1 Aggregation of Red Blood Cells: An Electrochemical and Colloid Chemical Problem Downloaded by 203.64.11.45 on November 10, 2016 | http://pubs.acs.org Publication Date: June 1, 1980 | doi: 10.1021/ba-1980-0188.ch001
SHU CHIEN Division of Circulatory Physiology and Biophysics, Department of Physiology, Columbia University, College of Physicians and Surgeons, New York, N Y 10032
The aggregation of red blood cells by macromolecular bridging involves energy balance at the cell surface. The energy of macromolecular binding to the cell membrane provides the primary aggregating energy. The disaggregating energies result from electrostatic repulsion between the sialic acids on cell surfaces and mechanical shearing stress. A portion of the net aggregation energy (aggregating energy minus disaggregating energies) is stored in the cell membrane as a change in strain energy. Experimental manipulations of these various forms of energies have provided evidence supporting the concept of energy balance at cell surfaces. The results indicate that an understanding of red cell aggregation requires an interdisciplinary approach by which principles in electrochemistry and colloid chemistry are applied to this biological system.
/ ^ e l l aggregation is an important phenomenon i n biology and medicine. ^ T h e aggregation of like cells gives rise to the structural basis of tissues and organs. The aggregation process involves interactions between membranes of adjacent cells. Since biological cell surfaces possess charged groups and the body fluids are composed of electrolyte solutions containing macromolecules, cell interactions entail electrochemical and colloid chemical factors. A n understanding of the role of these factors i n controlling cell aggregation would help to elucidate the physicochemi0-8412-0473-X/80/33-188-003$09.00/l © 1980 American Chemical Society
Blank; Bioelectrochemistry: Ions, Surfaces, Membranes Advances in Chemistry; American Chemical Society: Washington, DC, 1980.
Downloaded by 203.64.11.45 on November 10, 2016 | http://pubs.acs.org Publication Date: June 1, 1980 | doi: 10.1021/ba-1980-0188.ch001
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BIOELECTROCHEMISTRY: IONS, SURFACES, M E M B R A N E S
cal principles underlying many important physiological and pathological processes. These include the development, growth, and differentiation of cells, the functional behaviors of cells including secretion, contraction, and signal transmission, and the growth and metastasis of tumor cells. Among the various types of cells, the red blood cells ( R B C s ) are most easily obtainable and amenable to experimental manipulations, and the electrochemical properties of the R B C membrane are most clearly understood. Therefore the aggregation of R B C s provides an excellent model for studying cell-to-cell interactions in general. Of course, applying the knowledge gained from studies of R B C aggregation to other cell systems necessitates appropriate modifications by taking into account the structural and functional differences among different cell types. The whole blood of normal human subjects consists of a concentrated suspension of R B C s (approximately 40-45% by volume, w i t h white blood cells ( W B C s ) and platelets contributing only less than 1%) i n plasma (containing 7 g / d L proteins, i.e. albumin, globulins, and fibrinogen). Under normal conditions, the blood flowing through most parts of the circulatory system is subject to sufficiently high shear stresses to disperse the R B C s during flow. In regions of flow stagnation or when blood is examined in vitro in a stationary condition, extensive R B C aggregation occurs to form rouleaux. This R B C aggregation necessitates the presence of appropriate macromolecules in the suspending medium, such as fibrinogen and some globulin fractions present in the normal blood plasma or other polymers (e.g. dextrans). In the absence of such macromolecules, R B C s do not aggregate even i n the absence of flow. The physicochemical properties and the concentration of the macromolecules play a significant role i n affecting R B C aggregation, suggesting that R B C aggregation results from an interaction of the macromolecules with the R B C membrane surface ( J ) . O u r current model is that the energy which causes R B C aggregation results from the bridging of the surfaces of two adjacent cells by the adsorbed macromolecule (2). In order to attain a stable aggregation, the aggregating energy owing to macromolecular bridging ( E ) must overcome the disaggregating energies (3). Thus, the electrostatic repulsive energy ( E ) between two negatively charged cell surfaces brought into close range would cause disaggregation. As mentioned above, mechanical shearing also causes R B C disaggregation by supplying dispersing stress. R B C aggregation leads to an alteration i n the shape of the R B C s , and such cell deformation involves a change in membrane strain energy (E ) supplied by the aggregation energy. b
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The purpose of this chapter is to review the influences of electrochemical and colloid chemical factors on R B C aggregation from the viewpoint of the balance of these aggregating and disaggregating energies
Blank; Bioelectrochemistry: Ions, Surfaces, Membranes Advances in Chemistry; American Chemical Society: Washington, DC, 1980.
