Adhesion of human erythrocytes to glass: The nature of the interaction and the effect of serum and plasma

Studies of normal human erythrocyte adhesiveness to glass have demonstrated quantitative and qualitative differences between cell-glass and cell-cell contact interaction. Identifiable charged groups on the red cell surface were of minor importance in the adhesive process and it is postulated that direct cell-glass contact attraction involves nonpolar regions of the red cell surface. However, erythrocyte adhesion to glass in the presence of serum and plasma is affected by electrostatic forces. At least two critical factors are present in serum and plasma: a heat-stable factor(s) diminishing adhesion and a heat-labile factor(s) promoting cell-glass interaction. A postulate is presented concerning the role of soluble fibrin monomer complexes in promoting cell adhesion to glass.     

Platelet deposition onto fibrin-coated surfaces under flow conditions

Platelet deposition on fibrin-coated surfaces and release from these adherent platelets were studied in an in vitro flow system. When a mixed suspension of washed platelets and red cells flowed through a fibrin-coated glass tube, only platelets were deposited onto the fibrin-coated surfaces. The density of adhered platelets increased with flow time and decreased with distance from the tube inlet. The adhesion rate increased with increasing shear rates from 45 s-1 to 180 s-1. This adhesion process appears to fit a diffusion-limited mathematical model. Comparing with glass and other protein-coated surfaces such as collagen, fibrinogen, or albumin coated surfaces, the number of adhered platelet per unit area decreased in the following descending order: collagen, fibrin, fibrinogen, glass, albumin. On the other hand, the degree of release reaction from these platelets decreased by another order: collagen, glass, fibrinogen, fibrin. We observed little release from platelets that were in contact with a fibrin-coated surface. Our results suggest that platelets specifically adhere to fibrin-coated surface and that this interaction does not induce platelet release.     

Rheological behaviors of bovine blood forming artificial rouleaux

A purpose of the present study is to make an artificial rouleau of bovine red blood cells which is not capable of rouleau formation under physiological condition. Rheological behaviors of bovine blood forming artificial rouleaux were examined. The modification of cell surface by enzyme trypsin induced rouleau formation, whereas the modification of cell surface by neuraminidase did not cause any aggregate formation. The drastic elevation of the fibrinogen content in bovine red blood cells suspension also brought about the formation of rouleau. The value of dynamic rigidity modulus G' of bovine red blood cells in saline solution containing high concentration of fibrinogen is somewhat smaller than that of trypsin treated bovine red blood cells in plasma. The value of G' of trypsin treated bovine red blood cells in plasma first increased to a maximum value and then decreased with the time. It is supposed that the removal of macro-molecules from the cell surface facilitates the mutual approach of cells and causes the formation of rouleau which seems to be the same as that of human and horse bloods.     

Wettability of substrata controls cell-substrate and cell-cell adhesions

The maintenance of endothelial cell (EC) monolayer architecture requires stable adhesions not only between neighboring cells but also between cells and the extracellular matrix. While the influence of biomaterials surface wettability on cell-substratum adhesion is rather well studied, its impact on cell-cell cohesion has not been extensively investigated. In the present study a model system consisting of hydrophilic and hydrophobic glass pre-coated with fibronectin and fibrinogen was used to study the influence of surface wettability on both types of cell adhesions. It was demonstrated that the substrate wettability controls the adhesion and cytoskeletal organization of endothelial cells, which has an impact on the subsequent ability of cells to establish stable cell-cell cohesions. These effects were related to the accessibility of specific domains of the adsorbed proteins. While the hydrophobic substratum promoted cell-cell cohesion, on hydrophilic substrata cell-substrate adhesion was dominant. In addition, evidence for an influence of surface wettability on the cross talk between integrins and cadherins was found.     

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     

Purification and characterization of an anti-sticking factor from goat epididymal plasma that inhibits sperm--glass and sperm--sperm adhesions

An anti-sticking factor (ASF-I) that showed high affinity for inhibiting adhesion of spermatozoa to glass was isolated from goat epididymal plasma and characterized. The factor was purified approx. 5600-fold and showed a single protein band when examined by non-denaturation and SDS-polyacrylamide gel electrophoresis. The molecular mass and S20w value of ASF-I were approx. 47 kDa and 4.25 S. ASF-I at a concentration of 1 nM showed nearly maximal anti-sticking activity when approx. 60% of the intact spermatozoa were prevented from adhesion to glass and it showed a high degree of protein specificity. Studies with trypsin and glycosidases demonstrated that both the sugar and protein parts of the molecule are essential for its anti-sticking activity. Evidence has been presented to support the view that the outer surface of sperm possesses specific ASF-I receptors that bind to 125I-labelled ASF and mediate cell adhesion to glass. ASF-I also showed high affinity for inhibiting agglutination of corpus-epididymal spermatozoa. The ASF activity was found to be distributed in all the tissues tested and its specific activity was markedly higher in blood plasma than in the tissues. The results suggest that ASF may play an important biological role by serving as a specific inhibitor of cell-substratum and cell-cell adhesions.     

