Kinetics of specific and nonspecific adhesion of red blood cells on glass.

Fixed spherical human red blood cells suspended in 17% sucrose were allowed to adhere on either clean glass surfaces or glass surfaces preincubated with antibodies specific to a certain blood group antigen. The adhesion experiments were performed in an impinging jet apparatus, in which the cells are subjected to stagnation point flow. The objective of this study was to compare the efficiencies of nonspecific and specific (antigen-antibody mediated) adhesion of red blood cells on glass surfaces. The efficiency was defined as the ratio of the experimental adhesion rate to that calculated based on numerical solutions of the mass transfer equation, taking into account hydrodynamic interactions as well as colloidal forces. The efficiency for nonspecific adhesion was nearly unity at flow rates lower than 85 microliter/s (corresponding to a wall shear rate, Gw, of 30 s-1 at a radial distance of 110 microns from the stagnation point). The values of efficiency dropped at higher flow rates, due to an increase in the tangential force. The critical deposition concentration is found to occur at 120-150 mM NaCl, which is consistent with the theoretically predicted values. At low salt concentrations, the experimental values are higher than the theoretical ones. Similar discrepancies have been found in many colloidal systems. Introducing steric repulsion by adsorbing a layer of albumin molecules on the glass completely prevents nonspecific adhesion at flow rates below 60 microliter/s (Gw congruent to 15 s-1). The efficiency of specific adhesion depends both on the concentration of antibody molecules on the surface and the flow rate. Normal red cells adhere more readily through antigen-antibody bonds than fixed cells. Fixed spherical cells have a higher adhesion efficiency than fixed biconcave ones.     

Haemodynamics of angioaccess venous anastomoses

A large percentage of arteriovenous haemodialysis angioaccess loop grafts (AVLG) fail within the first year after surgery, the occlusive lesions being found predominantly at the venous anastomosis site. This paper presents a detailed flow dynamic study of the AVLG system using three elastic, transparent bench-top flow models, which were based on the geometry of silicone rubber casts obtained at different times from a chronic animal model. Each model thus represented a different stage of the lesion development. Flow visualization and laser Doppler anemometer surveys of the flow field confirmed that the hydrodynamic factors favour lesion development near the stagnation point opposite the anastomotic toe, where the momentum of the impinging jet stream, combined with the oscillating wall shear stress generated in the vicinity of the stagnation point, acts in both directions. The accumulation of tracer particles in the region of flow separation is believed to be a combined contribution from the hydraulic forces and the inward motion of the vessel wall. As these hydrodynamic factors are enhanced upon further development of the occlusive lesion, a vicious cycle may be formed.     

Role of blood cell-wall interactions in thrombogenesis and atherogenesis: a microrheological study

The relationship between blood flow and the localization of thrombosis and atherosclerosis in vivo was investigated using the approach and techniques of microrheology. The flow patterns and wall-adhesion of platelets were studied in the captive annular vortex formed at a sudden tubular expansion at various hematocrits in steady and pulsatile flow. The adhesion density exhibited a peak within the vortex and just downstream of the reattachment point, which is also a stagnation point. The peaks flattened out with increasing Reynolds number in steady flow and also in pulsatile flow. Platelet adhesion increased markedly with increasing hematocrit. The localization of adhesion peaks was explained by curvature of the streamlines carrying platelets to the wall on either side of the reattachment point. The relevance of these results to the circulation is that stagnation points are found in regions of disturbed flow at various sites in the arterial and venous circulations. This was shown in experiments using a technique whereby flow was visualized in isolated transparent natural blood vessels prepared from dogs and humans postmortem. In dog saphenous vein bileaflet valves, there was a large primary spiral vortex as well as a smaller secondary vortex, the latter acting as a trap and generator of thrombi. Recirculation zones also existed in the dog aorta at T-junctions of the celiac, cranial mesenteric and renal arteries. Finally, in the human carotid bifurcation, a large standing recirculation zone consisting of spiral secondary flows formed in the carotid sinus at physiological flow conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomechanics of cell rolling: shear flow, cell-surface adhesion, and cell deformability

The mechanics of leukocyte (white blood cell; WBC) deformation and adhesion to endothelial cells (EC) has been investigated using a novel in vitro side-view flow assay. HL-60 cell rolling adhesion to surface-immobilized P-selectin was used to model the WBC-EC adhesion process. Changes in flow shear stress, cell deformability, or substrate ligand strength resulted in significant changes in the characteristic adhesion binding time, cell-surface contact and cell rolling velocity. A 2-D model indicated that cell-substrate contact area under a high wall shear stress (20 dyn/cm2) could be nearly twice of that under a low stress (0.5 dyn/cm2) due to shear flow-induced cell deformation. An increase in contact area resulted in more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy that inputs to a cell decreased due to a flattened cell shape. The model also predicted a plateau of WBC rolling velocity as flow shear stresses further increased. Both experimental and computational studies have described how WBC deformation influences the WBC-EC adhesion process in shear flow.     

