytes and the erythrocyte sedimentation were examined for mixture of different kinds of the cells suspended in isotonic phosphate-buffered saline containing 1 or 2 g/dl dextran T-70 (MW = 70,400) and 4 g/dl albumin, using a low shear rheoscope and the Westergren method, respectively. The deformability of cells was decreased by treating with diamide, diazene-dicarboxylic acid bis[N,N-dimethylamide], and the sialic acid content of cells, i.e., the surface negative charge, was reduced by treating with neuraminidase. (1) The rate of rouleau formation was decreased in cells with decreased deformability, while it was increased in cells with reduced sialic acid content. The rate changed in proportion to the ratio of the modified cells to normal cells. (2) The erythrocyte sedimentation was also decreased in cells with decreased deformability, while it was increased in cells with reduced sialic acid content. Furthermore, the erythrocyte sedimentation changed almost proportionally to the ratio of the modified cells to normal cells. (3) When normal deformable cells were mixed with cells with decreased deformability, the deformable cells seemed to settle faster than the less deformable cells, though the difference was not significant. (4) When normal cells were mixed with cells with reduced sialic acid content, the cells with less sialic acid settled significantly faster than those with more sialic acid. The present experiment may conclude that erythrocyte aggregation is induced preferentially among more deformable cells and/or among less negatively charged cells with weaker electrostatic repulsive force, and then the formed aggregates settle faster.     

Lecithin and the erythrocyte factor in the blood sedimentation phenomenon

1. The erythrocyte factor in the blood sedimentation phenomenon is notinfluenced by changes in size or shape of the erythrocytes unless the surface isaltered by a hemolytic agent.

2. The erythrocyte factor varies considerably in health and disease. It is depletedwhen blood is stored under refrigeration.

3. Egg lecithin emulsions and particles of cinnabar covered with lecithin areflocculated by gelatin solutions. The phenomenon is similar to the formation ofrouleaux in gelatin solutions.

4. An attempt to explain rouleau formation as a flocculation reaction betweenplasma proteins and the lecitho-protein surface of the erythrocyte is made.

     

In situ atomic force microscopy study of Alzheimer's beta-amyloid peptide on different substrates: new insights into mechanism of beta-sheet formation

We have applied in situ atomic force microscopy to directly observe the aggregation of Alzheimer's beta-amyloid peptide (Abeta) in contact with two model solid surfaces: hydrophilic mica and hydrophobic graphite. The time course of aggregation was followed by continuous imaging of surfaces remaining in contact with 10-500 microM solutions of Abeta in PBS (pH 7.4). Visualization of fragile nanoscale aggregates of Abeta was made possible by the application of a tapping mode of imaging, which minimizes the lateral forces between the probe tip and the sample. The size and the shape of Abeta aggregates, as well as the kinetics of their formation, exhibited pronounced dependence on the physicochemical nature of the surface. On hydrophilic mica, Abeta formed particulate, pseudomicellar aggregates, which at higher Abeta concentration had the tendency to form linear assemblies, reminiscent of protofibrillar species described recently in the literature. In contrast, on hydrophobic graphite Abeta formed uniform, elongated sheets. The dimensions of those sheets were consistent with the dimensions of beta-sheets with extended peptide chains perpendicular to the long axis of the aggregate. The sheets of Abeta were oriented along three directions at 120 degrees to each other, resembling the crystallographic symmetry of a graphite surface. Such substrate-templated self-assembly may be the distinguishing feature of beta-sheets in comparison with alpha-helices. These studies show that in situ atomic force microscopy enables direct assessment of amyloid aggregation in physiological fluids and suggest that Abeta fibril formation may be driven by interactions at the interface of aqueous solutions and hydrophobic substrates, as occurs in membranes and lipoprotein particles in vivo.     

