Fibrinogen inhibits red cell adhesion to glass

Studies of human erythrocyte adhesion to glass have demonstrated consistently greater adhesion with serum-containing media than with a comparable concentration of plasma. This serum-plasma difference is explained by the adhesion-inhibiting property of plasma fibrinogen. The fibrinogen effect is probably mediated through its firm binding to glass, since no adsorption onto the red cell surface could be demonstrated. The ability of more red cells to adhere to a foreign surface after plasma coagulation (the formation of serum from plasma) may be significant in the red cell surface interactions necessary for the formation of a fibrin-red cell thrombus.     

Influence of flow velocity and cell concentration on dynamic adhesion of erythrocytes to glass surface

Comparative studies were carried out on dynamic adhesion of 51Cr-labelled erythrocytes to the surface of glass beads in the presence of serum in the medium (50 microng of protein/ml) and in protein-free medium. The influence of cell concentration (within the range 4 X 10(5) to 8 X 10(6)/ml) and of cellular flow velocity (within the range 1.5-0.4 cm/min) on the value of adhesion was investigated. It was found that when serum was present in the medium, the decisive influence on erythrocyte adhesion was exerted by the velocity with which the cells pass though the glass bead layer. Cell concentration under these conditions has only a very slight effect. When the medium does not contain serum, erythrocyte adhesion to the bead layer seems to depend on both cell concentration and flow velocity. Preliminary data were obtained concerning the release of 51Cr from the bead layer after erythrocyte adhesion.     

Locomotion and adhesion of polymorphonuclear leukocytes effects of the supporting substratum

Contact angle measurements have been used to correlate surface hydrophobicity of a supporting substratum with adhesion and locomotion of polymorphonuclear leukocytes. The binding of human serum albumin, a well-known chemokinetic substance, to hydrophilic glass slides gave rise to hydrophobic surfaces with adhesive properties conducive, to cell polarization thus allowing cell locomotion. Parallel contact angle and cell adhesion measurements suggested that albumin modified the cellsubstratum interaction by increasing the van der Waals forces of attraction and reducing the electrostatic forces. By allowing cells to adhere to a hydrophobic surface (siliconized glass), it was found that protein could be omitted from in vitro test systems for leukocyte locomotion. It is suggested that quantitatively equal cell adhesion values may, depending on the type of attraction forces working in adhesion to the substratum, result in different locomotion patterns.     

Locomotion and adhesion of polymorphonuclear leukocytes. Effects of the supporting substratum

Contact angle measurements have been used to correlate surface hydrophobicity of a supporting substratum with adhesion and locomotion of polymorphonuclear leukocytes. The binding of human serum albumin, a well-known chemokinetic substance, to hydrophilic glass slides gave rise to hydrophobic surfaces with adhesive properties conductive to cell polarization, thus allowing cell locomotion. Parallel contact angle and cell adhesion measurements suggested that albumin modified the cell-substratum interaction by increasing the van der Waals forces of attraction and reducing the electrostatic forces. By allowing cells to adhere to a hydrophobic surface (siliconized glass), it was found that protein could be omitted from in vitro test systems for leukocyte locomotion. It is suggested that quantitatively equal cell adhesion values may, depending on the type of attraction forces working in adhesion to the substratum, result in different locomotion patterns.     

A rapid and convenient preparation of [4-3H]NADP and stereospecifically tritiated NADP3H

The glass-binding properties of a number of purified glycoproteins capable of promoting attachment and spreading of a variety of types of animal cells in culture have been examined. Two such factors in human serum, fibronectin and serum spreading factor, exhibited strong affinities for glass beads and could be eluted from glass-bead columns under similar conditions. A number of other glycoproteins of human serum that do not promote cell adhesion did not bind to glass beads under conditions that resulted in binding of serum spreading factor or fibronectin. At a sufficiently low ratio of serum volume to glass-bead volume, human serum could be simultaneously depleted of serum spreading factor, fibronectin, and cell spreading-promoting activity by glass-bead affinity chromatography. Laminin, another cell spreading-promoting glycoprotein, possessed glass-binding properties similar to those of serum spreading factor and fibronectin while chondronectin, a fourth cell spreading-promoting factor of more limited specificity of biological activity and distribution in vivo, did not exhibit a strong interaction with glass beads under the same conditions. These observations suggest that glass-bead column affinity chromatography may prove useful as a general method for isolation and study of glycoprotein factors promoting attachment and spreading of cells in culture.     

