BACKGROUND: The interaction between endothelium and leukocytes plays a crucial role in ischemia-reperfusion injury. P-selectin, which is expressed on activated endothelium, mediates the first step in leukocyte adherence to the endothelium. This study examined the effects of a monoclonal antibody (mAb) against P-selectin on the recovery of cardiac function and myocardial neutrophil infiltration after ischemia.
METHODS AND RESULTS: Thirteen blood-perfused, isolated neonatal lamb hearts underwent 2 hours of hypothermic cardioplegic arrest and 2 hours of reperfusion. Immediately before reperfusion, mAb to P-selectin was administered to the perfusate (15 micrograms/mL) in 6 hearts (group P-sel). In control (n = 7), the same volume of saline was added. Isovolumic left ventricular function and coronary blood flow were measured. At 2 hours after reperfusion, myocardial myeloperoxidase activity, an index of neutrophil accumulation, was assayed. At 30 minutes of reperfusion, hearts treated with mAb to P-selectin achieved significantly greater recovery of maximum developed pressure (70 +/- 4% in control versus 77 +/- 2% in group P-sel, P < 0.01), maximum positive first derivative of pressure (dP/dt) (64 +/- 7% in control versus 73 +/- 5% in group P-sel, P < 0.05), and maximum negative dP/dt (61 +/- 6% in control versus 70 +/- 6% in group P-sel, P < 0.05) compared with control. Percent baseline of coronary blood flow was also significantly increased in group P-sel (135 +/- 40% in control versus 205 +/- 43% in group P-sel, P < 0.05). Myocardial myeloperoxidase activity was significantly lower (P < 0.05) in group P-sel (4.7 +/- 3.2) versus control (16.0 +/- 10.1). (Units are change in absorbance/min/g tissue.)
CONCLUSIONS: The functional blockade of P-selectin resulted in better recovery of cardiac function and attenuated neutrophil accumulation during early reperfusion. Strategies to block P-selectin mediated neutrophil adherence may have clinical application in improving myocardial function at early reperfusion.

Integrins are large, heterodimeric surface molecules of wide importance in cell adhesion. The N-terminal half of all integrin alpha-subunits contains seven weak sequence repeats of approximately 60 amino acids that are important in ligand binding and have been predicted to fold cooperatively into a single beta-propeller domain with seven beta-sheets. We provide evidence supporting this model with a mouse mAb to human Mac-1 (alphaM beta2, CD11b/CD18). This antibody, CBRM1/20, binds to amino acid residues that are in different repeats and are 94 residues apart in the primary structure in the loop between strands 1 and 2 of beta-sheet 5 and in the loop between strands 3 and 4 of beta-sheet 6. The 1-2 loops of beta-sheets 5-7 in integrins have EF hand-like Ca2+-binding motifs. CBRM1/20 binds to Mac-1 in the presence of Ca2+ or Sr2+ with an EC50 of 0.2 mM. Mg2+ or Mn2+ cannot substitute. Antibodies to other epitopes on the Mac-1 beta-propeller domain bind in the absence of calcium. mAb CBRM1/20 does not block ligand binding. Thus, the region on the lower surface of the beta-propeller domain to which mAb CBRM1/20 binds does not bind ligand and, furthermore, cannot bind other integrin domains, such as those of the beta-subunit.

An abundant, widely dispersed, extracellular sequence repeat that contains a consensus YWTD motif is shown here to occur in groups of six contiguous repeats. Thirteen lines of evidence, including experimental and computational data, predict with p<3x10(-9) that the repeats do not form tandem domains, but rather each group of six repeats folds into a compact beta-propeller structure. The six beta-sheets are arranged about a 6-fold pseudosymmetry axis, and each repeat contributes loops to the faces surrounding the pseudosymmetry axis. Seven different endocytic receptors that contain from one to eight YWTD beta-propeller domains act as lipoprotein, vitellogenin, and scavenger receptors. In the low density lipoprotein receptor (LDLR), the many mutations in familial hypercholesterolaemia that map to the YWTD domain can now be interpreted. In the extracellular matrix component nidogen, the YWTD domain functions to bind laminin. Three YWTD domains and interspersed fibronectin type III (FN3) domains constitute almost the entire extracellular domain of the sevenless and c-ros receptor tyrosine kinases. YWTD domains often are bounded by epidermal growth factor (EGF) modules, including in the EGF precursor itself. YWTD beta-propellers have a circular folding pattern that brings neighboring modules into close proximity, and may have important consequences for the architecture of multi-domain proteins.

