Mechanical anchoring strength of L-selectin, beta2 integrins, and CD45 to neutrophil cytoskeleton and membrane.

The strength of anchoring of transmembrane receptors to cytoskeleton and membrane is important in cell adhesion and cell migration. With micropipette suction, we applied pulling forces to human neutrophils adhering to latex beads that were coated with antibodies to CD62L (L-selectin), CD18 (beta2 integrins), or CD45. In each case, the adhesion frequency between the neutrophil and bead was low, and our Monte Carlo simulation indicates that only a single bond was probably involved in every adhesion event. When the adhesion between the neutrophil and bead was ruptured, it was very likely that receptors were extracted from neutrophil surfaces. We found that it took 1-2 s to extract an L-selectin at a force range of 25-45 pN, 1-4 s to extract a beta2 integrin at a force range of 60-130 pN, and 1-11 s to extract a CD45 at a force range of 35-85 pN. Our results strongly support the conclusion that, during neutrophil rolling, L-selectin is unbound from its ligand when the adhesion between neutrophils and endothelium is ruptured.     

Enhancement of L-selectin, but not P-selectin, bond formation frequency by convective flow

L-selectin-mediated leukocyte rolling has been proposed to require a high rate of bond formation compared to that of P-selectin to compensate for its much higher off-rate. To test this hypothesis, a microbead system was utilized to measure relative L-selectin and P-selectin bond formation rates on their common ligand P-selectin glycoprotein ligand-1 (PSGL-1) under shear flow. Using video microscopy, we tracked selectin-coated microbeads to detect the formation frequency of adhesive tether bonds. From velocity distributions of noninteracting and interacting microbeads, we observed that tether bond formation rates for P-selectin on PSGL-1 decreased with increasing wall shear stress, from 0.14 ± 0.04 bonds/μm at 0.2 dyn/cm² to 0.014 ± 0.003 bonds/μm at 1.0 dyn/cm². In contrast, L-selectin tether bond formation increased from 0.017 ± 0.005 bonds/μm at 0.2 dyn/cm² to 0.031 ± 0.005 bonds/μm at 1.0 dyn/cm². L-selectin tether bond formation rates appeared to be enhanced by convective transport, whereas P-selectin rates were inhibited. The transition force for the L-selectin catch-slip transition of 44 pN/bond agreed well with theoretical models (Pereverzev et al. 2005. Biophys. J. 89:1446-1454). Despite catch bond behavior, hydrodymanic shear thresholding was not detected with L-selectin beads rolling on PSGL-1. We speculate that shear flow generated compressive forces may enhance L-selectin bond formation relative to that of P-selectin and that L-selectin bonds with PSGL-1 may be tuned for the compressive forces characteristic of leukocyte-leukocyte collisions during secondary capture on the blood vessel wall. This is the first report, to our knowledge, comparing L-selectin and P-selectin bond formation frequencies in shear flow.     

Low spring constant regulates P-selectin-PSGL-1 bond rupture

Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, and tumor metastasis. Although the bond rupture has been well known at high loading rate r_f (≥10² pN/s), defined as the product of spring constant k and retract velocity v, how the low r_f (<10² pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap assay was used to quantify the bond rupture at r_f ≤ 20 pN/s with low k (∼10⁻³-10⁻² pN/nm) when P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) were respectively coupled onto two glass microbeads. Our data indicated that the bond rupture force f retained the similar values when r_f increased up to 20 pN/s. It was also found that f varied with different combinations of k and v even at the same r_f. The most probable force, f*, was enhanced with the spring constant when k < 47.0 × 10⁻³ pN/nm, indicating that the bond dissociation at low r_f was spring constant dependent and that bond rupture force depended on both the loading rate and the mechanical compliance of force transducer. These results provide new insights into understanding the P-selectin glycoprotein ligand 1 bond dissociation at low r_f or k.     

