Multiple Activation States of Integrin α4β1 Detected through Their Different Affinities for a Small Molecule Ligand

Abstract

We have used the highly specific α4β1 inhibitor 4-((N′-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study α4β1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of α4β1. Observed K Dvalues for BIO1211 binding ranged from a value of 20–40 nmin the non-activated state of the integrin that exists in 1 mm Mg2+, 1 mm Ca2+ to 100 pm in the activated state seen in 2 mmMn2+ to 18 pm when binding was measured after co-activation by 2 mm Mn2+ plus 10 μg/ml of the integrin-activating monoclonal antibody TS2/16. The large range inK D values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 × 10−4 s−1 to >140 × 10−4 s−1. Association rate constants varied only slightly under the same conditions, all falling in the narrow range from 0.9 to 2.7 × 106m−1 s−1. The further increase in affinity observed upon co-activation by divalent cations and TS2/16 compared with that observed at saturating concentrations of metal ions or TS2/16 alone indicates that the mechanism by which these factors bring about activation are distinct and identified a previously unrecognized high affinity state on α4β1that had not been detected by conventional assay methods. Similar changes in affinity were observed when the binding properties of vascular cell adhesion molecule-1 and CS1 to α4β1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin. We have used the highly specific α4β1 inhibitor 4-((N′-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study α4β1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of α4β1. Observed K Dvalues for BIO1211 binding ranged from a value of 20–40 nmin the non-activated state of the integrin that exists in 1 mm Mg2+, 1 mm Ca2+ to 100 pm in the activated state seen in 2 mmMn2+ to 18 pm when binding was measured after co-activation by 2 mm Mn2+ plus 10 μg/ml of the integrin-activating monoclonal antibody TS2/16. The large range inK D values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 × 10−4 s−1 to >140 × 10−4 s−1. Association rate constants varied only slightly under the same conditions, all falling in the narrow range from 0.9 to 2.7 × 106m−1 s−1. The further increase in affinity observed upon co-activation by divalent cations and TS2/16 compared with that observed at saturating concentrations of metal ions or TS2/16 alone indicates that the mechanism by which these factors bring about activation are distinct and identified a previously unrecognized high affinity state on α4β1that had not been detected by conventional assay methods. Similar changes in affinity were observed when the binding properties of vascular cell adhesion molecule-1 and CS1 to α4β1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin. Integrins comprise a large family of cell-surface receptors that mediate cell-cell and cell-matrix interactions in diverse biological settings (see Refs. 1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar and 2Ruoshlahti E. J. Clin. Invest. 1991; 87: 1-5Crossref PubMed Scopus (1494) Google Scholar for reviews). Each integrin is a two-chain heterodimer containing an α-chain and a β-chain. The leukocyte integrin α4β1 regulates cell migration into tissues during inflammatory responses and normal lymphocyte trafficking (3Hemler M.E. Elices M.J. Parker C. Takada Y. Immunol. Rev. 1990; 114: 45-65Crossref PubMed Scopus (290) Google Scholar, 4Lobb R.R. Hemler M.E. J. Clin. Invest. 1994; 94: 1722-1728Crossref PubMed Scopus (363) Google Scholar, 5Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6414) Google Scholar) and provides a key co-stimulatory signal supporting cell activation (6Damle N.K. Aruffo A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6403-6407Crossref PubMed Scopus (216) Google Scholar, 7Anwar A.R. Walsh G.M. Cromwell O. Kay A.B. Wardlaw A.J. Immunology. 1994; 82: 222-228PubMed Google Scholar, 8Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus Google Scholar, J. Immunol. Google Scholar, Y. J. Immunol. Google monoclonal R.R. Hemler M.E. J. Clin. Invest. 1994; 94: 1722-1728Crossref PubMed Scopus (363) Google Scholar) and inhibitor T.