Particle diameter influences adhesion under flow

The diameter of circulating cells that may adhere to the vascular endothelium spans an order of magnitude from approximately 2 microm (e.g., platelets) to approximately 20 microm (e.g., a metastatic cell). Although mathematical models indicate that the adhesion exhibited by a cell will be a function of cell diameter, there have been few experimental investigations into the role of cell diameter in adhesion. Thus, in this study, we coated 5-, 10-, 15-, and 20-microm-diameter microspheres with the recombinant P-selectin glycoprotein ligand-1 construct 19.ek.Fc. We compared the adhesion of the 19.ek.Fc microspheres to P-selectin under in vitro flow conditions. We found that 1) at relatively high shear, the rate of attachment of the 19.ek.Fc microspheres decreased with increasing microsphere diameter whereas, at a lower shear, the rate of attachment was not affected by the microsphere diameter; 2) the shear stress required to set in motion a firmly adherent 19.ek.Fc microsphere decreased with increasing microsphere diameter; and 3) the rolling velocity of the 19.ek.Fc microspheres increased with increasing microsphere diameter. These results suggest that attachment, rolling, and firm adhesion are functions of particle diameter and provide experimental proof for theoretical models that indicate a role for cell diameter in adhesion.     

Determination of binding strength and kinetics of binding initiation. A model study made on the adhesive properties of P388D1 macrophage-like cells

The adhesive properties of the mouse P388D1 macrophage-like line were explored. Cells were deposited in glass capillary tubes, and the kinetics of adhesion and spreading were studied. Binding involved the cell metabolism since it was decreased by cold, azide, or a divalent cation chelator. Glass-adherent cells were subjected to calibrated laminar shear flows with a highly viscous dextran solution. A tangential force of about 5 X 10(-3) dyn/cell was required to achieve substantial detachment. The duration of application of the shearing force strongly influenced cell-substrate separation when this was varied from 1-10 s. Further, this treatment resulted in marked cell deformation, with the appearance of an elongated shape. Hence, cell-substrate separation is a progressive process, and binding strength is expected to be influenced by cell deformability. The minimum time required for adhesion was also investigated by making cells adhere under flow conditions. The maximum flow rate compatible with adhesion was about 1000-fold lower than that required to detach glass-bound cells. A simple model was devised to provide a quantitative interpretation for the experimental results of kinetic studies. It is concluded that cell-to-glass adhesion required a cell-substrate contact longer than a few seconds. This first step of adhesion was rapidly followed by a large (about 1000-fold) increase of adhesion strength. It is therefore emphasized that adhesion is heavily dependent on the duration of cell-to-cell encounter, as well as the force used to remove so-called unbound cells.     

Shear modulation of intercellular contact area between two deformable cells colliding under flow

Shear rate has been shown to critically affect the kinetics and receptor specificity of cell-cell interactions. In this study, the collision process between two modeled cells interacting in a linear shear flow is numerically investigated. The two identical biological or artificial cells are modeled as deformable capsules composed of an elastic membrane. The cell deformation and trajectories are computed using the immersed boundary method (IBM) for shear rates of 100-400s(-1). As the two cells collide under hydrodynamic shear, large local cell deformations develop. The effective contact area between the two cells is modulated by the shear rate, and reaches a maximum value at intermediate levels of shear. At relatively low shear rate, the contact area is an enclosed region. As the shear rate increases, dimples form on the membrane surface, and the contact region becomes annular. The nonmonotonic increase of the contact area with the increase of shear rate from computational results implies that there is a maximum effective receptor-ligand binding area for cell adhesion. This finding suggests the existence of possible hydrodynamic mechanism that could be used to interpret the observed maximum leukocyte aggregation in shear flow. The critical shear rate for maximum intercellular contact area is shown to vary with cell properties such as radius and membrane elastic modulus.     