Multi-scale simulation of L-selectin–PSGL-1-dependent homotypic leukocyte binding and rupture

L-selectin–PSGL-1-mediated polymorphonuclear (PMN) leukocyte homotypic interactions potentiate the extent of PMN recruitment to endothelial sites of inflammation. Cell–cell adhesion is a complex phenomenon involving the interplay of bond kinetics and hydrodynamics. As a first step, a 3-D computational model based on the Immersed Boundary Method is developed to simulate adhesion-detachment of two PMN cells in quiescent conditions. Our simulations predict that the total number of bonds formed is dictated by the number of available receptors (PSGL-1) when ligands (L-selectin) are in excess, while the excess amount of ligands influences the rate of bond formation. Increasing equilibrium bond length results in a higher number of receptor–ligand bonds due to an increased intercellular contact area. On-rate constants determine the rate of bond formation, while off-rates control the average number of bonds by modulating bond lifetimes. Application of an external pulling force leads to time-dependent on- and off-rates and causes bond rupture. Moreover, the time required for bond rupture in response to an external force is inversely proportional to the applied load and decreases with increasing off-rate.     

Regulation of catch bonds by rate of force application

The current paradigm for receptor-ligand dissociation kinetics assumes off-rates as functions of instantaneous force without impact from its prior history. This a priori assumption is the foundation for predicting dissociation from a given initial state using kinetic equations. Here we have invalidated this assumption by demonstrating the impact of force history with single-bond kinetic experiments involving selectins and their ligands that mediate leukocyte tethering and rolling on vascular surfaces during inflammation. Dissociation of bonds between L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) loaded at a constant ramp rate to a constant hold force behaved as catch-slip bonds at low ramp rates that transformed to slip-only bonds at high ramp rates. Strikingly, bonds between L-selectin and 6-sulfo-sialyl Lewis X were impervious to ramp rate changes. This ligand-specific force history effect resembled the effect of a point mutation at the L-selectin surface (L-selectinA108H) predicted to contact the former but not the latter ligand, suggesting that the high ramp rate induced similar structural changes as the mutation. Although the A108H substitution in L-selectin eliminated the ramp rate responsiveness of its dissociation from PSGL-1, the inverse mutation H108A in P-selectin acquired the ramp rate responsiveness. Our data are well explained by the sliding-rebinding model for catch-slip bonds extended to incorporate the additional force history dependence, with Ala-108 playing a pivotal role in this structural mechanism. These results call for a paradigm shift in modeling the mechanical regulation of receptor-ligand bond dissociation, which includes conformational coupling between binding pocket and remote regions of the interacting molecules.     

Tyrosine Replacement of PSGL-1 Reduces Association Kinetics with P- and L-Selectin on the Cell Membrane

Binding of selectins to P-selectin glycoprotein ligand-1 (PSGL-1) mediates tethering and rolling of leukocytes on the endothelium during inflammation. Previous measurements obtained with a flow-chamber assay have shown that mutations of three tyrosines at the PSGL-1 N-terminus (Y46, Y48, and Y51) increase the reverse rates and their sensitivity to the force of bonds with P- and L-selectin. However, the effects of these mutations on the binding affinities and forward rates have not been studied. We quantified these effects by using an adhesion frequency assay to measure two-dimensional affinity and kinetic rates at zero force. Wild-type PSGL-1 has 2.2- to 8.5-fold higher binding affinities for P- and L-selectin than PSGL-1 mutants with two of three tyrosines substituted by phenylalanines, and 9.6- to 49-fold higher affinities than the PSGL-1 mutant with all three tyrosines replaced. In descending order, the affinity decreased from wild-type to Y48/51F, Y46/51F, Y46/48F, and Y46/48/51F. The affinity differences were attributed to major changes in the forward rate and minor changes in the reverse rate, suggesting that these tyrosines regulate the accessibility of PSGL-1 to P- and L-selectin via electrostatic interactions, which is supported by molecular-dynamics simulations. Our results provide insights into the structure-function relationship of receptor-ligand binding at a single-residue level.     