Correlation between the oscillatory and adhesion activities in erythroid cells

The attachment kinetics of erythroid cells, such as human erythrocytes, their saponin ghosts, and erythroleukemic cells K562 to a glass surface has been studied in the presence of substances inhibiting spontaneous fluctuations of cell membranes. It has been shown that wheat germ agglutinin (WGA) slows down the attachment kinetics of K562 cells, as is the case in intact erythrocytes. Concanavalin A (Con A), which inhibits the attachment of erythrocytes to glass does not affect the adhesion of K562 cells to glass due to the absence of band 3 proteins in the membranes of K562 cells. Both lectins slow down the adhesion rate of saponin ghosts of human erythrocytes, as it takes place in intact erythrocytes. Suramin and the anionic dye ANS bind specifically to the actin protofilaments of the erythrocyte skeleton and also inhibit cell adhesion to glass. At the same time, these substances do not affect the oscillatory and adhesion activities of intact erythrocytes due to the impermeability of erythrocyte membranes for these drugs. The results obtained allow the conclusion that inhibition of erythrocyte adhesion by lectins is due to lectin binding to different constituents of the erythrocyte membrane--sialic acid moieties of glycophorin in the case of WGA and band 3 proteins in the case of Con A. The most probable mechanism of erythrocyte and K562 cell attachment to glass is the formation of the so-called local contacts between cells and the glass surface. It is also suggested that the cell surface oscillations facilitate the formation of cell contacts.     

The suppression of rapid local surface oscillations in human erythrocytes slows and weakens their adhesion to glass

Rapid local oscillations of the erythrocyte surface with amplitude 200-300 nm are decreased by 10 times following addition of wheat germ agglutinin (10(-77) M). In this case the rate of erythrocyte adhesion to the cover glass is delayed approximately by 3-9 times. The total suppression of erythrocyte surface oscillations occurs in hypo-osmic solution or in a 0.01% solution of glutaraldehyde. It coincides with a two-fold decrease of erythrocyte adhesivity to the glass. It is suggested that the rapid erythrocyte surface oscillations may control the rate of cell adhesion to the substrate.     

A hypothesis of the disc-sphere transformation of the erythrocytes between glass surfaces and of related observations

Erythrocytes suspended at a low hematocrit in a non-buffered isotonic saline change from biconcave discs to spheres between glass surfaces of a slide and of a coverslip with the echinocyte as an intermediate. A pH increase is a major factor responsible for this disc-sphere transformation or glass effect. It is also observed between surfaces made of various polymers and of mica provided that the distance between them is controlled (0.1 mm). The glass effect is antagonized by serum, plasma, serum albumin, ammonium salts and CO2. It is not observed above a 1-2% hematocrit, but is enhanced by gamma-globulins. The sites of reappearance of the spicules are the same and the order of their disappearance is the inverse of the order of their reappearance during the repetitive cycle of the disc-sphere transformation and reversal when a small glass rod is alternatively approached near a site on the erythrocyte surface and withdrawn. A mechanism of erythrocyte shape control has been previously hypothesized in which Band 3 (AE1), the anion exchange protein, plays a central role. Specifically, decrease and increase of the ratio of its outward-facing conformation (Band 3o) and inward-facing conformation (Band 3i) contract and relax the membrane skeleton, promoting the echinocytosis and stomatocytosis, respectively. The Band 3o/Band 3i equilibrium ratio is determined by the Donnan equilibrium ratio of Cl-, HCO3- and H+ (r=Cl(i)-/Cl(o)-=HCO3i-/HCO3o-=Ho+/Hi+), increasing with it. The mechanism could explain by a change of the Donnan ratio the above observations with the assumptions that polymers are permeable to CO2 and that an unstirred layer slows the propagation of the change occurring at the site of approach of the glass rod to peripheral sites. The presence of HCO3- in serum or plasma may be the basis for the absence of the glass effect in these fluids.     

Non-adsorbing macromolecules promote endothelial adhesion of erythrocytes with reduced sialic acids

Background

Abnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium.

Methods

RBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500 kDa dextran with a flow chamber assay.

Results

Our results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction.

Conclusion

These results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids.

General significance

Our findings should therefore be of great value in understanding abnormal RBC–EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions.     

Deciphering the export pathway of malaria surface proteins

The intra-erythrocytic stages of Plasmodium falciparum assemble a unique protein trafficking system that targets parasite proteins to the red cell cytoplasm and cell surface. It is through this trafficking pathway that the primary virulence determinants of P. falciparum infections are targeted to the erythrocyte surface to mediate adhesion to host endothelial cells. A recent study has shown that SBP-1, a parasite protein associated with Maurer's clefts in the infected red cell cytosol, is essential for transport of the virulence factor PfEMP-1. This discovery sheds new light on the little-understood mechanisms that regulate protein trafficking in infected cells.     