Minimum energy analysis of membrane deformation applied to pipet aspiration and surface adhesion of red blood cells.

An experimental procedure is demonstrated which can be used to determine the interfacial free energy density for red cell membrane adhesion and membrane elastic properties. The experiment involves micropipet aspiration of a flaccid red blood cell and manipulation of the cell proximal to a surface where adhesion occurs. A minimum free energy method is developed to model the equilibrium contour of unsupported membrane regions and to evaluate the partial derivatives of the total free energy, which correspond to the micropipet suction force and the interfacial free energy density of adhesion. It is shown that the bending elasticity of the red cell membrane does not contribute significantly to the pressure required to aspirate a flaccid red cell. Based on experimental evidence, the upper bound for the bending or curvature elastic modulus of the red cell membranes is 10-12 ergs (dyn-cm). Analysis of the adhesion experiment shows that interfacial free energy densities for red cell adhesion can be measured from a lower limit of 10-4 ergs/cm2 to an upper limit established by the membrane tension for lysis of 5-10 ergs/cm2.     

Surface plasmon resonance detection of blood coagulation and platelet adhesion under venous and arterial shear conditions

A surface plasmon resonance (SPR) based flow chamber device was designed for real time detection of blood coagulation and platelet adhesion in platelet rich plasma (PRP) and whole blood. The system allowed the detection of surface interactions throughout the 6mm length of the flow chamber. After deposition of thromboplastin onto a section of the sensor surface near the inlet of the flow chamber, coagulation was detected downstream of this position corresponding to a SPR signal of 7 to 8 mRIU (7 to 8 ng/mm2). A nonmodified control surface induced coagulation 3.5 times slower. Platelet adhesion to gold and fibrinogen coated surfaces in the magnitude of 1.25 and 1.66 mRIU was also shown with platelets in buffer, respectively. SPR responses obtained with PRP and whole blood on surfaces that were methylated or coated with von Willebrand factor (vWF), fibrinogen, or collagen, coincided well with platelet adhesion as observed with fluorescence microscopy in parallel experiments. The present SPR detection equipped flow chamber system is a promising tool for studies on coagulation events and blood cell adhesion under physiological flow conditions, and allows monitoring of short-range surface processes in whole blood.     

Surface coat in steroid-secreting cells of the mouse ovary

Steroid-secreting cells (luteal, thecal and interstitial cells) of the mouse ovary have been studied with the ruthenium red method to stain the "cell coat". The results showed that a typical cell coat covers the entire surface of the plasma membrane except where the cells are connected by specific cell junctions. Further, particularly heavy concentration of ruthenium-red-reacting material was demonstrated in pericapillary and intercellular spaces. The differences in the thickness of the cell coat and its topographical modifications among different groups of steroidogenic cells may be related not only to intercellular adhesion and interactions but also concerned with a function of control in which the cell permeability is modified in relation with phenomena of cell recognition.     

Role of a major cell-substratum adhesion system in cell behavior and morphogenesis

Fibronectins are multifunctional glycoproteins involved in the embryogenesis and in multiple physiological processes. Most of their functions derive from interactions of these glycoproteins with the cell surface. Fibronectin and their cell surface receptor constitute the major cell-substratum adhesion that plays a key role in cell adhesion, cell spreading and cell motility, particularly during embryonic development. Additionally, other functions of fibronectins are due to their ability to ban a great variety of macromolecules contained in an extracellular matrix and in plasma. In particular, the interaction of fibronectins with the cell surface, collagen, fibrin and fibrinogen are involved in physiological processes such as hemostasis, wound healing and phagocytosis.     

Factors involved in cell adhesion to vascular endothelium

The adhesion of blood cells to endothelium can be studied in vitro using human endothelial cells in culture. This experimental model and radiometric techniques provide us with a simple system to quantify the adhesion of blood cells to endothelium. Normal human granulocytes isolated by density gradient adhere to normal endothelial cells in a proportion of 25%. Human promyelocytic cells (HL 60) induced by retinoic acid into mature cells adhere as well as normal granulocytes while the noninduced adhere poorly to endothelium. A small percentage of normal red cells attach to endothelial cells while red cells from patients with sickle cell anemia or diabetes mellitus have a significantly increased adhesion to endothelial cells (P greater than 0.001). This adhesion is statistically correlated with the extent and severity of vascular complications in diabetes mellitus (P less than 0.05). The addition of fibrinogen significantly increased (P less than 0.01) the adhesion of normal red cells, red cells from patients with sickle cell anemia or diabetes mellitus while gamma-globulins did not modify adhesion. Fibronectin potentiated the adhesion of normal red cells.     