Effect of Extracellular pH on Selectin Adhesion: Theory and Experiment

Selectins mediate circulatory leukocyte trafficking to sites of inflammation and trauma, and the extracellular microenvironments at these sites often become acidic. In this study, we investigated the influence of slightly acidic pH on the binding dynamics of selectins (P-, L-, and E-selectin) to P-selectin glycoprotein ligand-1 (PSGL-1) via computational modeling (molecular dynamics) and experimental rolling assays under shear in vitro. The P-selectin/PSGL-1 binding is strengthened at acidic pH, as evidenced by the formation of a new hydrogen bond (seen computationally) and the observed decrease in the rolling velocities of model cells. In the case of L-selectin/PSGL-1 binding dynamics, the binding strength and frequency increase at acidic pH, as indicated by the greater cell-rolling flux of neutrophils and slower rolling velocities of L-selectin-coated microspheres, respectively. The cell flux is most likely due to an increased population of L-selectin in the high-affinity conformation as pH decreases, whereas the velocities are due to increased L-selectin/PSGL-1 contacts. In contrast to P- and L-selectin, the E-selectin/PSGL-1 binding does not exhibit significant changes at acidic pH levels, as shown both experimentally and computationally.     

A direct comparison of selectin-mediated transient, adhesive events using high temporal resolution

Leukocyte capture and rolling on the vascular endothelium is mediated principally by the selectin family of cell adhesion receptors. In a parallel plate flow chamber, neutrophil rolling on purified selectins or a selectin-ligand substrate was resolved by high speed videomicroscopy as a series of ratchet-like steps with a characteristic time constant (Kaplanski, G., C. Farnarier, O. Tissot, A. Pierres, A.-M. Benoliel, M. C. Alessi, S. Kaplanski, and P. Bongrand. 1993. Biophys. J. 64:1922-1933; Alon, R., D. A. Hammer, and T. A. Springer. 1995. Nature (Lond.). 374:539-542). Under shear, neutrophil arrests due to bond formation events were as brief as 4 ms. Pause time distributions for neutrophils tethering on P-, E-, L-selectin, or peripheral node addressin (PNAd) were compared at estimated single bond forces ranging from 37 to 250 pN. Distributions of selectin mediated pause times were fit to a first order exponential, resulting in a molecular dissociation constant (k(off)) for the respective selectin as a function of force. At estimated single bond forces of 125 pN and below, all three selectin dissociation constants fit the Bell and Hookean spring models of force-driven bond breakage equivalently. Unstressed k(off) values based on the Bell model were 2.4, 2.6, 2.8, 3.8 s(-1) for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Bond separation distances (reactive compliance) were 0.39, 0.18, 1.11, 0.59 A for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Dissociation constants for L-selectin and P-selectin at single bond forces above 125 pN were considerably lower than either Bell or Hookean spring model predictions, suggesting the existence of two regimes of reactive compliance. Additionally, interactions between L-selectin and its leukocyte ligand(s) were more labile in the presence of flow than the L-selectin endothelial ligand, PNAd, suggesting that L-selectin ligands may have different molecular and mechanical properties. Both types of L-selectin bonds had a higher reactive compliance than P-selectin or E-selectin bonds.     

Replacing a lectin domain residue in L-selectin enhances binding to P-selectin glycoprotein ligand-1 but not to 6-sulfo-sialyl Lewis x

Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Here, we show that substituting one residue (A108H) in the lectin domain of L-selectin increased its force-free affinity for a glycosulfopeptide binding site (2-GSP-6) on P-selectin glycoprotein ligand-1 (PSGL-1) but not for a sulfated-glycan binding site (6-sulfo-sialyl Lewis x) on peripheral node addressin. The increased affinity of L-selectinA108H for 2-GSP-6 was due to a faster on-rate and to a slower off-rate that increased bond lifetimes in the absence of force. Rather than first prolonging (catching) and then shortening (slipping) bond lifetimes, increasing force monotonically shortened lifetimes of L-selectinA108H bonds with 2-GSP-6. When compared with microspheres bearing L-selectin, L-selectinA108H microspheres rolled more slowly and regularly on 2-GSP-6 at low flow rates. A reciprocal substitution in P-selectin (H108A) caused faster microsphere rolling on 2-GSP-6. These results distinguish molecular mechanisms for L-selectin to bind to PSGL-1 and peripheral node addressin and explain in part the shorter lifetimes of PSGL-1 bonds with L-selectin than P-selectin.     