Decreased hydrodynamic resistance in the two-phase flow of blood through small vertical tubes at low flow rates

The aggregation of red blood cells in blood flowing through small tubes at very low shear rates leads to the two-phase flow of an inner core of rouleaux surrounded by a cell-depleted peripheral layer. The formation of this layer is known to be accompanied by a decrease in hydrodynamic resistance to flow. To quantitate this effect, we measured the pressure gradient, flow rate, and the radius of the red blood cell core in suspensions flowing through tubes of 172-microns radius at mean linear flow rates (U) from 50 to 0.15 tube diameters.sec-1. Washed red blood cells were suspended in 1.5% buffered dextran 110 at hematocrits of 34-52%. Using syringe pumps, blood flowed from a stirred reservoir through a vertical 12-cm length of tube in either the upward or downward direction. The pressure drop was measured with transducers. Mean values in distributions in the core radius were obtained by analyzing cine films of flow taken through a microscope with flow in the upward direction, measuring the core radius at five equally spaced axial positions of the tube in each of 100 frames. At 34% and 46% hematocrit, the hydrodynamic resistance increased as U decreased from 50 sec-1, reaching a maximum at U-2 sec-1. It then decreased to a minimum at U less than 0.5 sec-1 as the red blood cell core formed in the tube, and the mean core radius/tube radius ratio decreased from 0.98 to 0.74 with marked axial fluctuations at the lower U. At higher hematocrits, both the increase and decrease in hydrodynamic resistance were greater. In a red blood cell albumin-saline suspension, where there is no aggregation of red blood cells and no two-phase flow, hydrodynamic resistance increases linearly with decreasing U. The experimental results were compared with the predictions of a two-phase steady-flow model, assuming axisymmetric flow of a core surrounded by cell-free suspending medium. Two models were considered, one in which the core is solid, the other in which the rheological properties of the suspension in the core are given by the Quemada equation. The effects of sedimentation of the core resulting in a zero net flow pressure gradient were taken into account. Provided that an experimentally extrapolated value for the zero pressure gradient was used, the Quemada-fluid model gave good agreement with the experimentally observed core radius as a function of U and hematocrit.     

Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network

A major activity of molecular chaperones is to prevent aggregation and refold misfolded proteins. However, when allowed to form, protein aggregates are refolded poorly by most chaperones. We show here that the sequential action of two Escherichia coli chaperone systems, ClpB and DnaK-DnaJ-GrpE, can efficiently solubilize excess amounts of protein aggregates and refold them into active proteins. Measurements of aggregate turbidity, Congo red, and 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid binding, and of the disaggregation/refolding kinetics by using a specific ClpB inhibitor, suggest a mechanism where (i) ClpB directly binds protein aggregates, ATP induces structural changes in ClpB, which (ii) increase hydrophobic exposure of the aggregates and (iii) allow DnaK-DnaJ-GrpE to bind and mediate dissociation and refolding of solubilized polypeptides into native proteins. This efficient mechanism, whereby chaperones can catalytically solubilize and refold a wide variety of large and stable protein aggregates, is a major addition to the molecular arsenal of the cell to cope with protein damage induced by stress or pathological states.     

Influence of Plasma Proteins on Erythrocyte Morphology and Sedimentation

The influence of plasma proteins on erythrocytes was studied by interference microscopy, scanning electron microscopy (SEM), and by Westergren erythrocyte sedimentation rate (ESR). Albumin kept erythrocytes dispersed as discoid spheres. Fibrinogen seemed responsible for the rouleaux phenomenon, but needed the co-influence of an immunoglobulin to induce rouleaux type of aggregates and high ESR. IgG, IgA and IgM caused immunologic type of aggregates. Albumin acted synergistically with fibrinogen and immunoglobulins. Normal blood contained a network of rouleaux, which probably explained the low normal ESR. High ESR was either due to rouleaux type aggregates where fibrinogen was dominant, or immunologic type aggregates where IgG, IgA or IgM were dominant proteins. Cold agglutinin disease showed normal blood morphology and normal ESR at 37°C and immunologic type aggregates and high ESR at 25°C.     

