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.     

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-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds.

Fixed spherical swollen human red blood cells of blood type B adhering on a glass surface through antigen-antibody bonds to monoclonal mouse antihuman IgM, adsorbed or covalently linked on the surface, were detached by known hydrodynamic forces created in an impinging jet. The dynamic process of detachment of the specifically bound cells was recorded and analyzed. The fraction of adherent cells remaining on the surface decreased with increasing hydrodynamic force. For an IgM coverage of 0.26%, a tangential force on the order of 100 pN was able to detach almost all of the cells from the surface within 20 min. After a given time of exposure to hydrodynamic force, the fraction of adherent cells remaining increased with time, reflecting an increase in adhesion strength. The characteristic time for effective aging was approximately 4 h. Results from experiments in which the adsorbed antibody molecules were immobilized through covalent coupling and from evanescent wave light scattering of adherent cells, imply that deformation of red cells at the contact area was the principal cause for aging, rather than local clustering of the antibody through surface diffusion. Experiments with latex beads specifically bound to red blood cells suggest that, instead of breaking the antigen-antibody bonds, antigen molecules were extracted from the cell membrane during detachment.     

Mechanisms of leukocyte adhesion

The interaction between granulocytes and endothelial walls may be influenced by the blood flow. This possibility was investigated by studying the influence of fluid flow on the adhesion and detachment of 51Cr-labeled rat granulocytes interacting with protein-coated glass surfaces. It is concluded that: i) Adhesion is markedly decreased when the wall shear rate becomes higher than about 20 s-1. ii) Pretreating glass with concanavalin A or polylysine significantly decreased adhesion, whereas fibronectin had little effect on binding. iii) Very high flow rates (about one thousandfold higher than those compatible with bond formation) were required to provoke substantial detachment of substrate-bound cells. iv) Coating glass with laminin or polylysine decreased binding strength whereas fibronectin or concanavalin A did not substantially influence this parameter. v) Exposing granulocytes to phorbol myristate acetate might increase the cell ability to form strong adhesions, whereas labile adhesion was unaffected or even decreased by this treatment.     

Towards a concept of thrombosis in accelerated flow: rheology, fluid dynamics, and biochemistry

The predilection sites of arterial thrombosis are characterized by local increase in wall shear stress, flow separation with eddy formation and stagnation point flow. The defenders of high shear, as well as those of low shear theory of thrombogenesis, point to correlations of predilection sites and the respective flow abnormalities. Experimental evidence is provided, that high shear rates can damage both red cells and platelets, that lysed red cells constitute a potent platelet stimulant, due to their content of adenine nucleotides, and that platelets do not adhere to surfaces unless transported onto them by convective motion, the effectiveness of the platelet-wall interaction being enhanced by platelet activation. Based on these facts, a resolution of the contrast between high and low shear theory of thrombosis is attempted in a way, that the different flow regimens, with blood cells sequentially passing them, are each considered important and interdependent steps on the way to thrombosis.     

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.     

Shear and time-dependent changes in Mac-1, LFA-1, and ICAM-3 binding regulate neutrophil homotypic adhesion

We examined the relative contributions of LFA-1, Mac-1, and ICAM-3 to homotypic neutrophil adhesion over the time course of formyl peptide stimulation at shear rates ranging from 100 to 800 s-1. Isolated human neutrophils were sheared in a cone-plate viscometer and the kinetics of aggregate formation was measured by flow cytometry. The efficiency of cell adhesion was computed by fitting the aggregate formation rates with a model based on two-body collision theory. Neutrophil homotypic adhesion kinetics varied with shear rate and was most efficient at 800 s-1, where approximately 40% of the collisions resulted in adhesion. A panel of blocking Abs to LFA-1, Mac-1, and ICAM-3 was added to assess the relative contributions of these molecules. We report that 1) LFA-1 binds ICAM-3 as its primary ligand supporting homotypic adhesion, although the possibility of other ligands was also detected. 2) Mac-1 binding to an unidentified ligand supports homotypic adhesion with an efficiency comparable to LFA-1 at low shear rates of approximately 100 s-1. Above 300 s-1, however, Mac-1 and not LFA-1 were the predominant molecules supporting cell adhesion. This is in contrast to neutrophil adhesion to ICAM-1-transfected cells, where LFA-1 binds with a higher avidity than Mac-1 to ICAM-1. 3) Following stimulation, the capacity of LFA-1 to support aggregate formation decreases with time at a rate approximately 3-fold faster than that of Mac-1. The results suggest that the relative contributions of beta2 integrins and ICAM-3 to neutrophil adhesion is regulated by the magnitude of fluid shear and time of stimulus over a range of blood flow conditions typical of the venular microcirculation.     

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.     

Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear

Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbalpha cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbalpha blocking antibody (AK2) and was not observed with CHO cells expressing GPIbbeta and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20-200 s-1), GPIb-IX was able to support cell rolling at both venous (150 s-1) and arterial (1500-10,500 s-1) shear rates. Studies with a mutant GPIbalpha receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000-6000 s-1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbalpha and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress.     