Determination of binding strength and kinetics of binding initiation

The adhesive properties of the mouse P388D1 macrophage-like line were explored. Cells were deposited in glass capillary tubes, and the kinetics of adhesion and spreading were studied. Binding involved the cell metabolism since it was decreased by cold, azide, or a divalent cation chelator. Glass-adherent cells were subjected to calibrated laminar shear flows with a highly viscous dextran solution. A tangential force of about 5×10⁻³ dyn/cell was required to achieve substantial detachment. The duration of application of the shearing force strongly influenced cell-substrate separation when this was varied from 1–10 s. Further, this treatment resulted in marked cell deformation, with the appearance of an elongated shape. Hence, cell-substrate separation is a progressive process, and binding strength is expected to be influenced by cell deformability. The minimum time required for adhesion was also investigated by making cells adhere under flow conditions. The maximum flow rate compatible with adhesion was about 1000-fold lower than that required to detach glass-bound cells. A simple model was devised to provide a quantitative interpretation for the experimental results of kinetic studies. It is concluded that cell-to-glass adhesion required a cell-substrate contact longer than a few seconds. This first step of adhesion was rapidly followed by a large (about 1000-fold) increase of adhesion strength. It is therefore emphasized that adhesion is heavily dependent on the duration of cell-to-cell encounter, as well as the force used to remove so-called unbound cells.     

Effects of wall shear stress and fluid recirculation on the localization of circulating monocytes in a three-dimensional flow model

There is a correlation between the location of early atherosclerotic lesions and the hemodynamic characteristics at those sites. Circulating monocytes are key cells in the pathogenesis of atherosclerotic plaques and localize at sites of atherogenesis. The hypothesis that the distribution of monocyte adhesion to the vascular wall is determined in part by hemodynamic factors was addressed by studying monocyte adhesion in an in vitro flow model in the absence of any biological activity in the model wall.

Suspensions of U937 cells were perfused (Re = 200) through an axisymmetric silicone flow model with a stenosis followed by a reverse step. The model provided spatially varying wall shear stress, flow separation and reattachment, and a three-dimensional flow pattern. The cell rolling velocity and adhesion rates were determined by analysis of videomicrographs. Wall shear stress was obtained by numerical solution of the equations of fluid motion. Cell adhesion patterns were also studied in the presence of chemotactic peptide gradients.

The cell rolling velocity varied linearly with wall shear stress. The adhesion rate tended to decrease with increasing local wall shear stress, but was also affected by the radial component of velocity and the dynamics of the recirculation region and flow reattachment. Adhesion was increased in the vicinity of chemotactic peptide sources downstream of the expansion site. Results with human monocytes were qualitatively similar to the U937 experiments.

Differences in the adhesion rates of U937 cells occurring solely as a function of the fluid dynamic properties of the flow field were clearly demonstrated in the absence of any biological activity in the model wall.     

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.     

Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin

Endothelial sequestration of circulating monocytes is a key event in early atherosclerosis. Hemodynamics is proposed to regulate monocyte-endothelial cell interactions by direct cell activation and establishment of flow environments that are conducive or prohibitive to cell-cell interaction. We investigated fluid shear regulation of monocyte-endothelial cell adhesion in vitro using a disturbed laminar shear system that models in vivo hemodynamics characteristic of lesion-prone vascular regions. Human endothelial cell monolayers were flow conditioned for 6 h before evaluation of monocyte adhesion under static and dynamic flow conditions. Results revealed a distinctive clustered cell pattern of monocyte adhesion that strongly resembles in vivo leukocyte adhesion in early- and late-stage atherosclerosis. Clustered monocyte cell adhesion correlated with endothelial cells coexpressing intercellular adhesion molecule-1 (ICAM-1) and E-selectin as result of a flow-induced, selective upregulation of E-selectin expression in a subset of ICAM-1-expressing cells. Clustered monocyte cell adhesion assayed under static conditions exhibited a spatial variation in size and frequency of occurrence, which demonstrates differential regulation of endothelial cell adhesiveness by the local flow environment. Dynamic adhesion studies conducted with circulating monocytes resulted in clustered cell adhesion only within the disturbed flow region, where the monocyte rate of motion is sufficiently low for cell-cell interaction. These studies provide evidence and reveal mechanisms of local hemodynamic regulation of endothelial adhesiveness and endothelial monocyte interaction that lead to localized monocyte adhesion and potentially contribute to the focal origin of arterial diseases such as atherosclerosis.     