Red blood cell size is important for adherence of blood platelets to artery subendothelium

The hematocrit is one of the main factors influencing platelet adherence to the vessel wall. Raising the hematocrit causes an increase of platelet accumulation of about an order of magnitude. Our studies concern the role of red cell size. We have studied this effect using an annular perfusion chamber, according to Baumgartner, with human umbilical arteries and a steady-flow system. Normal human red blood cells (MCV 95 cu mu) increased platelet adherence sevenfold, as the hematocrit increases from 0 to 0.6. Small erythrocytes from goats (MCV 25 cu mu) caused no increment in adherence in the same hematocrit range. Rabbit erythrocytes (MCV 70 cu mu) caused an intermediate increase in adherence. Red blood cells from newborns (MCV 110-130 cu mu) caused a larger increase in platelet adherence than normal red cells at hematocrit 0.4. These results were further confirmed with large red blood cells from two patients. Experiments with small red cells (MCV 70 cu mu) of patients with iron deficiency showed that platelet adherence was similar to normal red cells, provided the red cell diameter was normal. Small red blood cells of a patient with sideroblastic anemia caused decreased adherence. These data indicate that red cell size is of major importance for platelet adherence. Red cell diameter is more important than average volume. However, for size differences in the human range, the hematocrit remains the dominant parameter.     

Morphological properties of atmospheric aerosol aggregates

Ultrafine particles (smaller than about 0.1 microm) are often emitted from combustion and other high-temperature processes in the form of fractal-like aggregates composed of solid nanoparticles. Results of a study of atmospheric aggregates are reported. Particles were collected on transmission electron microscope grids fitted on the last two stages of a single-jet eight-stage low-pressure impactor for periods of a few minutes. Photomicrographs of transmission electron microscope grids from the impactor stages were analyzed to obtain the fractal dimension (D(f)) and prefactor (A) for aggregates. D(f) increased from near 1 to above 2 as the number of primary particles making up the aggregates increased from 10 to 180. Total particle concentrations in size ranges roughly equivalent to the low-pressure impactor stages were measured with a mobility analyzer and condensation particle counter. In one set of measurements, the fraction of the particles present as aggregates was about 60% for particles with aerodynamic diameters between 50 and 75 nm and 34% for the range 75 to 120 nm. The total aggregate concentration in the 50- to 120-nm size range was about 400 ml(-1). The primary particles that make up atmospheric aggregates are more polydisperse than soot aggregates generated from a single laboratory source, an ethane/oxygen flame. Most measurements were made in the Los Angeles area, where the aggregates may represent a signature for diesel emissions. Rural aggregate concentrations in the size range 50 to 120 nm were less than 1% of the concentrations at urban sites. The data will permit better estimates of atmospheric aggregate residence times, transport, and deposition in the lung, optical extinction, and heterogeneous nucleation.     

A novel approach for assessments of erythrocyte sedimentation rate

Introduction: Previous studies have shown that the dispersed phase of sedimenting blood undergoes dramatic structural changes: Discrete red blood cell (RBC) aggregates formed shortly after a settling tube is filled with blood are combined into a continuous network followed by its collapse via the formation of plasma channels, and finally, the collapsed network is dispersed into individual fragments. Based on this scheme of structural transformation, a novel approach for assessments of erythrocyte sedimentation is suggested. Methods: Information about erythrocyte sedimentation is extracted from time records of the blood conductivity measured after a dispersion of RBC network into individual fragments. Results: It was found that the sedimentation velocity of RBC network fragments correlates positively with the intensity of attractive intercellular interactions, whereas no effect of hematocrit (Hct) was observed. Conclusion: Thus, unlike Westergren erythrocyte sedimentation rate, sedimentation data obtained by the proposed method do not require correction for Hct.     