Margination and adhesion dynamics of tumor cells in a real microvascular network

In tumor metastasis, the margination and adhesion of tumor cells are two critical and closely related steps, which may determine the destination where the tumor cells extravasate to. We performed a direct three-dimensional simulation on the behaviors of the tumor cells in a real microvascular network, by a hybrid method of the smoothed dissipative particle dynamics and immersed boundary method (SDPD-IBM). The tumor cells are found to adhere at the microvascular bifurcations more frequently, and there is a positive correlation between the adhesion of the tumor cells and the wall-directed force from the surrounding red blood cells (RBCs). The larger the wall-directed force is, the closer the tumor cells are marginated towards the wall, and the higher the probability of adhesion behavior happen is. A relatively low or high hematocrit can help to prevent the adhesion of tumor cells, and similarly, increasing the shear rate of blood flow can serve the same purpose. These results suggest that the tumor cells may be more likely to extravasate at the microvascular bifurcations if the blood flow is slow and the hematocrit is moderate.     

Model experiments on platelet adhesion in stagnation point flow

Experiments with glass models of arterial branchings and bends, perfused with bovine platelet rich plasma (PRP), revealed platelet deposition being strongly dependent on fluid dynamic factors. Predilection sites of platelet deposits are characterized by flow vectors directed against the wall, so-called stagnation point flow. Thus collision of suspended particles with the wall, an absolute prerequisite for adhesion of platelets to surfaces even as thrombogenic as glass, appears mediated by convective forces. The extent of platelet deposition is correlated to the magnitude of flow components normal to the surface as well as to the state of biological activation of the platelets. The latter could be effective by an increase in hydrodynamically effective volume, invariably associated with the platelet shape change reaction to biochemical stimulants like ADP. The effect of altered rheological properties of platelets upon their deposition and of mechanical properties of surfaces was examined in a stagnation point flow chamber. Roughnesses in the order of 5 microns, probably by creating local flow disturbances, significantly enhance platelet adhesion, as compared to a smooth surface of identical chemical composition.     

Depletion interactions in polymer solutions promote red blood cell adhesion to albumin-coated surfaces

The effects of concentration and molecular weight of neutral dextrans on the adhesion of human red blood cells (RBC) to albumin-coated glass have been investigated using a parallel-plate flow chamber. Results indicate that the adhesion is markedly increased in the presence of 70 kDa and 500 kDa dextran, with this increase reflected by both the number of cells adhering and the strength of the adhesion. This increased adhesiveness is attributed to reduced surface concentrations of the large polymers and hence attractive forces due to depletion interaction. Depletion interaction brings the adjacent surfaces closer, leading to an increased number of binding sites available to the cell and thus more efficient and stronger adhesion of single cells. Our results suggest that depletion might play a role in other specific cell-cell or cell-surface interactions via initiating close contacts to allow specific binding.     

Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear.     

Thrombospondin inhibits adhesion of endothelial cells

Adsorption of thrombospondin to a substratum inhibits adhesion of endothelial cells to that substratum. Four hours after plating of cells on glass covered with thrombospondin, the number of cells bound per unit area was only 8% of that bound to fibronectin, and 20% of that which could bind to albumin. While on fibronectin cells assumed a well-spread configuration with time in culture, on thrombospondin they stayed completely round. On surfaces constructed by sequential incubation of glass with thrombospondin and fibronectin or other proteins, thrombospondin retained its inhibitory effect on cell adhesion. Fibronectin surfaces treated with thrombospondin lost 50% of their capacity to adhere endothelial cells. Cell spreading was also greatly impaired. These observations indicate that thrombospondin, which is a component of the extracellular matrix, can modulate adhesion of endothelial cells to the matrix.     

Enforced detachment of red blood cells adhering to surfaces: statics and dynamics

We investigated the mechanical strength of adhesion and the dynamics of unbinding of red blood cells to solid surfaces. Two different situations were tested: 1), native red blood cells nonspecifically adhered to glass surfaces coated with positively charged polymers and 2), biotinylated red blood cells specifically adhered to glass surfaces decorated with streptavidin, which has a high binding affinity for biotin. We used micropipette manipulation for forming and subsequently breaking the adhesive contact through a stepwise micromechanical procedure. Analysis of cell deformations provided the relation between force and contact radius, which was found to be in good agreement with theoretical predictions. We further demonstrated that the separation energy could be precisely derived from the measure of rupture forces and the cell shape. Finally, the dynamics of detachment was analyzed as a function of the applied force and the initial size of the adhesive patch. Our experiments were supported by original theoretical predictions, which allowed us to correlate the measured separation times with the molecular parameters (e.g., activation barrier, receptor-ligand characteristic length) derived from force measurements at the single bond level.     

Molecular dynamics of the transition from L-selectin- to beta 2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear.