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, J. Clin. Invest. 1994; 94: PubMed Scopus Google Scholar, S. Elices C. J. Clin. Invest. 1995; PubMed Scopus Google Scholar) have a for in cell adhesion by binding to of vascular cell adhesion molecule-1 used vascular cell adhesion 4-((N′-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic monoclonal metal adhesion high cell or the C. C. S. Cell. Full Text PDF PubMed Scopus Google Scholar, M.J. Takada Y. C. S. Hemler M.E. R.R. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar, E.A. A. M.J. J. PubMed Scopus Google Scholar, R.O. Cell. 1990; Full Text PDF PubMed Scopus Google of α4β1 is with is in an activated state that by cell the divalent and A. Hemler M.E. J. Immunol. 1991; Google Scholar, Y. S. 1990; PubMed Scopus Google Scholar, A. Hemler M.E. J. 268: Full Text PDF PubMed Google changes in affinity the is and the or is all divalent cations to the of by metal binding is and is not M.J. PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Ca2+ binding R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar, C. E. J. 1992; Full Text PDF PubMed Google which are in to T.A. Proc. Natl. Acad. Sci. U. S. A. 94: PubMed Scopus Google Scholar, A. Takada Y. Proc. Natl. Acad. Sci. U. S. A. 94: PubMed Scopus Google of the α-chain is of of about which are to in a in T.A. Proc. Natl. Acad. Sci. U. S. A. 94: PubMed Scopus Google Scholar, A. Takada Y. Proc. Natl. Acad. Sci. U. S. A. 94: PubMed Scopus Google The a of metal binding that with the of T.A. J. PubMed Scopus Google Scholar, M.J. PubMed Scopus Google is in as to for and the from and have been A. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, A. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Full Text Full Text PDF PubMed Scopus Google The a or Mn2+ at the of a binding A. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, A. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Full Text Full Text PDF PubMed Scopus Google The observed by the of the with the been to the in is to as the metal adhesion or (see Refs. M.J. PubMed Scopus Google Scholar and M.J. PubMed Scopus Google Scholar for the of in integrin is the are is of ligand binding by cations is further by the that concentrations of Ca2+ ligand indicating that of the an J. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. J. 1995; Full Text Full Text PDF PubMed Scopus Google a of highly α4β1 the ligand binding from the CS1 of as the for inhibitor R.R. PubMed Scopus Google is to the which the in Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google and ligand binding and cell adhesion Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, E.A. J. 1992; PubMed Scopus Google a that was was by with a A. C. S. A. R.R. J. Scopus Google used a of BIO1211 as a to α4β1 under states of The on the that different activation conditions have on the affinity of α4β1 for and that these differences in affinity are by changes in the dissociation rate of the was by as a The of the was as measured by on a and the the by was in with of and was to mm The was and at The binding affinity of the was from BIO1211 in the binding assay with as a R.R. A. 1995; PubMed Scopus Google Jurkat cells that had been for α4β1 by were in plus at in a cells that had been with the or and for high of and by were in the same with 1 10 μg/ml and μg/ml and cells were a of binding the cells were by with mm mm 2 10 mm at 2 × in and a The cells were further with to the and with at The cells were by in 100 of plus and to a containing of was by were in with a Each was in at the a of BIO1211 was to after the with to further the of cell and were in plus 2 mm as binding of to cells was at cell and in in the of metal were by from the of activation on binding were as the 1 mm 1 state K D 20–40 for binding of BIO1211 to a high at the BIO1211 concentrations at 10 in the assay the of that to by the specific of the from to binding at BIO1211 concentrations to 100 with on rate Jurkat cells were with 2 at for the and with a of BIO1211 to further binding by the were by and to rate Jurkat cells were with at for 1 of BIO1211 was and the cells were further for the were at and was measured by and dissociation are as a of the specific as a of The were