Are there two functionally distinguished Neu5Gc pools with respect to rouleau formation on the bovine red blood cell?

Bovine red blood cells (RBCs) do not exhibit any aggregation tendency in autologous plasma and, therefore, all bovine rouleaux obtained in vitro are regarded as artificial. The present study reports the bovine RBC rouleau formation by either bovine or human fibrinogen and Ca2+ ions. The phenomenon was induced through two-step cell incubation: in 0.9% NaCl and 1% bovine albumin at 37 degrees C for 30 min followed by 20 hrs incubation at 30 degrees C in the fresh solution supplemented with 10 mM glucose. Its mechanism is unknown. During the incubation the number of N-glycolylneuraminic acid molecules per cell decreased from 48.1 to 44.9 amoles, which accounted for 7%. The treatment of RBCs with V. cholerae sialidase under the same conditions resulted in a 94% drop in the Neu5Gc quantity and did not induce the rouleau formation in the same fibrinogen preparation. The preliminary results rise a question whether the bulk of sialic acid is required in the aggregation of bovine erythrocytes under static conditions. Only a minor pool of Neu5Gc seems to be responsible for suppression of the phenomenon.     

Oxygen transport studies of normal and sickle red cell suspensions in artificial capillaries

Oxygen transport from normal and sickle red cells was studied under known and carefully controlled conditions simulating the microcirculation. Oxygenated red cell suspensions became deoxygenated as they traversed silicone rubber artificial capillaries of 27 microns diameter. Oxygen saturation values of the flowing red cell suspensions were measured at several axial positions along the artificial capillary by use of a microspectrophotometric technique. Oxygen saturation decreased with increasing distance from the entrance of the artificial capillary and was influenced strongly by the flow rate. Under the same hematocrit and flow conditions, the rate of oxygen saturation decrease was significantly higher for the sickle red cells than that for the normal red cells. Similar results were obtained by use of a mathematical simulation of oxygen transport in the microcirculation for both normal and sickle red cells. Sickle red cells would be expected to have a higher diffusional resistance to oxygen transport than would normal red cells. However, the higher diffusional resistance is more than offset by the lower oxygen affinity of the sickle cells. The difference in oxygen affinity appears to account for the difference in oxygen transport rates between normal and sickle red cells.     

Biochemical and aggregation analysis of Bence Jones proteins from different light chain diseases

Deposition of immunoglobulin light chains is a result of clonal proliferation of monoclonal plasma cells that secrete free immunoglobulin light chains, also called Bence Jones proteins (BJP). These BJP are present in circulation in large amounts and excreted in urine in various light chain diseases such as light chain amyloidosis (AL), light chain deposition disease (LCDD) and multiple myeloma (MM). BJP from patients with AL, LCDD and MM were purified from their urine and studies were performed to determine their secondary structure, thermodynamic stability and aggregate formation kinetics. Our results show that LCDD and MM proteins have the lowest free energy of folding while all proteins show similar melting temperatures. Incubation of the BJP at their melting temperature produced morphologically different aggregates: amyloid fibrils from the AL proteins, amorphous aggregates from the LCDD proteins and large spherical species from the MM proteins. The aggregates formed under in vitro conditions suggested that the various proteins derived from patients with different light chain diseases might follow different aggregation pathways.     

Designing supramolecular porphyrin arrays that self-organize into nanoscale optical and magnetic materials