Single molecule characterization of P-selectin/ligand binding

P-selectin expressed on activated platelets and vascular endothelium mediates adhesive interactions to polymorphonuclear leukocytes (PMNs) and colon carcinomas critical to the processes of inflammation and blood-borne metastasis, respectively. How the overall adhesiveness (i.e. the avidity) of receptor/ligand interactions is controlled by the affinity of the individual receptors to single ligands is not well understood. Using single molecule force spectroscopy, we probed in situ both the tensile strength and off-rate of single P-selectin molecules binding to single ligands on intact human PMNs and metastatic colon carcinomas and compared them to the overall avidity of these cells for P-selectin substrates. The use of intact cells rather than purified proteins ensures the proper orientation and preserves post-translational modifications of the P-selectin ligands. The P-selectin/PSGL-1 interaction on PMNs was able to withstand forces up to 175 pN and had an unstressed off-rate of 0.20 s(-1). The tensile strength of P-selectin binding to a novel O-linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate was 2.78 s(-1). Monte Carlo simulations of receptor/ligand bond rupture under constant loading rate for both P-selectin/PSGL-1 and P-selectin/LS174T ligand binding give distributions and mean rupture forces that are in accord with experimental data. The pronounced differences in the affinity for P-selectin/ligand binding provide a mechanistic basis for the differential abilities of PMNs and carcinomas to roll on P-selectin substrates under blood flow conditions and underline the requirement for single molecule affinity measurements.     

The viscoelastic properties of microvilli are dependent upon the cell-surface molecule

We studied at nanometer resolution the viscoelastic properties of microvilli and tethers pulled from myelogenous cells via P-selectin glycoprotein ligand 1 (PSGL-1) and found that in contrast to pure membrane tethers, the viscoelastic properties of microvillus deformations are dependent upon the cell-surface molecule through which load is applied. A laser trap and polymer bead coated with anti-PSGL-1 (KPL-1) were used to apply step loads to microvilli. The lengthening of the microvillus in response to the induced step loads was fitted with a viscoelastic model. The quasi-steady state force on the microvillus at any given length was approximately fourfold lower in cells treated with cytochalasin D or when pulled with concanavalin A-coated rather than KPL-1-coated beads. These data suggest that associations between PSGL-1 and the underlying actin cytoskeleton significantly affect the early stages of leukocyte deformation under flow.     

Distinct sulfation requirements of selectins disclosed using cells that support rolling mediated by all three selectins under shear flow. L-selectin prefers carbohydrate 6-sulfation totyrosine sulfation,whereas p-selectin does not