to an by the rate is the the observed rate is the rate by the The of BIO1211 on dissociation rates was a range of concentrations from 100 to The dissociation were range of indicating that the ligand was on the rate of dissociation not was used to study α4β1 by the as a for Jurkat cells × in the were with of for 1 and an to all was for 10 the The cells were by and to under these conditions integrin that is not by the and is to the The was used for binding and dissociation constants were from α4β1 that after with was to the were by on a × at The was at 1 with the of in mm and conditions that the of BIO1211 from and not The was at and were The were with and by to on the were with of BIO1211 and an of BIO1211 with the and a The of the were used to The of to Jurkat cells and cells was under diverse conditions to the activation state of α4β1 binding to Jurkat cells was in the of 2 mmMn2+ 10 mm and 2 mm Mn2+ 10 μg/ml that α4β1. was under conditions by with mm (see and binding was observed to the cell The seen in is which was were to the specific binding was by from binding was for a that was to the same in the of divalent at a of α4β1 and on that the Jurkat cells used in these have of α4β1 the that under the different α4β1 were compared under the 1 mm 1 mm and 2 mm Mn2+ conditions by the as a The α4β1 in the activated and non-activated were of were to that affect the of the of on BIO1211 binding was observed under of the conditions when binding was compared at and at not the of or at concentrations to of the assay had on the binding assay not from Jurkat cells with indicating that the on the cell The BIO1211 was further by for the of that of in of the was by the indicating that only had that the K D values for binding of to cells of of Jurkat cells were that BIO1211 binding was not by differences in of α4β1 A. C. S. A. R.R. J. Scopus Google of the of in the BIO1211 as a metal that are to the concentrations of Mg2+, and Ca2+ as for were to an BIO1211 and these metal ions not The of seen in for binding of to Jurkat cells under conditions, that the affinity of BIO1211 for α4β1 was for non-activated for the activated integrin. to and to a of K the was BIO1211 The binding at 100 specific were for the 1 mm 1 state and the 2 mm Mn2+ state not on these binding a K 20–40 was for the 1 1 mm to K D values for activated states binding were D values were the of α4β1 in the observed and binding (see the binding were in a of the to and not used to of the affinity of BIO1211 for activated α4β1 was (see were in the 1 1 mm TS2/16 state under conditions that have been saturating for binding compared with activated states was not to in the of TS2/16 to TS2/16 had of the have with for the binding and dissociation of to α4β1 were measured under the conditions, as in 1 mm 1 mm Mg2+, Mn2+ 1 mm 1 10 μg/ml 2 and 2 mm Mn2+ 10 μg/ml TS2/16. a 10 of for all conditions Association rate constants from these all in the narrow range from 0.9 to 2.7 × 106m−1 s−1 in the α4β1 after 10 were to seen in the in for the rates of dissociation of BIO1211 from Jurkat cells are in the which were the different assay conditions, the dissociation rates varied a range and were highly on the activation state of the integrin. ranged from 0.17 × for the 2 mm Mn2+ TS2/16 state to >140 × 10−4 s−1 for the 1 mm 1 mm The rate constants from these and binding were used to K D which are in The the in K D values and in dissociation rates indicates that the affinity of BIO1211 for α4β1 is governed almost exclusively by K D values observed under conditions to ranged from pm in the Mg2+, Mn2+ state to 18 pm in the 2 mmMn2+ TS2/16 differences in affinity that from activation were not from the binding the K D values were the of α4β1 in the and were by the of the Similar were when were by the of BIO1211 to cell adhesion to CS1 or or to binding of to Jurkat cells with as the not of to α4β1 on Jurkat cells under states of mm 1 value of 20–40 was binding (see mm Mg2+, Mn2+ 1 mm were from the in is by a as in the mmMn2+ mmMn2+ mm and values for the binding of to α4β1 were from as in the to TS2/16 and were used at 10 rates for the were at and 100 mm were The value measured at concentrations been to the alone states