Tessellation of nine free-base porphyrins into a 3 x 3 array is accomplished by the self-assembly of 21 molecular entities of four different kinds, one central, four corner, and four side porphyrins with 12 trans Pd(II) complexes, by specifically designed and targeted intermolecular interactions. Strikingly, the self-assembly of 30 components into a metalloporphyrin nonamer results from the addition of nine equivalents of a first-row transition metal to the above milieu. In this case each porphyrin in the nonameric array coordinates the same metal such as Mn(II), Ni(II), Co(II), or Zn(II). This feat is accomplished by taking advantage of the highly selective porphyrin complexation kinetics and thermodynamics for different metals. In a second, hierarchical self-assembly process, nonspecific intermolecular interactions can be exploited to form nanoscaled three-dimensional aggregates of the supramolecular porphyrin arrays. In solution, the size of the nanoscaled aggregate can be directed by fine-tuning the properties of the component macrocycles, by choice of metalloporphyrin, and the kinetics of the secondary self-assembly process. As precursors to device formation, nanoscale structures of the porphyrin arrays and aggregates of controlled size may be deposited on surfaces. Atomic force microscopy and scanning tunneling microscopy of these materials show that the choice of surface (gold, mica, glass, etc.) may be used to modulate the aggregate size and thus its photophysical properties. Once on the surface the materials are extremely robust.     

Biochemical and aggregation analysis of Bence Jones proteins from different light chain diseases

Deposition of immunoglobulin light chains is a result of clonal proliferation of monoclonal plasma cells that secrete free immunoglobulin light chains, also called Bence Jones proteins (BJP). These BJP are present in circulation in large amounts and excreted in urine in various light chain diseases such as light chain amyloidosis (AL), light chain deposition disease (LCDD) and multiple myeloma (MM). BJP from patients with AL, LCDD and MM were purified from their urine and studies were performed to determine their secondary structure, thermodynamic stability and aggregate formation kinetics. Our results show that LCDD and MM proteins have the lowest free energy of folding while all proteins show similar melting temperatures. Incubation of the BJP at their melting temperature produced morphologically different aggregates: amyloid fibrils from the AL proteins, amorphous aggregates from the LCDD proteins and large spherical species from the MM proteins. The aggregates formed under in vitro conditions suggested that the various proteins derived from patients with different light chain diseases might follow different aggregation pathways.     

Determination of aggregation force in rouleaux by fluid mechanical technique

The shear stress needed to disaggregate red cell rouleaux was quantified in a flow channel under microscopic observation. Measurement were made on two-cell rouleaux formed in 4g% dextran with a mean molecular weight of 74,500. Stepwise increases in shear stress caused a progressive peeling of the top cell of the rouleau, and the percentage of separation as estimated from the exposed area of the bottom cell showed a sigmoidal relation with shear stress. The 50% separation occurred at a shear stress of 0.25 ± 0.01 dyn/cm² (mean ± SEM), which is approximately one-half of the value obtained in rotational measurements on bulk suspensions where the orientation of red cells is more random. This technique of studying stress-disaggregation relationships in a flow channel has the advantages that (a) the applied shear stress is parallel to the rouleau interface and (b) the degree and process of disaggregation can be followed directly by microscopic observation.     

Cartilage proteoglycan alterations in an experimentally induced model of rabbit osteoarthritis.

Size distribution of cartilage proteoglycans (PG) extracted from control and osteoarthritic rabbit knees after partial meniscectomy was analyzed. In normal control knees, about 30% of PG molecules were in aggregate form and average sedimentation constant was 60S. No aggregates were found in osteoarthritic cartilage, whether ulcer, rim about ulcer, or distant normal-appearing cartilage was examined. Weight average sedimentation constants for PG subunits were similar to controls, 15S. Up to 70% of guanidinium-extractable PG could be extracted from osteoarthritic cartilage by using 0.5M guanidine HC1 (GuHCl); sedimentation characteristics of extracted PG were similar to those using 4.0M GuHCl. Absence of aggregates is consistent with a disorder of link protein, hyaluronic acid, or PG subunit hyaluronic acid binding sites. Absence of subunit degradation was an enexpected finding. The demonstrated ability of 0.5 M GuHCl to extract large amounts of PG from osteoarthritic cartilage will allow study of cartilage proteoglycans in their native nondissociated state.     