l- and P-selectin are known to require sulfation in their ligand molecules. We investigated the significance of carbohydrate 6-sulfation and tyrosine sulfation in selectin-mediated cell adhesion. COS-7 cells were genetically engineered to express P-selectin glycoprotein ligand-1 (PSGL-1) or its mutant in various combinations with 6-O-sulfotransferase (6-Sul-T) and/or alpha1-->3fucosyltransferase VII (Fuc-T VII). The cells transfected with PSGL-1, 6-Sul-T, and Fuc-T VII cDNAs supported rolling mediated by all three selectins and provided the best experimental system so far to estimate kinetic parameters in selectin-mediated cell adhesion for all three selectins using the identical rolling substrate and to compare the ligand specificity of each selectin. L-selectin-mediated rolling was drastically impaired if the cells lacked carbohydrate 6-sulfation elaborated by 6-Sul-T, but not affected when PSGL-1 was replaced with a mutant lacking three tyrosine residues at its NH(2) terminus. L-selectin-mediated adhesion was also hardly affected by mocarhagin treatment of the cells, which cleaved a short peptide containing sulfated tyrosine residues from PSGL-1. In contrast, P-selectin-mediated rolling was abolished when PSGL-1 was either mutated or cleaved by mocarhagin at its NH(2) terminus, whereas the cells expressing PSGL-1 and Fuc-T VII but not 6-Sul-T showed only a modest decrease in P-selectin-mediated adhesion. These results indicate that L-selectin prefers carbohydrate 6-sulfation much more than tyrosine sulfation, whereas P-selectin favors tyrosine sulfation in the PSGL-1 molecule far more than carbohydrate 6-sulfation. E-selectin-mediated adhesion was sulfation-independent requiring only Fuc-T VII, and thus the three members of the selectin family have distinct requirements for ligand sulfation.     

Differences in zero-force and force-driven kinetics of ligand dissociation from beta-galactoside-specific proteins (plant and animal lectins, immunoglobulin G) monitored by plasmon resonance and dynamic single molecule force microscopy

Protein-carbohydrate interactions are involved in diverse regulatory processes. To help understand the mechanics and kinetics of dissociation of receptor-ligand complexes, we have analyzed the separation of lactose and the N-glycan chains of asialofetuin (ASF) from three lectins and an immunoglobulin G fraction by surface plasmon resonance at zero force and by atomic force microscopy with variations of the external force. While the (AB)2 agglutinins from Ricinus communis (RCA) and Viscum album (VAA) show structural homology, the homodimeric galectin-1 from bovine heart (BHL) has no similarity to the two plant lectins except for sharing this monosaccharide specificity. The beta-galactoside-binding immunoglobulin G (IgG) fraction from human serum provides a further model system with distinct binding-site architecture. The k(off) constants for the two plant agglutinins were independent of the nature of the ligand at 1.1-1.3 x 10(-3) s(-1), whereas the geometry of ligand and binding site presentation affected this parameter for BHL (0.5 x 10(-3) s(-1) for lactose and 1 x 10(-3) s(-1) for ASF) and IgG (1.3 x 10(-3) s(-1) for lactose and 0.55 x 10(-3) s(-1) for ASF). When assessing comparatively the rupture forces at a loading rate of 3 nN/s with lactose as ligand, 34 +/- 6 pN (BHL), 36 +/- 4 pN (IgG), 47 +/- 7 pN (VAA), and 58 +/- 9 pN (RCA) were measured. For the same loading rate the rupture forces for the receptor-ASF interactions were found to be 37 +/- 3 pN (BHL), 43 +/- 5 pN (VAA), 45 +/- 6 pN (IgG), and 65 +/- 9 pN (RCA). The variation of the pulling velocity revealed in all cases a linear dependence between the rupture force and the natural logarithm of the loading rate. Performing probability density and Monte Carlo calculations, the potential barrier widths, which determine the inverse dynamic dependence with the rate of force elevation, increased from 4 A (RCA) and 7 A (VAA and IgG) to 10 A (BHL) for the receptor-lactose interactions. Presenting ASF as ligand potential widths of 4 A for RCA and IgG and 6 A for VAA and BHL were obtained. Since the dissociation kinetics at zero force apparently cannot predict the behavior in force-driven experiments, these results reveal new insights into biological functions. The dissociation kinetics under force helps to explain the difference in the toxic potency of VAA and RCA and points to a function of the galectin in cis-crosslinking and in transient trans-bridging.     

