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.     

Modeling the reversible kinetics of neutrophil aggregation under hydrodynamic shear.

Neutrophil emigration into inflamed tissue is mediated by beta 2-integrin and L-selectin adhesion receptors. Homotypic neutrophil aggregation is also dependent on these molecules, and it provides a model system in which to study adhesion dynamics. In the current study we formulated a mathematical model for cellular aggregation in a linear shear field based on Smoluchowski's two-body collision theory. Neutrophil suspensions activated with chemotactic stimulus and sheared in a cone-plate viscometer rapidly aggregate. Over a range of shear rates (400-800 s-1), approximately 90% of the single cells were recruited into aggregates ranging from doublets to groupings larger than sextuplets. The adhesion efficiency fit to these kinetics reached maximum levels of > 70%. Formed aggregates remained intact and resistant to shear up to 120 s, at which time they spontaneously dissociated back to singlets. The rate of cell disaggregation was linearly proportional to the applied shear rate, and it was approximately 60% lower for doublets as compared to larger aggregates. By accounting for the time-dependent changes in adhesion efficiency, disaggregation rate, and the effects of aggregate geometry, we succeeded in predicting the reversible kinetics of aggregation over a wide range of shear rates and cell concentrations. The combination of viscometry with flow cytometry and mathematical analysis as presented here represents a novel approach to differentiating between the effects of hydrodynamics and the intrinsic biological processes that control cell adhesion.     

Dynamics of neutrophil aggregation in couette flow revealed by videomicroscopy: effect of shear rate on two-body collision efficiency and doublet lifetime

During inflammation, neutrophil capture by vascular endothelial cells is dependent on L-selectin and beta(2)-integrin adhesion receptors. One of us (S.I.S.) previously demonstrated that homotypic neutrophil aggregation is analogous to this process in that it is also mediated by these receptors, thus providing a model for studying the dynamics of neutrophil adhesion. In the present work, we set out to confirm the hypothesis that cell-cell adhesion via selectins serves to increase the lifetimes of neutrophil doublets formed through shear-induced two-body collisions. In turn, this would facilitate the engagement of more stable beta(2)-integrin bonds and thus increase the two-body collision efficiency (fraction of collisions resulting in the formation of nonseparating doublets). To this end, suspensions of unstimulated neutrophils were subjected to a uniform shear field in a transparent counter-rotating cone and plate rheoscope, and the formation of doublets and growth of aggregates recorded using high-speed videomicroscopy. The dependence of neutrophil doublet lifetime and two-body collision-capture efficiency on shear rate, G, from 14 to 220 s(-1) was investigated. Bond formation during a two-body collision was indicated by doublets rotating well past the orientation predicted for break-up of doublets of inert spheres. A striking dependence of doublet lifetime on shear rate was observed. At low shear (G = 14 s(-1)), no collision capture occurred, and doublet lifetimes were no different from those of neutrophils pretreated with a blocking antibody to L-selectin, or in Ca(++)-depleted EDTA buffers. At G > or = 66 s(-1), doublet lifetimes increased, with increasing G reaching values twice those for the L-selectin-blocked controls. This correlated with capture efficiencies in excess of 20%, and, at G > or = 110 s(-1), led to the rapid formation of large aggregates, and this in the absence of exogenous chemotactic stimuli. Moreover, the aggregates almost completely broke up when the shear rate was reduced below 66 s(-1). Partial inhibition of aggregate formation was achieved by blocking beta(2)-integrin receptors with antibody. By direct observation of the shear-induced interactions between neutrophils, these data reveal that steady application of a threshold level of shear rate is sufficient to support homotypic neutrophil aggregation.     