Oligospecificity of the cellular adhesion receptor Mac-1 encompasses an inducible recognition specificity for fibrinogen

Mitogenesis, cellular aggregation, and motility follow upon the interaction of fibrinogen with certain defined cell surface receptors. In addition to circulating platelets and vascular endothelium, monocytes express what appears to be a receptor for fibrinogen. Evidence is presented here that the leukocyte adhesion receptor Mac-1 can be specifically induced to bind fibrinogen with characteristics immunochemically and functionally distinct from the established Arg-Gly-Asp-directed fibrinogen receptors. The competence of Mac-1 as a fibrinogen receptor is a general property of cells of monocyte and myeloid lineage acquired after maturational changes of some regions of the alpha subunit of Mac-1 during the process of cell differentiation. This ligand recognition specificity of Mac-1 is lacking for the resting cell. Rather, induction of fibrinogen binding capacity of Mac-1 is due to a cellular response to selected agonists characterized by inducing rapid transients of cytosolic Ca2+. Although different in activation pathways and recognition specificity, Mac-1 exhibits an oligospecific ligand versatility characteristic of other homologous Arg-Gly-Asp-directed adhesion receptors.     

Role of IIb-IIIa-like glycoproteins in cell-substratum adhesion of human melanoma cells

The platelet fibrinogen receptor, glycoprotein complex IIb-IIIa, was isolated from human platelets by lectin and monoclonal antibody affinity chromatography and a polyclonal antiserum (anti-IIb-IIIa) was generated and used to probe for the presence and function of IIb-IIIa-like molecules in two adherent human cell lines. Both C32 melanoma cells and WI38 fibroblasts expressed a IIb-IIIa-like complex on their surface as indicated by immunoprecipitation of detergent extracts of surface radiolabeled cells. When added to cells plated in medium containing 10% serum, the anti-IIb-IIIa antiserum perturbed the adhesion of C32 melanoma cells, but not of WI38 fibroblasts. In a serum-free system, anti-IIb-IIIa antibodies inhibited attachment and spreading of C32 cells to fibrinogen, vitronectin, and fibronectin adsorbed to glass. Anti-IIb-IIIa had no effect on the attachment and spreading of WI38 cells to the extracellular matrix proteins, however. Thus, the IIb-IIIa-like complex appears to play a predominant role in cell-substratum adhesion of C32 cells, but not WI38 cells, and may result from the fact that, on a protein basis, the C32 melanoma cells express approximately 3 times more complex on their surface than do WI38 fibroblasts. The results suggest that the relative abundance of a particular adhesion receptor on the cell surface may govern its importance to cell-substratum adhesion.     

可溶性血纤蛋白促进细胞伸展及其作用机制

可溶性血纤蛋白（ｓｏｌｕｂｌｅｆｉｂｒｉｎ,ＳＦ）为血纤蛋白单体和血纤蛋白原１∶２的复合物．现已知在血液凝固系统被激活的病理状态下,存在于循环血液中,然而它的生理作用仍然不明．首次发现细胞能在固定的ＳＦ上伸展,并能被外源性ＳＦ及精氨酸－甘氨酸－天冬氨酸（ＲＧＤ）合成肽所抑制,但不能被血纤蛋白原和血纤蛋白单体所抑制,提示ＳＦ形成后其结构变化是引起细胞伸展的关键．片段Ｘ（缺乏ＲＧＤ２序列的血纤蛋白原片段）与血纤蛋白单体形成的复合物,使细胞伸展活性明显减低,提示在ＳＦ结构中,血纤蛋白原的ＲＧＤ２序列在细胞伸展中起重要作用．同时发现ＤＩＣ患者血浆中的ＳＦ也具有细胞伸展活性．ＳＦ作为一个粘附分子在体内血栓形成过程中起重要调节作用     

Cell surface fibronectin and oncogenic transformation

Fibronectin is a large glycoprotein at the cell surface of many different cell types; a related protein is present in plasma. Fibronectin is a dimer of 230,000-dalton subunits and also occurs in larger aggregates; it forms fibrillar networks at the cell surface, between cells and substrata and between adjacent cells, and it is not a typical membrane protein. Cell surface fibronectin is reduced in amount or absent on transformed cells and in many cases its loss correlates with acquisition of tumorigenicity and, in particular, metastatic ability. Exceptions to the correlations with transformation and tumorigenicity exist. Loss of fibronectin and the resulting reduced adhesion appear to be involved in pleiotrpoic alterations in cell behavior and may be responsible for several aspects of the transformed phenotype in vitro. Fibronectin interacts with other macromolecules (collagen/gelatin, fibrin/fibrinogen, proteoglycans) and is apparently connected to microfilaments inside the cell.     