Platelet deposition in non-parallel flow

This paper deals with flow- and surface-related aspects of primary hemostasis. It investigates the influence of both shear stress and changes in surface reactivity on platelet adhesion. For this purpose, a mathematical model based on the Navier-Stokes equations and on particle conservation is developed. Several vessel geometries of physiological relevance are considered, such as stagnation point flow, sudden expansion and t-junction. Model parameters have been optimized to fit corresponding experimental data. When platelet adhesion was assumed independent of shear, numerically predicted spatial platelet distribution did not match these data at all. However, when adhesion was assumed shear-dependent, better agreement was achieved. Further improvement was obtained when changes in surface reactivity due to platelet adhesion were taken into account. This was done by coupling platelet flux conditions to ordinary differential equations for the evolution of surface-bound platelets. Existence of weak solutions is shown for generalized parabolic systems having such boundary conditions. This, together with proofs for uniqueness and positivity of solutions, guarantees mathematical well posedness of the presented model. Limitations due to the complexity of the hemostatic system are discussed, as well as possible applications in practice. The findings of this paper contribute to understand the roles of flow and surface in primary hemostasis, which is of paramount interest in bioengineering and clinical practice.     

Nano-to-micro scale dynamics of P-selectin detachment from leukocyte interfaces. III. Numerical simulation of tethering under flow

Transient capture of cells or model microspheres from flow over substrates sparsely coated with adhesive ligands has provided significant insight into the unbinding kinetics of leukocyte:endothelium adhesion complexes under external force. Whenever a cell is stopped by a point attachment, the full hydrodynamic load is applied to the adhesion site within an exceptionally short time-less than the reciprocal of the hydrodynamic shear rate (e.g., typically <0.01 s). The decay in numbers of cells or beads that remain attached to a surface has been used as a measure of the kinetics of molecular bond dissociation under constant force, revealing a modest increase in detachment rate at growing applied shear stresses. On the other hand, when detached under steady ramps of force with mechanical probes (e.g., the atomic force microscope and biomembrane force probe), P-selectin:PSGL-1 adhesion bonds break at rates that increase enormously under rising force, yielding 100-fold faster off rates at force levels comparable to high shear. The comparatively weak effect of force on tether survival in flow chamber experiments could be explained by a possible partition of the load amongst several bonds. However, a comprehensive understanding of the difference in kinetic behavior requires us to also inspect other factors affecting the dynamics of attachment-force buildup, such as the interfacial compliance of all linkages supporting the adhesion complex. Here, combining the mechanical properties of the leukocyte interface measured in probe tests with single-bond kinetics and the kinetics of cytoskeletal dissociation, we show that for the leukocyte adhesion complex P-selectin:PSGL-1, a detailed adhesive dynamics simulation accurately reproduces the tethering behavior of cells observed in flow chambers. Surprisingly, a mixture of 10% single bonds and 90% dimeric bonds is sufficient to fully match the data of the P-selectin:PSGL-1 experiments, with the calculated decay in fraction of attached cells still appearing exponential.     

The forward rate of binding of surface-tethered reactants: effect of relative motion between two surfaces

The reaction of molecules confined to two dimensions is of interest in cell adhesion, specifically for the reaction between cell surface receptors and substrate-bound ligand. We have developed a model to describe the overall rate of reaction of species that are bound to surfaces under relative motion, such that the Peclet number is order one or greater. The encounter rate between reactive species is calculated from solution of the two-dimensional convection-diffusion equation. The probability that each encounter will lead to binding depends on the intrinsic rate of reaction and the encounter duration. The encounter duration is obtained from the theory of first passage times. We find that the binding rate increases with relative velocity between the two surfaces, then reaches a plateau. This plateau indicates that the increase in the encounter rate is counterbalanced by the decrease in the encounter duration as the relative velocity increases. The binding rate is fully described by two dimensionless parameters, the Peclet number and the Damk?hler number. We use this model to explain data from the cell adhesion literature by incorporating these rate laws into "adhesive dynamics" simulations to model the binding of a cell to a surface under flow. Leukocytes are known to display a "shear threshold effect" when binding selectin-coated surfaces under shear flow, defined as an increase in bind rate with shear; this effect, as calculated here, is due to an increase in collisions between receptor and ligand with increasing shear. The model can be used to explain other published data on the effect of wall shear rate on the binding of cells to surfaces, specifically the mild decrease in binding within a fixed area with increasing shear rate.     