The chemokine stromal cell-derived factor 1, SDF-1, is an important regulator of leukocyte and hematopoietic precursor migration and pre-B cell proliferation. The receptor for SDF-1, CXCR4, also functions as a coreceptor for T-tropic HIV-1 entry. We find that mice deficient for CXCR4 die perinatally and display profound defects in the hematopoietic and nervous systems. CXCR4-deficient mice have severely reduced B-lymphopoiesis, reduced myelopoiesis in fetal liver, and a virtual absence of myelopoiesis in bone marrow. However, T-lymphopoiesis is unaffected. Furthermore, the cerebellum develops abnormally with an irregular external granule cell layer, ectopically located Purkinje cells, and numerous chromophilic cell clumps of abnormally migrated granule cells within the cerebellar anlage. Identical defects are observed in mice lacking SDF-1, suggesting a monogamous relationship between CXCR4 and SDF-1. This receptor-ligand selectivity is unusual among chemokines and their receptors, as is the function in migration of nonhematopoietic cells.

The transient regulation of very late antigen (VLA)-4 avidity by CC chemokines may promote chemotaxis of monocytes across VCAM-1-bearing barriers, whereas late and prolonged activation of VLA-5 may mediate subsequent localization in the extracellular matrix. We demonstrate that interactions of VLA-4 with VCAM-1, fibronectin, or a 40-kDa fragment but not a 120-kDa fragment of fibronectin supported the lateral random migration of isolated blood monocytes induced by CC chemokines, termed chemokinesis. This effect was optimal at intermediate substrate concentrations. Moreover, coimmobilization of VCAM-1 with ICAM-1 allowed better migration than ICAM-1 alone. Chemokinesis on VCAM-1 appeared to be associated with transient regulation of VLA-4 avidity by CC chemokines, given that locking VLA-4 in a high avidity state markedly inhibited migration and the locomotion rate was inversely correlated with the adhesive strength of VLA-4 to VCAM-1 following stimulation with monocyte chemoattractant protein-1. Induction of VCAM-1 expression by endothelial activation with IL-4 improved chemokinesis and lateral migration toward a monocyte chemoattractant protein-1 or a monocyte inflammatory protein-1alpha gradient on endothelium and increased transendothelial chemotaxis of monocytes by a VLA-4-dependent mechanism. In contrast, endothelial activation with IL-4 did not affect the time required for diapedesis of monocytes itself. Hence, VCAM-1 may facilitate transendothelial chemotaxis by supporting lateral migration of attached monocytes along endothelium.

The kinetics of rolling and transient adhesions through selectins may depend on the kinetics and mechanical properties of the selectin:ligand bond, as well as on cellular properties including receptor-anchoring to the cell membrane and cytoskeleton. Kinetics are known to depend on the selectin and may also be ligand dependent. Here, we study the kinetics of transient and rolling interactions of leukocytes with L-selectin immobilized on a substrate. Remarkably, all properties examined are similar to those seen when the sidedness is opposite, i.e., when the L-selectin ligand is on the substrate and when the ligand is isolated from HEV rather than present on leukocytes. The similar properties include rolling velocity, a threshold shear stress above 0.4 dyn/cm2 required to support rolling, a k degreesoff of 7.0 to 6.8 s-1 for the L-selectin tether bond, and a mechanical bond length of 0.24 to 0.20 A. Our results argue against a model in which L-selectin shedding mediates rolling. Furthermore, the fast and force-resistant kinetic properties suggest that L-selectin is specialized dynamically for tethering leukocytes to vessel walls and adherent leukocytes.

During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewisx (sLex)-containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLex on HEV using a panel of mAbs specific for sLex and sLex-related structures, and have examined the function of different sLex-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLex mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLex, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLex on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease-resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30%, whereas the sLex mAb 2H5 blocks binding by approximately 60% and a combination of MECA-79 and 2H5 mAb blocks binding by 75%. We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV.

Interactions between adhesion molecules on two different cells differ from interactions between receptors and soluble ligands in that the adhesion molecule interaction (bond) is often subjected to force. It is widely assumed by cell biologists that the 'strength' of a bond is a simple function of the affinity of one adhesion molecule for the other, whereas biophysicists suggest that bonds have 'mechanical properties' that affect their strength. Mechanical properties are a function of the shape of the energy landscape related to bond formation and dissociation, whereas affinity is related only to the net energy change. Mechanical properties determine the amount by which the kinetics and affinity of bonds are altered by applied force. To date there has been no experimental manipulation of an adhesion molecule that has been shown to affect mechanical properties. L-selectin is an adhesion molecule that mediates lymphocyte binding to, and rolling on, high endothelial venules; these are prerequisites for the emigration of lymphocytes from the bloodstream into lymph nodes. Here we report a selective and reversible chemical modification of a mucin-like ligand that alters the mechanical properties of its bond with L-selectin. The effect of force on the rate of bond dissociation, that is, on a mechanical property, is altered, whereas there is little or no effect of the modification on the rate of bond dissociation in the absence of force. Moreover, the puzzling requirement for hydrodynamic shear flow above a threshold level for L-selectin interactions is dramatically altered.