Stiffness of normal and pathological erythrocytes studied by means of atomic force microscopy

During recent years, atomic force microscopy has become a powerful technique for studying the mechanical properties (such as stiffness, viscoelasticity, hardness and adhesion) of various biological materials. The unique combination of high-resolution imaging and operation in physiological environment made it useful in investigations of cell properties. In this work, the microscope was applied to measure the stiffness of human red blood cells (erythrocytes). Erythrocytes were attached to the poly-L-lysine-coated glass surface by fixation using 0.5% glutaraldehyde for 1 min. Different erythrocyte samples were studied: erythrocytes from patients with hemolytic anemias such as hereditary spherocytosis and glucose-6-phosphate-dehydrogenase deficiency patients with thalassemia, and patients with anisocytosis of various causes. The determined Young's modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that the Young's modulus of pathological erythrocytes was higher than in normal cells. Observed differences indicate possible changes in the organization of cell cytoskeleton associated with various diseases.     

Use of cell contour analysis to evaluate the affinity between macrophages and glutaraldehyde-treated erythrocytes.

Recently, several authors evaluated the affinity between lipid bilayers or erythrocyte membranes by analyzing the deformation of cells or vesicles they brought into close contact using micromanipulators. In the present report, we extend this approach in a study of the adhesive properties of rough nucleated cells. Rat peritoneal macrophages were made to bind human red cells modified with glutaraldehyde or glutaraldehyde and polylysine. Conjugates were examined with electron microscopy, and photomicrographs were digitized for quantification of cell surface roughness in and out of adhesion areas. Also, macrophages were subjected to micropipette aspiration to find a relationship between apparent surface tension and area increase. Assuming that this increase was a direct consequence of a smoothing of the cell surface on the submicrometer scale, the actual affinity between macrophages and erythrocytes was estimated. The obtained values ranged between 8.4 X 10(-5) and 18.2 X 10(-5) J/m2. It is concluded that cell surface roughness may be an important parameter of cell adhesion and perhaps deformation. This is made amenable to experimental study by the present approach.     

Plasmodium iophurae: cationized ferritin staining, an electron microscope cytochemical method for differentiating malarial parasite and host cell membranes

The surface membranes of erythrocyte-free Plasmodium lophurae and its host cell, the duckling erythrocyte, stain differentially when exposed to cationized ferritin (CF). At low CF concentrations (0.18 mg/ml) only the outer surface of the red cell stains, whereas at the standard concentration (0.7 mg/ml) both the red cell and the parasitophorous vacuolar membranes (PVM) were stained on their outer faces. By using a high CF concentration (3.7 mg/ml), the parasite's plasma membrane (PM) could be distinguished from that of the PVM: The former did not bind CF, whereas the latter was stained on its outer surface. At this level of CF the red cell membrane stained on both faces if these surfaces were exposed to stain. Although the PVM is formed by red cell endocytosis of the parasite, it can be distinguished from the membrane of the erythrocyte as well as that of the PM.     

Influence of collagen gel on the orientation of epithelial cell polarity: follicle formation from isolated thyroid cells and from preformed monolayers

The influence of collagen gels on the orientation of the polarity of epithelial thyroid cells in culture was studied under four different conditions. (a) Isolated cells cultured on the surface of a collagen gel formed a monolayer. The apical pole was in contact with the culture medium and the basal membrane was attached to the substratum. (b) Isolated cells embedded inside the gel organized within 8 into follicles. The basal pole was in contact with collagen and the apical pole was oriented towards the interior of the follicular lumen. (c) Cells were first organized into floating vesicles, structures in which the apical surface is in contact with the culture medium, and the vesicles were embedded inside the collagen gel. After 3 d, cell polarity was inverted, the apical pole being oriented towards the cavity encompassed by cells. Vesicles had been transformed into follicles. (d) Monolayers formed on collagen gels as in a were overlaid with a second layer of collagen, which was polymerized in contact with the apical cell surface. A disorganization of the continuous pavement occurred within 24 h; cells attached to the upper layer of collagen and reorganized into follicles in the collagen sandwich within 4-8 d. A similar process occurred when the monolayer was grown on plastic and overlaid with collagen, or grown on collagen and covered with small pieces of glass cover slips. No reorganization was observed between two glass surfaces. In conclusion, first, a basal pole was always formed in the area of contact between the cell membrane and an adhesive surface and, second, the interaction of a preformed apical pole with an adhesive surface was not compatible with the stability of this domain of the plasma membrane. The interaction of the cell membrane with extracellular components having adhesive properties appears to be a determinant factor in the orientation and stabilization of epithelial cell polarity.     

