RESUMEN

Integrin alpha4beta1 plays an important role in inflammatory processes by regulating the migration of leukocytes into inflamed tissues. Previously, we identified BIO5192 [2(S)-{[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino}-4-[4-methyl-2(S)-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino)-pentanoylamino]-butyric acid], a highly selective and potent (K(D) of 9 pM) small molecule inhibitor of alpha4beta1. Although BIO5192 is efficacious in various animal models of inflammatory disease, high doses and daily treatment of the compound are needed to achieve a therapeutic effect because of its relatively short serum half-life. To address this issue, polyethylene glycol modification (PEGylation) was used as an approach to improve systemic exposure. BIO5192 was PEGylated by a targeted approach in which derivatizable amino groups were incorporated into the molecule. Two sites were identified that could be modified, and from these, five PEGylated compounds were synthesized and characterized. One compound, 2a-PEG (K(D) of 19 pM), was selected for in vivo studies. The pharmacokinetic and pharmacodynamic properties of 2a-PEG were dramatically improved relative to the unmodified compound. The PEGylated compound was efficacious in a rat model of experimental autoimmune encephalomyelitis at a 30-fold lower molar dose than the parent compound and required only a once-a-week dosing regimen compared with a daily treatment for BIO5192. Compound 2a-PEG was highly selective for alpha4beta1. These studies demonstrate the feasibility of PEGylation of alpha4beta1-targeted small molecules with retention of activity in vitro and in vivo. 2a-PEG, and related compounds, will be valuable reagents for assessing alpha4beta1 biology and may provide a new therapeutic approach to treatment of human inflammatory diseases.

Asunto(s)

Antiinflamatorios , Integrina alfa4beta1/antagonistas & inhibidores , Oligopéptidos/farmacología , Compuestos de Fenilurea/farmacología , Polietilenglicoles/farmacología , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacocinética , Antiinflamatorios/farmacología , Adhesión Celular , Diseño de Fármacos , Encefalomielitis Autoinmune Experimental/complicaciones , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Humanos , Inyecciones Intravenosas , Inyecciones Subcutáneas , Células Jurkat , Mediciones Luminiscentes , Recuento de Linfocitos , Proteína Básica de Mielina/toxicidad , Oligopéptidos/síntesis química , Oligopéptidos/farmacocinética , Parálisis/etiología , Parálisis/prevención & control , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/farmacocinética , Polietilenglicoles/farmacocinética , Ratas , Ratas Endogámicas Lew , Relación Estructura-Actividad

RESUMEN

Integrin alpha 4 beta 1 plays an important role in inflammatory processes by regulating the migration of lymphocytes into inflamed tissues. Here we evaluated the biochemical, pharmacological, and pharmacodynamic properties and efficacy in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, of two types of alpha 4 beta 1 inhibitors, the anti-rat alpha 4 monoclonal antibody TA-2 and the small molecule inhibitor BIO5192 [2(S)-[[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino]-4-[4-methyl-2(S)-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino)-pentanoylamino]-butyric acid]. TA-2 has been extensively studied in rats and provides a benchmark for assessing function. BIO5192 is a highly selective and potent (KD of <10 pM) inhibitor of alpha 4 beta 1. Dosing regimens were identified for both inhibitors, which provided full receptor occupancy during the duration of the study. Both inhibitors induced leukocytosis, an effect that was used as a pharmacodynamic marker of activity, and both were efficacious in the EAE model. Treatment with TA-2 caused a decrease in alpha 4 integrin expression on the cell surface, which resulted from internalization of alpha 4 integrin/TA-2 complexes. In contrast, BIO5192 did not modulate cell surface alpha 4 beta 1. Our results with BIO5192 indicate that alpha 4 beta 7 does not play a role in this model and that blockade of alpha 4 beta 1/ligand interactions without down-modulation is sufficient for efficacy in rat EAE. BIO5192 is highly selective and binds with high affinity to alpha 4 beta 1 from four of four species tested. These studies demonstrate that BIO5192, a novel, potent, and selective inhibitor of alpha 4 beta 1 integrin, will be a valuable reagent for assessing alpha 4 beta 1 biology and may provide a new therapeutic for treatment of human inflammatory diseases.

Asunto(s)

Encefalomielitis Autoinmune Experimental/metabolismo , Integrina alfa4beta1/antagonistas & inhibidores , Linfocitos/efectos de los fármacos , Oligopéptidos/farmacología , Compuestos de Fenilurea/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Endocitosis , Femenino , Humanos , Integrina alfa4beta1/inmunología , Integrina alfa4beta1/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Parálisis/etiología , Ratas , Ratas Endogámicas Lew

RESUMEN

We characterized the early effects of anti-very late antigen (VLA-4) and its counterligand vascular cell adhesion molecule-1 (VCAM-1) antibody therapy on T cell infiltration and apoptosis in adoptive transfer experimental autoimmune neuritis of female Lewis rats. At the peak of disease, animals were treated with anti-VCAM-1 monoclonal antibody (mAb), anti-VLA-4 mAb, or the respective isotype mAb controls 18, 12, or 6 h before perfusion. Anti-VCAM-1 led to a rapid, significant increase of apoptotic T cells in the sciatic nerve with a maximum after 6 h, preceding the significant decrease of T cell infiltration seen after 18 h. This was accompanied by a significant reduction in mRNA levels for IFN-gamma and inducible nitric oxide synthase. The results for anti-VLA-4 treatment showed a similar trend. The early increase of T cell apoptosis following disruption of VLA-4/VCAM-1 interaction may reflect a novel signaling component of proapoptotic pathways.