It has been hypothesized that the intercellular adhesion receptors used by normal cells could also be operative in the spreading of circulating malignant cells to target organs. In the present work, we show that genetic ablation of the ICAM-1 gene confers resistance to T cell lymphoma metastasis. Following i.v. inoculation of LFA-1-expressing malignant T lymphoma cells, we found that ICAM-1-deficient mice were almost completely resistant to the development of lymphoid malignancy compared with wild-type control mice that developed lymphoid tumors in the kidneys, spleen, and liver. Histologic examinations confirmed that ICAM-1-deficient mice, in contrast to wild-type mice, had no evidence of lymphoid infiltration in these organs. The effect of ICAM-1 on T cell lymphoma metastasis was observed in two distinct strains of ICAM-1-deficient animals. Nonetheless, lymphoma cells migrated with the same efficiency to target organs in both normal and ICAM-1-deficient mice, indicating not only that ICAM-1 expression by the host is essential in lymphoma metastasis, but also that this is so at stages subsequent to homing and extravasation into target organs. These results point to posthoming events as a focus of future investigation on the control of metastasis mediated by ICAM-1.

The circulation and migration of leukocytes are critical for immune surveillance and immune response to infection or injury. The key step of leukocyte recruitment involves the adhesion between immunoglobulin superfamily (IgSF) proteins on endothelium and integrin molecules on leukocyte surfaces. Some of the IgSF members are subverted as virus receptors. Four crystal structures of N-terminal two-domain fragments of these IgSF proteins have been determined: intercellular adhesion molecule-1 (ICAM-1), ICAM-2, vascular adhesion molecule-1 (VCAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). An acidic residue near the bottom of domain 1 plays a key role in integrin binding. For ICAM-1 and ICAM-2, this glutamic acid residue is located on a flat surface, complementary to the flat surface of the I domain of the integrin to which they bind, lymphocyte function-associated antigen-1 (LFA-1). For VCAM-1 and MAdCAM-1, the acidic residue is aspartic acid, and it resides on a protruded CD loop which may be complementary to a more pocket-like structure in the alpha 4 integrins to which they bind, which lack I domains. A number of unique structural features of this subclass of IgSF have been identified which are proposed to consolidate the domain structure to resist force during adhesion to integrins. Different mechanisms are proposed for the different CAMs to present the integrin-binding surface toward the opposing cell for adhesion, and prevent cis interaction with integrins on the same cell. Finally, CD4 and ICAM-1 are compared in the context of ligand binding and virus binding, which shows how human immunodeficiency virus and rhinovirus fit well with the distinct structural feature of their cognate receptors.

BACKGROUND: Mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is a cell adhesion molecule that is expressed on the endothelium in mucosa, and guides the specific homing of lymphocytes into mucosal tissues. MAdCAM-1 belongs to a subclass of the immunoglobulin superfamily (IgSF), the members of which are ligands for integrins. Human MAdCAM-1 has a unique dual function compared to other members in the same subclass in that it binds both the integrin alpha4beta7, through its two IgSF domains, and a selectin expressed on leukocytes, via carbohydrate sidechains. The structure determination of the two IgSF domains and comparison to the N-terminal two-domain structures of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecules (ICAM-1 and ICAM-2) allow us to assess the molecular basis of the interactions between integrins and their preferred ligands.
RESULTS: The crystal structure of a fragment containing the two IgSF domains of human MAdCAM-1 has been determined to 2.2 A resolution. The structure of MAdCAM-1 reveals two separate integrin-recognition motifs. The key integrin-binding residue, Asp42, resides in the CD loop of domain 1; a buried arginine residue (Arg70) plays a critical role in maintaining the conformation of this loop. The second binding site is associated with an unusual long D strand in domain 2. The D and E strands extend beyond the main body of the domain, forming a negatively charged beta ribbon unique to MAdCAM-1. This ribbon is located on the same face as the key aspartate residue in domain 1, consistent with evidence that it is involved in integrin binding.
CONCLUSIONS: The structural comparison of MAdCAM-1 to other members of the same IgSF subclass reveals some interesting features. Firstly, MAdCAM-1, like VCAM-1, has the key integrin-binding residue located on the protruding CD loop of domain 1 and binds to an integrin that lacks an I domain. This is in contrast to ICAM-1 and ICAM-2 where the key residue is located at the end of the C strand on a flat surface and which bind to integrins that contain I domains. Secondly, architectural differences in the CD loops of MAdCAM-1 and VCAM-1 cause an 8 A shift in position of the critical aspartate residue, and may partly determine their binding preference for different integrins. Finally, the unusual charge distribution of the two-domain fragment of MAdCAM-1 is predicted to orient the molecule optimally for integrin binding on the top of its long mucin-like stalk.