Neutrophil string formation: hydrodynamic thresholding and cellular deformation during cell collisions

Neutrophils unexpectedly display flow-enhanced adhesion (hydrodynamic thresholding) to L-selectin in rolling or aggregation assays. We report that the primary collision efficiency (epsilon) of flowing neutrophils with preadhered neutrophils on intercellular adhesion molecule-1 (ICAM-1) or fibrinogen also displayed a maximum of epsilon approximately 0.4-0.45 at a wall shear rate of 100 s(-1), an example of thresholding. Primary collision lifetime with no detectable bonding decreased from 130 to 10 ms as wall shear rate increased from 30 to 300 s(-1), whereas collision lifetimes with bonding decreased from 300 to 100 ms over this shear range using preadhered neutrophils on ICAM-1, with similar results for fibrinogen. Antibodies against L-selectin, but not against CD11a, CD11b, or CD18, reduced epsilon at 100 s(-1) by >85%. High resolution imaging detected large scale deformation of the flowing neutrophil during the collision at 100 s(-1) with the apparent contact area increasing up to approximately 40 microm(2). We observed the formation of long linear string assemblies of neutrophils downstream of neutrophils preadhered to ICAM-1, but not fibrinogen, with a maximum in string formation at 100 s(-1). Secondary capture events to the ICAM-1 or fibrinogen coated surfaces after primary collisions were infrequent and short lived, typically lasting from 500 to 3500 ms. Between 5 and 20% of neutrophil interactions with ICAM-1 substrate converted to firm arrest (>3500 ms) and greatly exceeded that observed for fibrinogen, thus defining the root cause of poor string formation on fibrinogen at all shear rates. Additionally, neutrophils mobilized calcium after incorporation into strings. Static adhesion also caused calcium mobilization, as did the subsequent onset of flow. To our knowledge, this is the first report of 1). hydrodynamic thresholding in neutrophil string formation; 2). string formation on ICAM-1 but not on fibrinogen; 3). large cellular deformation due to collisions at a venous shear rate; and 4), mechanosensing through neutrophil beta(2)-integrin/adhesion. The increased contact area during deformation was likely responsible for the hydrodynamic threshold observed in the primary collision efficiency since no increase in primary collision lifetime was detected as shear forces were increased (for either surface coating).     

Kinetics of beta2-integrin and L-selectin bonding during neutrophil aggregation in shear flow.

Activated neutrophils aggregate in a shear field via bonding of L-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) followed by a more stable bonding of LFA-1 (CD11a/CD18) to intercellular adhesion molecule 3 (ICAM-3) and Mac-1 (CD11b/CD18) to an unknown counter receptor. Assuming that the Mac-1 counter receptor is ICAM-3-like in strength and number, rate processes were deconvoluted from neutrophil homoaggregation data for shear rates (G) of 100-3000 s-1 with a two-body hydrodynamic collision model (. Biophys. J. 73:2819-2835). For integrin-mediated aggregation (characteristic bond strength of 5 microdynes) in the absence of L-selectin contributions, an average forward rate of kf = 1.57 x 10(-12) cm2/s predicted the measured efficiencies for G = 100-800 s-1. For a selectin bond formation rate constant equal to the integrin bond formation rate constant, the colloidal stability of unactivated neutrophils was satisfied for a reverse rate of the L-selectin-PGSL bond corresponding to an average bond half-life of 10 ms at a characteristic bond strength of 1 microdyne. Colliding neutrophils initially bridged by at least one L-selectin-PSGL-1 bond were calculated to rotate from 8 to 50 times at G = 400 to 3000 s-1, respectively, before obtaining mechanical stability in sheared fluid of either 0.75 or 1.75 cP viscosity. Thus for G > 400 s-1, the interaction time needed for the rotating aggregates to become stable was relatively constant at 52.5 +/- 8.5 ms, largely independent of shear rate or shear stress. Aggregation data and the colloidal stability criterion can provide a consistent set of forward and reverse rate constants and characteristic bond strengths for a known time-dependent stoichiometry of receptors on cells interacting in a shear flow field.     