Direct observation of membrane tethers formed during neutrophil attachment to platelets or P-selectin under physiological flow

Adhesion and subsequent aggregation between neutrophils and platelets is dependent upon the initial binding of P-selectin on activated platelets to P-selectin glycoprotein ligand 1 (PSGL-1) on the microvilli of neutrophils. High speed, high resolution videomicroscopy of flowing neutrophils interacting with spread platelets demonstrated that thin membrane tethers were pulled from neutrophils in 32 +/- 4% of the interactions. After capture by spread platelets, neutrophil membrane tethers (length of 5.9 +/- 4.1 microm, n = 63) were pulled at an average rate of 6-40 microm/s as the wall shear rate was increased from 100-250 s(-1). The average tether lifetime decreased significantly (P < 0.001) from 630 to 133 ms as the shear rate was increased from 100 s(-1) (F(bond) = 86 pN) to 250 s(-1) (F(bond) = 172 pN), which is consistent with P-selectin/PSGL-1 bond dynamics under stress. Tether formation was blocked by antibodies against P-selectin or PSGL-1, but not by anti-CD18 antibodies. During neutrophil rolling on P-selectin at 150 s(-1), thin membrane tethers were also pulled from the neutrophils. The characteristic jerking motion of the neutrophil coexisted with tether growth (8.9 +/- 8.8 microm long), whereas tether breakage (average lifetime of 3.79 +/- 3.32 s) caused an acute jump in the rolling velocity, proving multiple bonding in the cell surface and the tether surface contact area. Extremely long membrane tethers (>40 microm) were sometimes pulled, which detached in a flow-dependent mechanism of microparticle formation. Membrane tethers were also formed when neutrophils were perfused over platelet monolayers. These results are the first visualization of the often hypothesized tethers that shield the P-selectin/PSGL-1 bond from force loading to regulate neutrophil rolling during inflammation and thrombosis.     

The Kinetics of L-selectin Tethers and the Mechanics of Selectin-mediated Rolling

Two mechanisms have been proposed for regulating rolling velocities on selectins. These are (a) the intrinsic kinetics of bond dissociation, and (b) the reactive compliance, i.e., the susceptibility of the bond dissociation reaction to applied force. To determine which of these mechanisms explains the 7.5–11.5-fold faster rolling of leukocytes on L-selectin than on E- and P-selectins, we have compared the three selectins by examining the dissociation of transient tethers. We find that the intrinsic kinetics for tether bond dissociation are 7–10-fold more rapid for L-selectin than for E- and P-selectins, and are proportional to the rolling velocities through these selectins. The durations of pauses during rolling correspond to the duration of transient tethers on low density substrates. Moreover, applied force increases dissociation kinetics less for L-selectin than for E- and P-selectins, demonstrating that reactive compliance is not responsible for the faster rolling through L-selectin. Further measurements provide a biochemical and biophysical framework for understanding the molecular basis of rolling. Displacements of tethered cells during flow reversal, and measurements of the distance between successive pauses during rolling provide estimates of the length of a tether and the length of the adhesive contact zone, and suggest that rolling occurs with as few as two tethers per contact zone. Tether bond lifetime is an exponential function of the force on the bond, and the upper limit for the tether bond spring constant is of the same order of magnitude as the estimated elastic spring constant of the lectin–EGF unit. Shear uniquely enhances the rate of L-selectin transient tether formation, and conversion of tethers to rolling adhesions, providing further understanding of the shear threshold requirement for rolling through L-selectin.     

P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells

Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L- selectin/IgM heavy chain (mu) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34+ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and leukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P- and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/mu chimera to inhibit L-selectin/mu binding to leukocytes and by the complete inhibition of both selectin interactions with myeloid cells treated with mocarhagin, a cobra venom metalloproteinase that cleaves the amino terminus of PSGL-1 at Tyr-51. Finally, the abrogation of L- and P-selectin binding to myeloid cells treated with a polyclonal antibody, raised against a peptide corresponding to the amino acid residues 42-56 of PSGL-1, indicated that L- and P-selectin interact with a domain located at the amino- terminal end of PSGL-1. The ability of the anti-PSGL-1 mAb PL-1 to inhibit L- and P-selectin binding to KG-1 cells further supported that possibility. Thus, apart from being involved in neutrophil rolling on P- and E-selectin, PSGL-1 also plays a critical role in mediating neutrophil attachment to adherent neutrophils. Interaction between L- selectin and PSGL-1 may be of major importance for increasing leukocyte recruitment at inflammatory sites.     

Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1

P-, E- and L-selectin constitute a family of cell adhesion receptors that mediate the initial tethering and rolling of leukocytes on inflamed endothelium as a prelude to their firm attachment and extravasation into tissues. The selectins bind weakly to sialyl Lewisx (SLe(X))-like glycans, but with high-affinity to specific glycoprotein counterreceptors, including PSGL-1. Here, we report crystal structures of human P- and E-selectin constructs containing the lectin and EGF (LE) domains co-complexed with SLe(X). We also present the crystal structure of P-selectin LE co-complexed with the N-terminal domain of human PSGL-1 modified by both tyrosine sulfation and SLe(X). These structures reveal differences in how E- and P-selectin bind SLe(X) and the molecular basis of the high-affinity interaction between P-selectin and PSGL-1.     

Quantitative analysis of platelet alpha v beta 3 binding to osteopontin using laser tweezers

To determine whether platelet adhesion to surfaces coated with the matrix protein osteopontin requires an agonist-induced increase in the affinity of the integrin alpha v beta 3 for this ligand, we used laser tweezers to measure the rupture force between single alpha v beta 3 molecules on the platelet surface and osteopontin-coated beads. Virtually all platelets stimulated with 10 microM ADP bound strongly to osteopontin, producing rupture forces as great as 100 piconewtons (pN) with a peak at 45-50 pN. By contrast, 90% of unstimulated, resting non-reactive platelets bound weakly to osteopontin, with rupture forces rarely exceeding 30-35 pN. However, approximately 10% of unstimulated platelets, resting reactive platelets, exhibited rupture force distributions similar to stimulated platelets. Moreover, ADP stimulation resulted in a 12-fold increase in the probability of detecting rupture forces >30 pN compared with resting non-reactive platelets. Pre-incubating stimulated platelets with the inhibitory prostaglandin E1, a cyclic RGD peptide, the monoclonal antibody abciximab, or the alpha v beta 3-specific cyclic peptide XJ735 returned force histograms to those of non-reactive platelets. These experiments demonstrate that ADP stimulation increases the strength of the interaction between platelet alpha v beta 3 and osteopontin. Furthermore, they indicate that platelet adhesion to osteopontin-coated surfaces requires an agonist-induced exposure of alpha v beta 3-binding sites for this ligand.     

炎症反应中选择素粘附分子与配体间相互作用的研究

在炎症反应中,白细胞在内皮细胞上滚动由选择素分子与其配体分子相互作用所导致,选择素分子有3种,P选择素分子（P—selectin）、E选择素分子（E—selectin）、L选择素分子（L—selectin）,选择素分子与其对应的P-选择素糖蛋白配体-1（PSGL-1）的相互作用起着重要的作用。用等离子共振、流动腔、原子力显微镜等技术能定量分析选择素分子与其配体分子相互作用的动力学反应。     

Two Barriers or not? Dynamic Force Spectroscopy on the Integrin α7β1 Invasin Complex

Dynamic force spectroscopy was used to test force-induced dissociation of the complex between the integrin α₇β₁ and the bacterial protein invasin. Both proteins were used in truncated forms comprising the respective binding sites. Using the biomembrane force-probe, the bond system was exposed to 14 different loading rates ranging from 18 pN/s to 5.3 nN/s. At each rate, bond rupture spectra were collected. Median forces ranged from 8 to 72 pN. These showed two linear regimes when plotted against the logarithm of the force-loading rate. However, a statistical analysis of the full rupture force spectra including the detection limits of the setup showed that all measured data are well described by dissociation over a single barrier.