The alphaM subunit of integrin Mac-1 contains several distinct regions in its extracellular segment. The N-terminal region has been predicted to fold into a beta-propeller domain composed of seven beta-sheets each about 60 amino acid residues long, with the I-domain inserted between beta-sheets 2 and 3. The structure of the C-terminal region is unknown. We have used monoclonal antibodies (mAbs) as probes to study the dependence of the structure of different regions of the alphaM subunit on association with the beta2 subunit in the alphaM/beta2 heterodimer. All of the mAbs to the I-domain immunoprecipitated the unassociated alphaM precursor and reacted with the alphaM subunit expressed alone on the surface of COS cells. By contrast, four mAbs to the beta-propeller domain did not react with the unassociated alphaM precursor nor with the uncomplexed alphaM subunit expressed on COS cell surface. The four mAbs were mapped to three subregions in three different beta-sheets, making it unlikely that each recognized an interface between the alpha and beta subunits. These results suggest that folding of different beta-propeller subregions is coordinate and is dependent on association with the beta2 subunit. The segment C-terminal to the beta-propeller domain, residues 599-1092, was studied with nine mAbs. A subset of four mAbs that reacted with the alphaM/beta2 complex but not with the unassociated alphaM subunit were mapped to one subregion, residues 718-759, and five other mAbs that recognized both the unassociated and the complexed alphaM subunit were localized to three other subregions, residues 599-679, 820-882, and 943-1047. This suggests that much of the region C-terminal to the beta-propeller domain folds independently of association with the beta2 subunit. Our data provide new insights into how different domains in the integrin alpha and beta subunits may interact.

Two subpopulations of human T lymphocytes expressing different antigen receptors, alpha/beta and gamma/delta, emigrate into inflamed tissues in distinctive patterns. We compared the transmigration of alpha/beta and gamma/delta T cells to C-C and C-X-C chemokines using an in vitro transendothelial chemotaxis assay. The C-C chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta stimulated similar, dose-dependent chemotaxis of purified gamma/delta T cells, whereas MCP-1, RANTES, and MIP-1alpha produced greater chemotaxis of purified alpha/beta T cells than MIP-1beta. In contrast, the C-X-C chemokines interleukin (IL)-8 and interferon-gamma inducible protein-10 (IP-10) did not promote chemotaxis of either alpha/beta or gamma/delta T cells. Three gamma/delta T cell clones with differing CD4 and CD8 phenotypes also migrated exclusively to C-C chemokines. Phenotypic analysis of mononuclear cells that transmigrated from an input population of unfractionated peripheral blood mononuclear cells confirmed the results with purified gamma/delta T cells. Our data demonstrate that human peripheral blood alpha/beta and gamma/delta T cells can transmigrate to MCP-1, RANTES, MIP-1alpha, and MIP-1beta, and suggest that both T lymphocyte subpopulations share the capacity to emigrate in response to C-C chemokines during inflammation.

The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-adapted human immunodeficiency virus type 1 (HIV-1) isolates. Here we show that murine CXCR-4 can support the entry of some of these HIV-1 isolates. Differences between mouse and human CXCR-4 in the ability to function as an HIV-1 receptor are determined by sequences in the second extracellular loop of the CXCR-4 protein.