value of 20–40 was binding (see were from the in is by a as in the in a and values for the binding of to α4β1 were from as in the to TS2/16 and were used at 10 rates for the were at and 100 mm were The value measured at concentrations been to the alone states the metal ions have on the study in binding was as a of The dissociation rates were highly on the and from × 10−4 s−1 to × to × 10−4 s−1 to × 10−4 s−1 to × 10−4 s−1 when dissociation rates were measured in the of and K D was as a of the a that the were of a high concentrations of Mg2+, binding to saturating at an affinity that the value seen at 2 mm concentrations of a of the dissociation rate to that seen with Mg2+, at a of the metal not in the of TS2/16 from to 10 μg/ml had on K D not indicating that 10 μg/ml was a saturating the affinity for binding of BIO1211 to α4β1 in the of divalent cations was with 2 mm a further increase in affinity was observed were with 2 mm Mn2+ that the by which TS2/16 and divalent cations about activation were from these are in the of 2 mm Mn2+ alone and 2 mm Mn2+ K Dvalues of 100 and 18 were D value from activation by TS2/16 alone 1 mm Mg2+, 1 was increase in affinity was seen after of the 10 alone state with TS2/16 D and pm for the 10 mm TS2/16 and 10 mm TS2/16 the K after co-activation by divalent cations plus TS2/16 was by the and of divalent D values varied with metal concentrations in a to the observed in the divalent alone the TS2/16 to a in is from the dissociation in to the seen after co-activation by TS2/16 plus divalent the dissociation rate observed in the of 2 mm Mn2+ plus 10 mm not that seen with 2 mmMn2+ indicating that activation by Mn2+ and was not not the non-activated 1 mm 1 mm the affinity of BIO1211 for α4β1 was the for the activated the for the state was to that observed under conditions (see dissociation was to an of and the only a to K D (see The of K D with the of K 20–40 that was from the in that the affinity of BIO1211 for α4β1 is highly to the activation state of by on the assay α4β1 affinity states are on metal the of on dissociation of the under from are in and of the 2 mm Mn2+ alone state as in a of from the complex, after the rate of was not as as from the non-activated in a of BIO1211 from the 2 mm Mn2+ TS2/16 the rate of was in the of TS2/16 when the 10 mm alone or 2 mm Mn2+ alone activated states were with constants for the dissociation of under mm Mn2+ not the or of a of the of BIO1211 the mm not the or of a of the of BIO1211 the mm Ca2+ 1 mm Ca2+ of Jurkat cells × in with the were with for 2 at BIO1211 was in the same with or 10 mm as and the cells were further for and The cells were and was measured by BIO1211 were to or (see by as in values were from the dissociation to a and are rate constants for the and of not the or of a of the of BIO1211 the in a of Jurkat cells × in with the were with for 2 at BIO1211 was in the same with or 10 mm as and the cells were further for and The cells were and was measured by BIO1211 were to or (see by as in values were from the dissociation to a and are rate constants for the and of of the dissociation for the of BIO1211 from the divalent plus state was that a to for the for the of BIO1211 was observed for in the of 1 mm 1 TS2/16 and with (see the large of on the of BIO1211 from the divalent alone and divalent plus the of on the 1 mm 1 mm TS2/16 state was only a increase in the rate of the of had that the dissociation of BIO1211 from a on the that had been for the a of the in that was not for all of with the of the dissociation that BIO1211 from divalent alone activated α4β1 a and the that the dissociation of BIO1211 from the 1 mm 1 TS2/16 state was the by the to the that about of the integrin-BIO1211 for the 1 mm 1 TS2/16 state was in the binding states that were by divalent plus TS2/16 was that was to a and to the dissociation that during the of the the of these states for the of BIO1211 after TS2/16 was by the on α4β1 that had been on and in an rates for the of BIO1211 from that were in the of 2 mm Mn2+ or 2 mm Mn2+ TS2/16 were to values observed on and when the same were with TS2/16 the rate of of BIO1211 from the not The of TS2/16 in the was on about of the BIO1211 was from cells after in the only about was in the and of the were in the The of Ca2+ on are in the of 2 mm Ca2+ was as as at of BIO1211 a to for the Ca2+ had of an on the divalent alone or TS2/16 states (see for activation by 1 mm Ca2+ in only a on and × for the Mg2+, Mn2+ mm and 2 mmMn2+ the in observed that the from 1 to 10 mm slightly BIO1211 the of Ca2+ to the of BIO1211 from the 2 mm Mn2+ TS2/16 state is with the model on for in which high concentrations of as a inhibitor of ligand binding J. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. J. 1995; Full Text Full Text PDF PubMed Scopus Google the observed for Ca2+ on states that Ca2+ have a range of on Hemler M.E. J. Full Text Full Text PDF PubMed Scopus Google Scholar) have further differences α4β1 and in to study is to the Ca2+ and metal binding and these are to ligand a of high affinity and rate of the used as a for interactions in which binding is used as a for Jurkat cells are with and to a with and under these conditions integrin that is not by the and is to the from of are in for binding was seen with and CS1 at concentrations that are with binding constants for α4β1 A. C. S. A. R.R. J. Scopus Google Scholar, R.R. A. 1995; PubMed Scopus Google of the binding was that the a to for the as had been previously seen in binding A. S. 1995; PubMed Scopus Google The binding used to the for the for a ligand C. Scopus Google Scholar) and from the seen for the binding was by a the for dissociation of the was only for the Mn2+ which identified a in as a of integrin for BIO1211 under these as a have as a for α4β1 the in the 2 mm Mn2+ alone and 2 mmMn2+ TS2/16 states is specific to BIO1211 or a property of α4β1 to with and CS1 binding to Jurkat cells under the same conditions for in and CS1 were to differences the 2 mm and 2 mm Mn2+ TS2/16 the that BIO1211 of the properties of α4β1 and that the different activation states of α4β1 are to the binding of Mn2+ TS2/16 high affinity state is a property of α4β1 that binding of and mm Mn2+ 10 μg/ml mm Mn2+ mm 1 mm of Jurkat cells × in the were for 1 at with of or CS1 was and the were further 10 The cells were by and binding by for and CS1 were from the values for of BIO1211 for BIO1211 were for the 2 mm Mn2+ and 2 mm Mn2+ TS2/16 states from on and rates and for the 1 mm 1 mm state from binding as in the not in a of Jurkat cells × in the were for 1 at with of or CS1 was and the were further 10 The cells were by and binding by for and CS1 were from the values for of BIO1211 for BIO1211 were for the 2 mm Mn2+ and 2 mm Mn2+ TS2/16 states from on and rates and for the 1 mm 1 mm state from binding as in the not We have used as a model LDV-containing ligand to study α4β1 integrin binding was under conditions that affect the activation state of D values were observed that ranged from a value of 20–40 in the 1 mm Mg2+, 1 mm Ca2+ state to 100 pm in the 2 mm Mn2+ state to 18 pm in a identified state detected when binding was measured in the of 2 mmMn2+ 10 μg/ml TS2/16. The differences in affinity were almost exclusively by changes in the dissociation rate of the had to affinity differences non-activated and activated the large in affinity the activated states was a in affinity for BIO1211 was detected the Mg2+, Mn2+ mm state and the 2 mmMn2+ TS2/16 that the in the integrin affinity state are to and binding under conditions and that were seen for these of the activated that activation was not by a high affinity state by a of that was by changes in the assay is from the in the affinity from to to to to pm by high concentrations of Mg2+, BIO1211 affinity and dissociation rate to the values seen at 2 mm Mn2+ that the seen with and Mn2+ at of the same affinity state of the integrin. was by the that further increase in affinity was observed when that had been with 2 mmMn2+ were with 10 concentrations of a of the dissociation rate to that seen with Mg2+, with the seen at concentrations of Mn2+ not the of metal of a of states of the integrin. the the states is as as states for the in are to the of affinity states the of 10 mm TS2/16 or 2 mm Mn2+ a further increase in affinity was The of activation by divalent cations and by TS2/16 on the K D for binding indicates that