Macroaggregate Formation in Optimal Additive Red Cells

The formation of macroaggregates weighing up to 9 g was observed in optimal additive red cells. Such aggregates, with a mean wet weight of 3 g, formed progressively during storage and were present in up to 85% of units. They were composed of leucocyte and platelet debris, together with some fibrin. Macroaggregates formation was halved by less stringent centrifugation during preparation and was reduced by use of an optimal additive system in which the additive solution contained citrate. Extra mixing during transfer of the additive solution only delayed aggregate formation. Partial leucocyte depletion or addition of 200,000 KIU of the enzyme inhibitor aprotinin did not prevent macroaggregate formation.     

Evidence that folliculo-stellate cells do not impede the permeability of intercellular spaces to molecular diffusion in three-dimensional aggregate cell cultures of rat anterior pituitary

The permeability of intercellular spaces within the anterior pituitary (AP) and the influence of folliculo-stellate (FS) cells on compartmentalization within this tissue, has become a matter of debate. In reaggregated pituitary cell cultures as well as in the AP in situ the intercellular gaps and follicle-like structures remain accessible to molecular diffusion, whereas in some studies FS cells were reported to form tight epithelia that impede macromolecular transport through the spaces between the epithelial cells. In the present study the permeability of AP cell reaggregates was examined using fluorescent BSA as a tracer. Using confocal scanning laser microscopy a direct visualization of the permeation process was achieved. Quantitative estimation of the effective diffusion coefficient (Deff) for fluorescein-BSA within the aggregates was obtained using the fluorescence photobleaching recovery technique. Deff was 1.33 +/- 0.31 x 10(-7) cm2/sec (mean +/- SD) in aggregates from 14-day-old female rats and 2.45 +/- 0.55 x 10(-7) cm2/sec in aggregates from adult female rats. These values are about three times lower than in free solution. Calculation of the time-dependent concentration distribution inside the aggregate for a Deff = 2 x 10(-7) cm2/sec revealed that the concentration of the fluorescent tracer in the center of the aggregate reaches 90% of the concentration outside the aggregate after 0.5 min for aggregates with a radius of 50 microns and 6 min for aggregates with a radius of 150 microns. Aggregates enriched in FS cells, in which we previously showed a sustained inhibition of secretory responses to stimulatory and inhibitory agents as compared to total population aggregates, showed a diffusion coefficient (Deff = 1.85 +/- 0.77 x 10(-7) cm2/sec) which was not significantly different from that in the total population aggregates. The present study shows that AP cell aggregates are fully permeable to diffusing molecules within minutes and that in a three-dimensional tissue configuration FS cells, which were reported to tonically inhibit AP hormone release in response to various secretagogues, do not impede molecular diffusion to an extent which would account for sustained inhibition of hormone release.     

Effects of fibrinogen, globulin, albumin and hematocrit on the kinetics of erythrocyte aggregation in man

The effects of fibrinogen, globulin, albumin, and packed blood cell volume (hematocrit) on the kinetics of erythrocyte aggregation (RBC-A) after stasis were quantitatively evaluated by recording the changes of light transmission through the blood in transparent tubing with an apparatus consisting of a light-emitting diode and a silicon photodiode. The measurements were done on samples of heparinized whole blood, suspensions of erythrocytes containing various concentrations of fibrinogen (0.5 to 25 g/L) or gamma globulin (25 to 150 g/L), and blood of various hematocrit values prepared from three samples of whole blood having different concentrations of fibrinogen. The half-time of the increase of light transmission after the flow-stop was used as an index of the kinetics of RBC-A (T1/2). This simple and rapid method for estimating erythrocyte aggregation had excellent reproducibility (N = 51, r = 0.99). The values of T1/2 showed significant negative correlations with the concentrations of fibrinogen and globulin but a positive correlation with that of albumin. The T1/2 value decreased markedly and then remained almost constant with increase in the value of hematocrit, but the influence of hematocrit on T1/2 was markedly dependent on the concentration of fibrinogen.