Vinculin and Cell-Cell Adhesion

Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, γ227 and γ229, and a reconstituted γ229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins α- and β-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines, BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculinnegative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted γ229 and γ227 cells that were formed in suspension during a two-hour static incubation at 37°C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.     

Estimate of the sticking probability for cells in uniform shear flow with adhesion caused by specific bonds

A theory is developed for the aggregation rate of cells in uniform shear flow when the cell-cell adhesion is mediated by bonds between specific molecules on the cell surfaces such as antigen and antibody or lectin and carbohydrate. The theory is based on estimates of the frequency and duration of cell-cell collisions and of the number of bonds formed and required to hold the cells together. For high shear rates, the sticking probability is a function of a single dimensionless parameter, A, that is proportional to G-2, with G the shear rate. For low shear rates, the sticking probability is a function of a second dimensionless parameter, A' proportional to G-1. In either case the rate of cell-cell sticking is a maximum when A (or A') congruent to 1.0. For small values of A (or A') the cells collide frequently, but do not stick, whereas for large values of A (or A') the cells collide infrequently, but stick with larger probability. Studies in Couette viscometer or other flow having approximately uniform shear can test these models.     

Platelet adhesion onto the wall of a flow chamber with an obstacle

In the present study, the data of the initial adhesion of platelets onto the wall of a flow chamber with an obstacle in steady human blood flows were obtained. The flowfields and the distribution of stress-related factors were simulated numerically by a finite volume method and the fluid dynamic effect on the platelet adhesion is discussed. In addition to the wall shear effect, the normal stress effect was also taken into account. A parameter Vn/¦Vt¦ was devised to assess the combined effect of both shear and normal forces in platelet adhesion. It was found that the peak adhesion occurred next to, but not on, the impingement point on the obstacle where the value of Vn/¦Vt¦ was negative. In these regions, direct impact played a major role in platelet adhesion. On the other hand, near the separation point before the obstacle where Vn/¦Vt¦ was insignificant, the mechanism was believed to be different from that in the direct impact region. Denser adhesion there might be caused by the accumulation and frequent collision of particles due to flow retardation and/or detour of the flow path. Interestingly, relatively low adhesion was found inside the recirculation regions. These results show that the normal stress effect (impingement) should be considered in platelet adhesion in addition to the shear effect.     

The deformation of an adherent leukocyte under steady shear flow: a numerical study

Leukocyte adhesion is a pathophysiological process in which the balance between hemodynamic and adhesion forces (molecular bonds) plays a key role. In this work, we studied the deformation of an adherent leukocyte and calculated the forces exerted on it. Three model cells were proposed, considering the leukocyte as a single drop, a compound drop, and a nucleus drop, representing a cell without nucleus, a cell with a nucleus, and a nucleus only, respectively. These model cells were supposedly adherent to a smooth substrate under steady shear flow. Our numerical results showed that all three model cells deformed in function of the initial contact angle, capillary number, and Reynolds number. The single drop was the most deformable, while the nucleus drop was the most resistant to the external flow. Each of the model cells showed maximum cell deformation at a high Reynolds number. The distribution of pressure on the cell confirmed the existence of a high-pressure region downstream of the drop, which retarded further deformation of the cell and provided a positive lift force on the drop. The consideration of a highly viscous nucleus can correct the over evaluation of the cell deformation in a flow.     

A free boundary problem modeling thrombus growth

Recently, neglect of either shear stress, surface saturation, or thrombus growth in mathematical models of platelet deposition has been identified as leading cause of inability to match experimental evidence. While the consideration of shear stress is necessary to obtain at least some qualitative agreement, purely shear-dependent approaches yield notable quantitative discrepancies. In a previous paper, the author demonstrated that surface saturation significantly improves model predictions. However, discrepancies still persist when thrombus growth is neglected. Therefore, the present work develops a free boundary problem which takes this into account. Numerical simulations are performed using the level set method. The results agree well with measurements in stagnation point flow and tubular expansions, which demonstrates the coupling of flow, platelet adhesion, and aggregate growth in primary hemostasis.