the of activation by divalent and by antibody are The are about the the mm Mn2+ and the affinity state observed with or Mn2+ plus TS2/16. the observed differences in the and dissociation rates 2 mm Mn2+ TS2/16 and 10 mm TS2/16 that the affinity state to the of a of was further by in which dissociation rates were measured in the of 2 TS2/16 × 10−4 10 mm TS2/16 × and 100 mm TS2/16 0.17 × The changes in the dissociation rate of the that were observed as was from 2 to 10 from 10 to 100 mm are to the changes observed at the same concentrations in the of TS2/16 values for the TS2/16 states were values for the alone observed a with a of of the binding are in the in the distinct and by which divalent cations and TS2/16 α4β1 high affinity were with BIO1211 was the rate of on the of for α4β1 to a indicating that the integrin is with BIO1211 is at a the for α4β1 not a and a indicating that of to the The rate for the was to that for from the divalent alone state after with the rate for the was to that for the divalent plus TS2/16 state in the of Similar were of were activated by TS2/16 alone or by TS2/16 plus divalent indicating that the dissociation were not to the high affinity that the divalent cations are not to to the same in the different activated states of α4β1 or that a different the are to these is not activation with TS2/16 in the integrin or to the that dissociation after divalent and states provides for the that the bring about activation of α4β1 in distinct the mechanism by which the affinity states of are is a large of the as a key M.J. PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. J. 1995; Full Text Full Text PDF PubMed Scopus Google to the same on the and as the have been in which the cations with or with ligand for binding A. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, A. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, R.R. PubMed Scopus Google Scholar, T.A. C. Cell. 1994; Full Text PDF PubMed Scopus Google or in which binding regulates the of the integrin to the M.J. PubMed Scopus Google the model only the and not the rate constants for ligand binding are by changes in the activation indicating that the is for binding under all states of study a the from the of was used as a model for the the ligand binding for Mn2+ binding T.A. C. Cell. 1994; Full Text PDF PubMed Scopus Google We for as a for α4β1 have been for integrin activation is the for the and of divalent cations and TS2/16 on α4β1 activation is that the on the and the on the of α4β1 affinity on divalent cations on the α-chain the of the affinity on TS2/16 binding on the β-chain. of the on the in the and of metal ions affect of these and the affinity of α4β1 for TS2/16 or not a of the antibody on affinity a by antibody binding that ligand binding that metal binding at the which affect ligand the not to BIO1211 binding at a the of α-chain A. Takada Y. J. 1995; PubMed Scopus Google Scholar) that are at these key to the of these distinct on ligand binding and integrin activation a of changes the integrin that to the activation of changes have been in are or ligand binding M.J. PubMed Scopus Google Scholar, M.J. J. Immunol. Google BIO1211 is a and to the same of that A. C. S. A. R.R. J. Scopus Google The of BIO1211 for of the of of antibody is from the for in is a which is as a inhibitor of interactions that not leukocyte M.J. J. Immunol. Google is a inhibitor of binding R.R. A. 1995; PubMed Scopus Google that on BIO1211 binding and that ligand binding binding at the The of the to differences in α4β1 activation that the to an for of have been used to study α4β1 R.R. A. 1995; PubMed Scopus Google Scholar, R.R. S. S. C. C. 1991; PubMed Scopus Google Scholar, A. Hemler M.E. J. 268: Full Text PDF PubMed Google the with about ligand binding that were not from these conventional assay methods. were the that activation is not by a state by distinct states that to a range of and that the affinity differences are to dissociation rates of the integrin-ligand for α4β1 and that in further the with integrin We for the TS2/16 Hemler for the and and the and for the cell We and for on the properties of BIO1211 in diverse in which values were adhesion and binding with as