Prostaglandin E2 suppresses β1-integrin expression via E-prostanoid receptor in human monocytes/macrophages

β₁-Integrins mediate cell attachment to different extracellular matrix proteins, intracellular proteins, and intercellular adhesions. Recently, it has been reported that prostaglandin E₂ (PGE₂) has anti-inflammatory properties such as inhibition of the expression of adhesion molecules or production of chemokines. However, the effect of PGE₂ on the expression of β₁-integrin remains unknown. In this study, we investigated the effects of PGE₂ on the expression of β₁-integrin in the human monocytic cell line THP-1 and in CD14⁺ monocytes/macrophages in human peripheral blood. For this, we examined the role of four subtypes of PGE₂ receptors and E-prostanoid (EP) receptors on PGE₂-mediated inhibition. We found that PGE₂ significantly inhibited the expression of β₁-integrin, mainly through EP₄ receptors in THP-1 cells and CD14⁺ monocytes/macrophages in human peripheral blood. We suggest that PGE₂ has anti-inflammatory effects, leading to the inhibited expression of β₁-integrin in human monocytes/macrophages, and that the EP₄ receptor may play an important role in PGE₂-mediated inhibition.     

Distinct role of hydrodynamic shear in leukocyte-facilitated tumor cell extravasation

Previously, we found polymorphonuclear neutrophils (PMNs) increased melanoma cell extravasation under flow conditions (Intl J Cancer 106: 713–722, 2003). In this study, we characterized the effect of hydrodynamic shear on PMN-facilitated melanoma extravasation using a novel flow-migration assay. The effect of shear stress and shear rate on PMN-facilitated melanoma extravasation was studied by increasing the medium viscosity with dextran to increase shear stress independently of shear rate. Under fixed shear rate conditions, melanoma cell extravasation did not change significantly. In contrast, the extravasation level increased at a fixed shear stress but with a decreasing shear rate. PMN-melanoma aggregation and adhesion to the endothelium via ₂-integrin/intracellular adhesion molecule-1 (ICAM-1) interactions were also studied. Lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, whereas Mac-1 (CD11b/CD18) affected prolonged PMN-melanoma aggregation. Blockage of E-selectin or ICAM-1 on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. We have found PMN-melanoma adhesion is correlated with the inverse of shear rate, whereas the PMN-endothelial adhesion correlated with shear stress. Interleukin-8 (IL-8) also influenced PMN-melanoma cell adhesion. Functional blocking of the PMN IL-8 receptors, CXCR1 and CXCR2, decreased the level of Mac-1 upregulation on PMNs while in contact with melanoma cells and reduced melanoma extravasation. We have found PMN-facilitated melanoma adhesion to be a complex multistep process that is regulated by both microfluid mechanics and biology. neutrophil; melanoma; shear stress; shear rate; ₂-integrins; intracellular adhesion molcule-1; CXCR1/2; adhesion; migration     

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion

Background

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β₂-integrin activation and function in neutrophilic firm adhesion to endothelium.

Methods

Human polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β₂-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β₂-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined.

Results

Human neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly.

Conclusions

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion.

General significance

This is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.     

Fluid shear- and time-dependent modulation of molecular interactions between PMNs and colon carcinomas

This study compares the effects offluid shear on the kinetics, adhesion efficiency, stability, andmolecular requirements of polymorphonuclear leukocyte (PMN) binding totwo colon adenocarcinoma cell-lines, theCD54-negative/sLe^x-bearing LS174T cells and theCD54-expressing/sLe^x-low HCT-8 cells. The efficiency ofPMN-colon carcinoma heteroaggregation decreases with increasing shear,with PMNs binding HCT-8 more efficiently than LS174T cells at low shear(50-200 s¹). In the low shear regime, CD11b issufficient to mediate PMN binding to LS174T cells. In contrast, bothCD11a and CD11b contribute to PMN-HCT-8 heteroaggregation, with CD54 onHCT-8 cells acting as a CD11a ligand at early time points. At highshear, only PMN-LS174T heteroaggregation occurs, which is initiated byPMN L-selectin binding to a sialylated, O-linked, protease-sensitiveligand on LS174T cells. PMN-LS174T heteroaggregation is primarilydependent on the intercellular contact duration (or shear rate),whereas PMN-HCT-8 binding is a function of both the intercellularcontact duration and the applied force (or shear stress). Cumulatively, these studies suggest that fluid shear modulates the kinetics andmolecular mechanisms of PMN-colon carcinoma cell aggregation.