1.
CHTEN
Aggregation of Red Blood Cells Table I.
Dextran Fractions Dx 20 Dx 40 D x 70 or 80 D x 150 D x 500 Dx2000
b
5
Molecular Weight of Dextrans M
M /M '
w
20 42 75 141 450 2000
X X X X X X
w
10 10 10 10 10 10
3 3 3 3 3 3
n
1.24 1.63 1 54 1.64 2.54 ND°
° The ratio of M « to M» gives an indication of the dispersion of molecular weight distribution. M « / K T = 1 for monodispersion. Dx 70 and Dx 80 have been used interchangeably in this chapter for the same dextran fraction with a KT« of 75,000. Not determined. N
b
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at the cell surface. Most of the studies to be presented involve R B C aggregation by dextrans, which are neutral polymers of glucose synthesized by bacterial action (4). The availability of various dextran fractions with controlled molecular size (see Table I) renders them particularly suited for investigations on R B C aggregation. Aggregation of Normal RBCs in Dextrans Quantitative Relationship Between RBC Aggregation and the Molecular Weight and Concentration of Dextrans. The aggregation of normal R B C s i n suspending media containing dextrans w i t h various molecular weights and concentrations has been quantified by microscopic counting (5), determination of erythrocyte sedimentation rate ( 6 , 7 , 8 ) , viscometry at low shear rates (8,9) and measurements by optical methods (10,11,12). In Figure 1 the various aggregation indices are plotted against the logarithm of the molar concentration of dextrans (3). N o aggregation occurs with D x 20. R B C s can be aggregated by D x 40, but only to a slight degree; furthermore, it occurs only in a narrow range of D x 40 concentrations. W h e n higher-molecular-weight dextrans are used, the degree of cell aggregation becomes more prominent. The molar concentration of dextrans required to induce aggregation varies inversely with the molecular size (see Figure 1). For each dextran molecular size, R B C aggregation and dextran concentration have a bell-shaped relationship, i.e., w i t h progressive increases in dextran concentration, there is first an aggregation phase to reach a peak aggregation and then a disaggregation phase at high dextran concentrations. The cell aggregation curves in different dextran fractions resemble one another more closely when plotted against the weight concentration of dextrans (e.g. i n grams per deciliter). F o r all fractions studied the maximum aggregation occurs at a dextran concentration of approximately 4 g / d L (see Figure 2). As
Blank; Bioelectrochemistry: Ions, Surfaces, Membranes Advances in Chemistry; American Chemical Society: Washington, DC, 1980.
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BIOELECTROCHEMISTRY:
Downloaded by 203.64.11.45 on November 10, 2016 | http://pubs.acs.org Publication Date: June 1, 1980 | doi: 10.1021/ba-1980-0188.ch001
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DEXTRAN
IONS, SURFACES, M E M B R A N E S
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Figure 1. Effects of the concentration of dextrans with various molecular weights on three indices of RBC aggregation (16). MAI indicates the average number of RBCs in each aggregation unit counted under the microscope. ESR is the maximum rate of sedimentation of erythrocytes in a calibrated tube, with corrections made for changes in viscosity and density of the suspending medium following the addition of dextrans. The relative viscosity (rj ) is the ratio of the viscosity of RBC suspension to that of the suspending medium at a shear rate fo 0.1 sec' . The RBC concentration of the suspension was 1% for MAI and 45% for ESR and rj measurements. The vertical bars represent SEM. (A), Dx 40; (O), Dx 80; (M), Dx 150; (A), Dx 500; (%), Dx 2000. r
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Blank; Bioelectrochemistry: Ions, Surfaces, Membranes Advances in Chemistry; American Chemical Society: Washington, DC, 1980.
1.
CHTJEN
Aggregation of Red Blood Cells
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