Blood platelet surface interactions on fibrinogen under flow as viewed with fluorescent video-microscopy

The interaction of fluorescently labeled blood platelets with fibrinogen-coated glass was studied in Poiseuille flow at 3 wall shear rates, 40, 80 and 944 s-1. Observations were made via video-microscopy at a distance of 0.5 cm from a tube's entrance over a 1370 microns 2 portion of luminal area. The rates of arrival and detachment, and the net rate of adhesion of cells increased nonlinearly with flow rate. The fraction of arriving cells, first contacts, which adhered without subsequent movement and the fraction of arriving cells which adhered, moved to new positions and then remained adherent, were maximal at 80 s-1. For platelets which adhere and then move to a number of new positions, the likelihood of permanent adhesion is greater than 85 percent. The adhesion process is one in which 40-60 percent of cells permanently adhere on first contact with an additional 30 percent adhering after several moves along the surface. Cells contacting where a platelet was previously adherent had a greater chance of adhering than they would on an unaltered fibrinogen surface. The efficiency of platelet adhesion is greater for second contacts than for first contacts on unaltered fibrinogen coated surface.     

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.     

Light-induced Adhesion of Spirogyra Cells to Glass

Adhesion of Spirogyra (tentatively, Spirogyra fluviatilis) cells to glass is described. The cells of an algal filament can adhere to a substrate only when they are located at the end of the filament. Rapid adhesion is induced by blue-violet light (blue adhesion) as well as by temperature shift (about 6 C → about 22 C) or shaking (dark adhesion). Adherent cells detach in 1 hour in the absence of one of these stimuli. Slow adhesion is induced by red light (red adhesion) 1 hour after irradiation, and may be controlled by phytochrome. A cell once caused to adhere by red light does not release from the glass.     

A new feasible method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen

Plasma fibrinogen participates in several physiological and pathological events thus becoming a useful studying material in biomedical research. Here we report a new convenient method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen. Clumping factor A (ClfA) is a cell wall-anchored surface protein of S. aureus bacteria that binds with a high affinity to the fibrinogen gamma chain C-terminus via a segment encompassing the residues 221-550. This activity of ClfA (ClfA(221-550)) was produced in fusion to the C-terminus of glutathione-S-transferase (GST) with recombinant technology and used as an affinity ligand to capture plasma fibrinogen. GST-ClfA(221-550) fusion protein was immobilized onto the glutathione-conjugated beads packed in a plastic column by its GST part. Then, this affinity column was loaded with citrated and heparinized human plasma. After washing out unbound proteins, column-captured fibrinogen was specifically eluted down with a citrate buffer solution (50mM, pH 5.6). Purified human fibrinogen exhibited the ability to support platelet adhesion and aggregation and formed fibrin clot by thrombin, indicating that ClfA(221-550)-purified human fibrinogen is a functionally active product. We also found that both the rat and mouse fibrinogens could be purified as well as human fibrinogen with this method. By virtue of its simplicity and feasibility, ClfA(221-550)-based method would be very useful to the investigators who need fibrinogen to perform their studies.     

Association of the actin cytoskeleton with glass-adherent proteins in mouse peritoneal macrophages

Summary— When mouse peritoneal macrophages adherent to glass surface were removed by treatment with triethanolamine and Nonidet P-40, fine thread structures of unique loops were left behind on glass at the sites of cell adhesion. To examine the ultrastructural relationship between such looped threads and cytoskeletal components in glass-adherent macrophages, we successfully used the ‘zinc method’ to remove most of the cytoplasm including nuclei and to expose the cytoskeleton associated with the ventral plasma membrane. The cytoskeleton was seen to be mainly composed of actin filaments forming dense networks. The network contained scattered star-like foci from which actin filaments radiated. When the ventral plasma membrane-cytoskeleton complex was further treated with Nonidet P-40, the membrane was dissolved to expose the glass surface with actin foci persisting on glass. When the complex was removed by further treatment with Nonidet P-40 and DNase I, the looped threads became visible. Confocal laser microscopy of glass-adherent macrophages stained with fluorescent phalloidin showed the preferential distribution of F-actin in the ventral cytoplasm along the plasma membrane, where intense fluorescent spots were also scattered. Confocal interference reflection microscopy revealed densely populated dark dots and striae of focal contact, which corresponded in overall distribution to actin foci and looped threads. These observations suggest that actin cytoskeleton is closely associated with looped threads to reinforce cell adhesion to glass.