     

Shear stress and shear rate differentially affect the multi-step process of leukocyte-facilitated melanoma adhesion

Previous studies have shown that neutrophils (PMNs) facilitate melanoma cell extravasation [M.J. Slattery, C. Dong, Neutrophils influence melanoma adhesion and migration under flow conditions, Intl. J. Cancer 106 (2003) 713–722] Little is known, however, about the specific interactions between PMNs, melanoma and the endothelium (EC) or the molecular mechanism involved under flow conditions. The aim of this study is to investigate a “two-step adhesion” hypothesis that involves initial PMN tethering on the EC and subsequent melanoma cells being captured by tethered PMNs. Different effects of hydrodynamic shear stress and shear rate were analyzed using a parallel-plate flow chamber. Results indicate a novel finding that PMN-facilitated melanoma cell arrest on the EC is modulated by shear rate, which is inversely-proportional to cell–cell contact time, rather than by the shear stress, which is proportional to the force exerted on formed bonds. β₂ integrins/ICAM-1 adhesion mechanisms were examined and the results indicate LFA-1 and Mac-1 cooperate to mediate the PMN–EC–melanoma interactions under shear conditions. In addition, endogenously produced IL-8 contributes to PMN-facilitated melanoma arrest on the EC through the CXC chemokine receptors 1 and 2 (CXCR1 and CXCR2) on PMN. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines and PMN–melanoma adhesion in the recruitment of metastatic cancer cells to the EC.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

LFA-1/ICAM-3 mediates neutrophil homotypic aggregation under fluid shear stress

We found that human neutrophils undergo homotypic aggregation by loading the physiological range of fluid shear stress (12–30 dynes/cm²). Under the fluid shear stress, an increase of intracellular Ca²⁺ concentration of neutrophils was observed. This increase of intracellular Ca²⁺ concentration was caused by Ca²⁺ influx, and the blockage of the flux by NiCl₂ suppressed the neutrophil homotypic aggregation. Furthermore, this neutrophil aggregation under fluid shear stress was completely inhibited by pretreatment with antibody against LFA-1 or ICAM-3. These results suggested that NiCl₂-sensitive Ca²⁺ channel played an important role in LFA-1/ICAM-3-mediated neutrophil homotypic aggregation under fluid shear stress. © 1996 Wiley-Liss, Inc.     

The effects of chemotactic factors on the adhesiveness of rabbit neutrophil granulocytes.

A range of chemotactic factors has been shown to affect the adhesion of rabbit peritoneal neutrophil granulocytes to cultured endothelial cells and to serum-coated glass. At chemotactically optimal concentrations, α_s-casein, β-casein, alkali denatured human serum albumin (HSA) and several synthetic formyl-peptides reduced the number of adherent neutrophils after 30 min to around 50% of control values. These effects were still observed after neutrophils, but not endothelium or serum-coated glass had been exposed to chemotactic factors and washed before use in assays. Two non-chemotactic analogues, native HSA and a non-formyl-peptide were ineffective. The dose responses for adhesion after 30 min in the presence of α_s-casein and formyl-methionyl-leucyl-phenylalanine (FMLP) were found to be inversely related to those for migration towards these substances. After incubation for 60 min in high (10^?8–10^?7 M) concentrations of FMLP, neutrophil adhesion was found to be enhanced. Neutrophil aggregation was also affected by the presence of chemotactic factors in a similar time- and dose-dependent manner to the adhesion to substratum assays. Using FMLP, it was also shown that the timing of the adhesive changes depended on the concentration of chemotactic factor present.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

Fluid shear stress induces actin polymerization in human neutrophils

We have previously reported that a physiological range of shear stress induces neutrophil homotypic aggregation mediated by lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-3 (ICAM-3) interactions. To further characterize the homotypic aggregation, actin polymerization was investigated in neutrophils stimulated by shear stress in comparison with formyl-methionyl-leucyl-phenylalanine (fMLP). In fMLP-stimulated neutrophils, actin polymerization was localized in the pseudopods, and this reaction was not mediated by a cytosolic level of Ca²⁺. In contrast to fMLP stimulation, the actin polymerization induced by shear stress in a cone-plate viscometer was localized in cell-cell contact regions, and this polymerization required the increase of intracellular Ca²⁺. This shear stress-induced actin polymerization was not observed when neutrophils were pretreated with anti-LFA-1 or anti-ICAM-3 antibody. In conclusion, LFA-1 and ICAM-3 interaction mediated by the increase of [Ca²⁺]i generated the intercellular signal in order to accumulate F-actin in the cell-cell contact regions. © 1996 Wiley-Liss, Inc.     