Homotypic adhesion o2 neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes depend on L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand hydrodynamic stresses. Using cone-plate viscometry to apply a uniform linear shear field to suspensions of neutrophils, we conducted a detailed examination of the effect of shear rate and shear stress on the kinetics of cell aggregation. A collisional analysis based on Smoluchowski's flocculation theory was employed to fit the kinetics of aggregation with an adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency with shear was dependent on L-selectin, and peak efficiency was maintained over a relatively narrow range of shear rates (400-800 s-1) and shear stresses (4-7 dyn/cm2). When L-selectin was blocked with antibody, beta 2-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s-1). The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to stable adhesion.     

A novel strategy to obtain a hyaluronan monolayer on solid substrates

The aim of this study was to find a novel simple method to obtain polysaccharide ultrathin layers on solid substrates to investigate the interaction between the surface and the biological environment. A Hyaluronan (Hyal) monolayer with a well-defined chemistry was obtained by exploiting the capability of organosilanes to spontaneously adhere onto glass surfaces. A silane alkylic chain was conjugated with Hyal, and the derivatized polysaccharide was allowed to spontaneously adhere onto a glass surface. The elemental analysis of the modified polysaccharide demonstrated that one out of five disaccharide units was conjugated with the alkyl silane chain, corresponding to a substitution degree of the carboxylate groups of approximately 20%. The film of the modified polysaccharide was characterized by means of X-ray photoelectron spectroscopy (XPS), water contact angle, and atomic force microscopy (AFM) measurements. XPS analysis demonstrated that we obtained a Hyal layer with a thickness of about 2.0 nm corresponding to a Hyal monolayer. The Hyal-coated surfaces appeared to be rather smooth and highly hydrophilic and showed significant resistance to nonspecific cell adhesion.     

Use of a modified Robbins device to directly compare the adhesion of Staphylococcus epidermidis RP62A to surfaces

Staphylococcus epidermidis is a frequent cause of infection associated with the use of biomedical devices. Flow cell studies of the interaction between bacteria and surfaces do not generally allow direct comparison of different materials using the same bacterial suspension. The use of a modified Robbins Device (MRD) to compare the adhesion to different surfaces of Staph. epidermidis RP62A grown in continuous culture was investigated. Adhesion to glass was compared with siliconized glass, plasma-conditioned glass, titanium, stainless steel and Teflon. Attachment to siliconized glass was also compared with glass under differing ionic strength, and divalent cation concentrations. Both the differences in numbers adhering and changes in adhesion (slope) through the MRD were compared. There was a trend towards higher numbers adhering to the discs at the in-flow end of the MRD than at the outflow end, probably reflecting depletion of adherent bacteria in the interacting stream. Adhesion of Staph. epidermidis RP62A to siliconized glass and Teflon was reduced when compared to glass with increasing flow rates. Adhesion to stainless steel was not affected by flow rate and titanium gave a different slope of adhesion through the MRD when compared with glass, suggesting an interaction with different sub-populations within the interacting stream. Differences between siliconized glass and glass at flow rates of 300 ml h-1 were abolished by the addition of calcium or EDTA and reduced by the addition of magnesium. Increasing ionic strength reduced the statistical significance of the differences between glass and siliconized glass. Pre-conditioning of glass with pooled human plasma reduced adhesion compared with untreated glass and again gave a different slope to glass. The MRD linked to a chemostat can be used to compare directly bacterial adhesion to potential biomaterials. Variable depletion of the interacting stream should be taken into account in the interpretation of results. Divalent cation concentration, substrate properties and flow rate were important determinants of the comparative adhesion of Staph. epidermidis RP62A to surfaces.     

Efficiency of platelet adhesion to fibrinogen depends on both cell activation and flow

The kinetics of adhesion of platelets to fibrinogen (Fg) immobilized on polystyrene latex beads (Fg-beads) was determined in suspensions undergoing Couette flow at well-defined homogeneous shear rates. The efficiency of platelet adhesion to Fg-beads was compared for ADP-activated versus "resting" platelets. The effects of the shear rate (100-2000 s(-1)), Fg density on the beads (24-2882 Fg/microm(2)), the concentration of ADP used to activate the platelets, and the presence of soluble fibrinogen were assessed. "Resting" platelets did not specifically adhere to Fg-beads at levels detectable with our methodology. The apparent efficiency of platelet adhesion to Fg-beads readily correlated with the proportion of platelets "quantally" activated by doses of ADP, i.e., only ADP-activated platelets appeared to adhere to Fg-beads, with a maximal adhesion efficiency of 6-10% at shear rates of 100-300 s(-1), decreasing with increasing shear rates up to 2000 s(-1). The adhesion efficiency was found to decrease by only threefold when decreasing the density of Fg at the surface of the beads by 100-fold, with only moderate decreases in the presence of physiologic concentrations of soluble Fg. These adhesive interactions were also compared using activated GPIIbIIIa-coated beads. Our studies provide novel model particles for studying platelet adhesion relevant to hemostasis and thrombosis, and show how the state of activation of the platelet and the local flow conditions regulate Fg-dependent adhesion.     

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.