Mapping the binding domains involved in the interaction between the Plasmodium falciparum knob-associated histidine-rich protein (KAHRP) and the cytoadherence ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1).

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) clusters at electron-dense knob-like structures on the surface of malaria-infected red blood cells and mediates their adhesion to the vascular endothelium. In parasites lacking knobs, vascular adhesion is less efficient, and infected red cells are not able to immobilize successfully under hemodynamic flow conditions even though PfEMP1 is still present on the exterior of the infected red cell. We examined the interaction between the knob-associated histidine-rich protein (KAHRP), the parasite protein upon which knob formation is dependent, and PfEMP1, and we show evidence of a direct interaction between KAHRP and the cytoplasmic region of PfEMP1 (VARC). We have identified three fragments of KAHRP which bind VARC. Two of these KAHRP fragments (K1A and K2A) interact with VARC with binding affinities (K(D(kin))) of 1 x 10(-7) M and 3.3 x 10(-6) M respectively, values comparable to those reported previously for protein-protein interactions in normal and infected red cells. Further experiments localized the high affinity binding regions of KAHRP to the 63-residue histidine-rich and 70-residue 5' repeats. Deletion of these two regions from the KAHRP fragments abolished their ability to bind to VARC. Identification of the critical domains involved in interaction between KAHRP and PfEMP1 may aid development of new therapies to prevent serious complications of P. falciparum malaria.     

Rheological approach to the analysis of blood coagulation in endothelial cell-coated tubes: Activation of the intrinsic reaction on the erythrocyte surface

Coagulation of blood in cultured endothelial cell-coated tubes was examined using a theological technique. Coagulation of recalcified, platelet-free plasma in contact with an endothelial cell monolayer did not occur within the experimental time period (more than 150 min). The endothelial cell surface did not activate the intrinsic coagulation reaction or the extrinsic coagulation reaction initiated by tissue factor. The time of onset of coagulation in platelet-free plasma supplemented with erythrocytes was nearly the same as that of whole blood (31.2 ± 5.5 min), which was shorter than that for platelet-rich plasma (54.3 ± 14.3 min) and platelet-free plasma supplemented with granulocytes (58.3 ± 6.3 min). In factor VII-, XI- or XII- deficient, platelet-free plasma supplemented with erythrocytes, the time of onset of coagulation was about 30 min. The coagulation of factor IX-deficient, platelet-free plasma supplemented with erythrocytes, however, did not occur within the experimental time period. These data suggest that activation of factor IX on the erythrocyte surface is capable of activating the intrinsic coagulation system.     

Affinity of red blood cell membrane for particle surfaces measured by the extent of particle encapsulation.

An experimental technique and a simple analysis are presented that can be used to quantitate the affinity of red blood cell membrane for surfaces of small beads or microsomal particles up to 3 micrometers Diam. The technique is demonstrated with an example of dextran-mediated adhesion of small spherical red cell fragments to normal red blood cells. Cells and particles are positioned for contact by manipulation with glass micropipets. The mechanical equilibrium of the adhesive contact is represented by the variational expression that the decrease in interfacial free energy due to a virtual increase in contact area is balanced by the increase in elastic energy of the membrane due to virtual deformation. The surface affinity is the reduction in free energy per unit area of the interface associated with the formation of adhesive contact. From numerical computations of equilibrium configurations, the surface affinity is derived as a function of the fractional extent of particle encapsulation. The range of surface affinities for which the results are applicable is increased over previous techniques to several times the value of the elastic shear modulus. It is shown that bending rigidity of the membrane has little effect on the analytical results for particles 1--3 micrometers Diam and that results are essentially the same for both cup- and disk-shaped red cells. A simple analytical model is shown to give a good approximation for surface affinity (normalized by the elastic shear modulus) as a function of the fractional extent of particle encapsulation. The model predicts that a particle would be almost completely vacuolized for surface affinities greater than or equal to 10 times the elastic shear modulus. Based on an elastic shear modulus of 6.6 x 10(-3) dyn/cm, the range for the red cell-particle surface affinity as measured by this technique is from approximately 7 x 10(-4) to 7 x 10(-2) erg/cm2. Also, an approximate relation is derived for the level of surface affinity necessary to produce particle vacuolization by a phospholipid bilayer surface which possesses bending rigidity and a fixed tension.     