The leukocyte, or beta2, integrins are heterodimeric cell surface molecules that share a common beta subunit, and have unique alpha subunits. LFA-1 is the predominant beta2 integrin on lymphocytes, and plays an important role in many lymphocyte functions; however, most studies of the cytoplasmic regions of LFA-1 have been performed in transfected epithelial cells, such as COS, in part because no lymphoid cell lines deficient in the LFA-1 alpha subunit have been described. To address structure-function studies of beta2 integrins in relevant cell types, two T lymphoblastoid cell clones that completely lack cell surface LFA-1, J-(beta2).7 and SK-(beta2).7, derived from Jurkat and SKW3, respectively, were prepared by chemical mutagenesis and selection. Biosynthetic labeling and immunoprecipitation showed that the J-(beta2).7 clone did not translate any LFA-1 alpha subunit protein, while the SK-(beta2).7 cells did not synthesize any beta2 subunit protein. Northern blot analysis of poly(A+) RNA from these cells revealed an absence of the corresponding mRNA in each case. By transfection analysis, only the alpha subunit reconstituted LFA-1 expression in the J-(beta2).7 cells, while only the beta subunit restored cell surface LFA-1 expression in the SK-(beta2).7 cells. Functional studies with the parental cell lines, the J-(beta2).7 and SK-(beta2).7 cells, and the transfectants showed that all binding of Jurkat and SKW3 cells to purified ICAM-1 is mediated by LFA-1, and the reconstituted LFA-1 expressed by the J-(beta2).7 and SK-(beta2).7 transfected cells is regulated normally.

Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38- or CD34+/DR-) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.

C5a > IL-8 > Gro-alpha, in agreement with previous results in neutrophils on the Ca2+-mobilizing response. These results may have important implications for the behavior of these cell types in inflammatory sites."]" data-sheets-userformat="[null,null,8961,[null,0],null,null,null,null,null,null,null,3,0,null,null,null,9]">At inflammatory sites in vivo, leukocytes may confront multiple, competing chemoattractive signals. We found significant differences between eosinophils and neutrophils in transendothelial chemotaxis to a chemoattractant diffusing from the lower chamber, when a chemoattractant that binds to another receptor is present at uniform concentration. The transendothelial migration of eosinophils to FMLP, C5a, RANTES, or MCP-3 was totally inhibited by the presence of the homologous chemoattractant, and only RANTES and MCP-3 showed mutual inhibition. C5a and to a lesser extent FMLP chemokinetically stimulated migration to RANTES and MCP-3, without stimulating random migration. Results with neutrophils contrasted. The presence of FMLP not only abrogated neutrophil transmigration to FMLP but also strongly decreased chemotaxis to C5a, IL-8, and Gro-alpha. Similarly, C5a inhibited neutrophil chemotaxis to IL-8 and Gro-alpha. IL-8 almost totally abrogated chemotaxis to Gro-alpha, but Gro-alpha only moderately inhibited chemotaxis to IL-8. Neither IL-8 nor Gro-alpha significantly inhibited transmigration to FMLP or C5a. Actin polymerization in eosinophils and neutrophils was desensitized by the same combinations of chemoattractants that desensitized chemotaxis. We conclude that eosinophils have at least three noninterfering receptor-signal transduction pathways for chemotaxis and actin polymerization. In contrast, the signaling pathways for FMLP, C5a, and IL-8/Gro-alpha in neutrophils are heterologously cross-desensitized, with a hierarchy of resistance to competing signals of FMLP > C5a > IL-8 > Gro-alpha, in agreement with previous results in neutrophils on the Ca2+-mobilizing response. These results may have important implications for the behavior of these cell types in inflammatory sites.

Recognition by integrin proteins on the cell surface regulates the adhesive interactions between cells and their surroundings. The structure of the 'I' domain that is found in some but not all integrins, has been determined. However, the only integrin ligands for which structures are known, namely fibronectin and VCAM-1, are recognized by integrins that lack I domains. The intercellular adhesion molecules ICAM-1, 2 and 3 are, like VCAM-1, members of the immunoglobulin superfamily (IgSF), but they are recognized by an I domain-containing integrin, lymphocyte-function-associated antigen 1 (LFA-1, or CD11a/CD18). Here we present the crystal structure of the extracellular region of ICAM-2. The glutamic acid residue at position 37 is critical for LFA-1 binding and is proposed to coordinate the Mg2+ ion in the I domain; this Glu 37 is surrounded by a relatively flat recognition surface and lies in a beta-strand, whereas the critical aspartic acid residue in VCAM-1 and fibronectin lie in protruding loops. This finding suggests that there are differences in the architecture of recognition sites between integrins that contain or lack I domains. A bend between domains 1 and 2 of ICAM-2 and a tripod-like arrangement of N-linked glycans in the membrane-proximal region of domain 2 may be important for presenting the recognition surface to LFA-1. A model of ICAM-1 based on the ICAM-2 structure provides a framework for understanding its recognition by pathogens.

It was previously shown that mutations of integrin alpha4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in alpha4beta1-dependent cell adhesion. Some reports have suggested that alpha-chain "EF-hand" sites may interact directly with ligands. However, we show here that mutations of three different alpha4 "EF-hand" sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on alpha4beta1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin alpha4 "EF-hand-like" sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.