Hypotonicity induces L-selectin shedding in human neutrophils

Expression levels of adhesion molecules on neutrophils are affectedunder various conditions, including ischemia, possibly becauseof associated increases in cell volume. We examined the effects of cellswelling in hypotonic media on the level of L-selectin (CD62L) and₂-integrin (CD18) on human neutrophils. In hypotonic media, neutrophils shed L-selectin. The shedding was greatly reduced by30 µM RO31-9790, the metalloprotease (sheddase) inhibitor. Hypotonicity-induced L-selectin shedding was also time and tonicity dependent. Decreasing tonicity caused increased shedding. In 0.6× medium (0.6× the normal tonicity of 300 mosmol/kgH₂O),shedding increased over a 2-h period, after which >70% of theneutrophils had lost L-selectin. In contrast to L-selectin, the levelof ₂-integrin on the neutrophil surface was notsignificantly affected. Thus L-selectin shedding, which occurs onneutrophil activation and is usually accompanied by₂-integrin upregulation, was selectively induced byhypotonicity without a corresponding effect on₂-integrin.

     

Influence of cavitation and high shear stress on HSA aggregation behavior

Neither the influence of high shear rates nor the impact of cavitation on protein aggregation is fully understood. The effect of cavitation bubble collapse‐derived hydroxyl radicals on the aggregation behavior of human serum albumin (HSA) was investigated. Radicals were generated by pumping through a micro‐orifice, ultra‐sonication, or chemically by Fenton's reaction. The amount of radicals produced by the two mechanical methods (0.12 and 11.25 nmol/(L min)) was not enough to change the protein integrity. In contrast, Fenton's reaction resulted in 382 nmol/(L min) of radicals, inducing protein aggregation. However, the micro‐orifice promoted the formation of soluble dimeric HSA aggregates. A validated computational fluid dynamic model of the orifice revealed a maximum and average shear rate on the order of 10⁸ s^?1 and 1.2 × 10⁶ s^?1, respectively. Although these values are among the highest ever reported in the literature, dimer formation did not occur when we used the same flow rate but suppressed cavitation. Therefore, aggregation is most likely caused by the increased surface area due to cavitation‐mediated bubble growth, not by hydroxyl radical release or shear stress as often reported.     

Platelet adhesive dynamics. Part II: high shear-induced transient aggregation via GPIbalpha-vWF-GPIbalpha bridging

A three-dimensional multiscale computational model, platelet adhesive dynamics (PAD), is developed and applied in Part I and Part II articles to characterize and quantify key biophysical aspects of GPIbα-von-Willebrand-factor (vWF)-mediated interplatelet binding at high shear rates, a necessary and enabling step that initiates shear-induced platelet aggregation. In this article, an adhesive dynamics model of the transient aggregation of two unactivated platelets via GPIbα-vWF-GPIbα bridging is developed and integrated with the three-dimensional hydrodynamic flow model discussed in Part I. Platelet binding efficiencies predicted by PAD are in good agreement with platelet aggregation behavior observed experimentally, as documented in the literature. Deviations from average vWF ligand size or healthy GPIbα-vWF-A1 binding kinetics are observed in simulations to have significant effects on the dynamics of transient platelet aggregation, i.e., the efficiency of platelet aggregation and characteristics of bond failure, in ways that typify diseased conditions. The GPIbα-vWF-A1 bond formation rate is predicted to have piecewise linear dependence on the prevailing fluid shear rate, with a sharp transition in fluid shear dependency at 7200 s⁻¹. Interplatelet bond force-loading is found to be complex and highly nonlinear. These results demonstrate PAD as a powerful predictive modeling tool for elucidating platelet adhesive phenomena under flow.     

Two-dimensional kinetics of beta 2-integrin and ICAM-1 bindings between neutrophils and melanoma cells in a shear flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta 2-integrin (lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta 2-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta 2-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.