Surface molecules on HaCaT keratinocytes after interaction with non‐thermal atmospheric pressure plasma

Non‐thermal atmospheric‐pressure plasmas have been developed that will be used in future for several purposes, e.g. medicine. Living tissues and cells are at the focus of plasma treatment, e.g. to improve wound healing, or induce apoptosis and growth arrest in tumour cells. Detailed investigations of plasma‐cell interactions are needed. Cell surface adhesion molecules as integrins, cadherins or the EGFR (epidermal growth factor receptor) are of importance in wound healing and also for development of cancer metastasis. This study has focused on measurement of cell surface molecules on human HaCaT keratinocytes (human adult low calcium temperature keratinocytes) promoting adhesion, migration and proliferation as one important feature of plasma‐cell interactions. HaCaT keratinocytes were treated with plasma by a surface dielectric barrier discharge in air. Cell surface molecules and induction of intracellular ROS (reactive oxygen species) were analysed by flow cytometry 24 h after plasma treatment. Besides a reduction of cell viability a significant down‐regulation of E‐cadherin and the EGFR expression occurred. The influence on α₂‐ and β₁‐integrins was less pronounced, and expression of ICAM‐1 (intercellular adhesion molecule 1) was unaffected. The extent of effects depended on the exposure time of cells to the plasma and the treatment regimen. Intracellular level of ROS detected by the fluorescent dye H₂DCFDA (2′,7′‐dichlorodihydrofluorescein diacetate) increased by plasma treatment, but it was neither dependent on the treatment time nor related to the different treatment regimens. Two‐dimensional cultures of HaCaT keratinocytes appear to be a suitable method of investigating plasma‐cell interactions.     

Characteristics of new Staphylococcus aureus-RBC adhesion mechanism independent of fibrinogen and IgG under hydrodynamic shear conditions

Staphylococcus aureus infection begins when bacterial cells circulating in blood adhere to components of the extracellular matrix or endothelial cells of the host and initiate colonization. S. aureus is known to exhibit extensive interactions with platelets. S. aureus is also known to bind to red blood cells (RBCs) in the presence of plasma proteins, such as fibrinogen and IgG. Herein we report a new binding mechanism of S. aureus to RBC independent of those plasma proteins. To characterize the new adhesion mechanism, we experimentally examine the binding kinetics and molecular constituents mediating the new adhesive interactions between S. aureus and RBCs under defined shear conditions. The results demonstrate that the receptors for fibrinogen (clumping factor A) and IgG (protein A) of S. aureus are not involved in the adhesion. S. aureus binds to RBCs with maximal adhesion at the shear rate 100 s^–1 and decreasing adhesion with increasing shear. The heteroaggregates formed after shear are stable when subjected to the shear rate 2,000 s^–1, indicating that intercellular contact time rather than shear forces controls the adhesion at high shear. S. aureus binding to RBC requires plasma, and 10% plasma is sufficient for maximal adhesion. Plasma proteins involved in the cell-cell adhesion, such as fibrinogen, fibronectin, von Willebrand factor, IgG, thrombospondin, laminin, and vitronectin are not involved in the observed adhesion. The extent of heteroaggregation is dramatically reduced on RBC treatment with trypsin, chymotrypsin, or neuraminidase, suggesting that the receptor(s) mediating the heteroaggregation process is a sialylated glycoprotein on RBC surface. Adhesion is divalent cation dependent and also blocked by heparin. This work demonstrates a new mechanism of S. aureus-RBC binding under hydrodynamic shear conditions via unknown RBC sialoglycoprotein(s). The binding requires plasma protein(s) other than fibrinogen or IgG and does not involve the S. aureus adhesins clumping factor A or protein A. adhesion; red blood cell     

Serum lipoproteins promote efficient presentation of the malaria virulence protein PfEMP1 at the erythrocyte surface

The virulence of the malaria parasite Plasmodium falciparum is related to its ability to express a family of adhesive proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) at the infected red blood cell surface. The mechanism for the transport and delivery of these adhesins to the erythrocyte membrane is only poorly understood. In this work, we have used specific immune reagents in a flow cytometric assay to monitor the effects of serum components on the surface presentation of PfEMP1. We show that efficient presentation of the A4 and VAR2CSA variants of PfEMP1 is dependent on the presence of serum in the bathing medium during parasite maturation. Lipid-loaded albumin supports parasite growth but allows much less efficient presentation of PfEMP1 at the red blood cell surface. Analysis of the serum components reveals that lipoproteins, especially those of the low-density lipoprotein fraction, promote PfEMP1 presentation. Cytoadhesion of infected erythrocytes to the host cell receptors CD36 and ICAM-1 is also decreased in infected erythrocytes cultured in the absence of serum. The defect appears to be in the transfer of PfEMP1 from parasite-derived structures known as the Maurer's clefts to the erythrocyte membrane or in surface conformation rather than a down-regulation or switching of particular PfEMP1 variants.