Asunto(s)

Antialérgicos/antagonistas & inhibidores , Antialérgicos/farmacología , Apoptosis/efectos de los fármacos , Integrinas/antagonistas & inhibidores , Neuritis Autoinmune Experimental/fisiopatología , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/farmacología , Animales , Apoptosis/fisiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Integrina alfa4beta1 , Neuritis Autoinmune Experimental/patología , Ratas , Ratas Endogámicas Lew , Transducción de Señal/fisiología , Linfocitos T/patología , Linfocitos T/fisiología , Factores de Tiempo

RESUMEN

We have used the highly selective alpha(4)beta(1) inhibitor 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino)-pentanoylamino]-butyric acid (BIO7662) as a model ligand to study alpha(4)beta(1) integrin-ligand interactions on Jurkat cells. Binding of [(35)S]BIO7662 to Jurkat cells was dependent on the presence of divalent cations and could be blocked by treatment with an excess of unlabeled inhibitor or with EDTA. K(D) values for the binding of BIO7662 to Mn(2+)-activated alpha(4)beta(1) and to the nonactivated state of the integrin that exists in 1 mm Mg(2+), 1 mm Ca(2+) were <10 pm, indicating that it has a high affinity for both activated and nonactivated integrin. No binding was observed on alpha(4)beta(1) negative cells. Through an analysis of the metal ion dependences of ligand binding, several unexpected findings about alpha(4)beta(1) function were made. First, we observed that Ca(2+) binding to alpha(4)beta(1) was stimulated by the addition of BIO7662. From solution binding studies on purified alpha(4)beta(1), two types of Ca(2+)-binding sites were identified, one dependent upon and the other independent of BIO7662 binding. Second, we observed that the metal ion dependence of ligand binding was affected by the affinity of the ligand for alpha(4)beta(1). ED(50) values for the metal ion dependence of the binding of BIO7762 and the binding of a lower affinity ligand, BIO1211, differed by 2-fold for Mn(2+), 30-fold for Mg(2+), and >1000-fold for Ca(2+). Low Ca(2+) (ED(50) = 5-10 microm) stimulated the binding of BIO7662 to alpha(4)beta(1). The effects of microm Ca(2+) closely resembled the effects of Mn(2+) on alpha(4)beta(1) function. Third, we observed that the rate of BIO7662 binding was dependent on the metal ion concentration and that the ED(50) for the metal ion dependence of BIO7662 binding was affected by the concentration of the BIO7662. These studies point to an even more complex interplay between metal ion and ligand binding than previously appreciated and provide evidence for a three-component coupled equilibrium model for metal ion-dependent binding of ligands to alpha(4)beta(1).

Asunto(s)

Integrinas/química , Integrinas/metabolismo , Iones , Ligandos , Receptores Mensajeros de Linfocitos/química , Receptores Mensajeros de Linfocitos/metabolismo , Benzoatos/farmacología , Calcio/metabolismo , Calcio/farmacología , Cationes , Dipéptidos/farmacología , Relación Dosis-Respuesta a Droga , Ácido Edético/farmacología , Humanos , Integrina alfa4beta1 , Integrinas/antagonistas & inhibidores , Células Jurkat , Cinética , Magnesio/farmacología , Manganeso/farmacología , Modelos Químicos , Compuestos de Fenilurea/farmacología , Unión Proteica , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Factores de Tiempo

RESUMEN

BACKGROUND AND PURPOSE: The present study was performed to determine the role of alpha4 (CD49d), a member of the integrin family of adhesion molecules, in ischemic brain pathology. METHODS: Male spontaneously hypertensive rats (SHR) or Sprague-Dawley rats underwent 60-minute middle cerebral artery occlusion (MCAO) followed by 23-hour reperfusion. Animals were injected intravenously with 2.5 mg/kg anti-rat alpha4 antibody (TA-2) or isotype control antibody (anti-human LFA-3 IgG(1), 1E6) 24 hours before MCAO. Infarct volume was quantified by staining of fresh tissue with tetrazolium chloride and myeloperoxidase activity measured in SHR tissue homogenates 24 hours after MCAO. In SHR, mean arterial blood pressure was recorded before and after MCAO in animals treated with TA-2 and 1E6. Fluorescence-activated cell sorting analysis was performed on peripheral blood leukocytes before and after MCAO. RESULTS: TA-2 treatment significantly reduced total infarct volume by 57.7% in normotensive rats (1E6, 84.2+/-11.5 mm(3), n=17; TA-2, 35.7+/-5.9 mm(3), n=16) and 35.5% in hypertensive rats (1E6, 146.6+/-15.5 mm(3), n=15; TA-2, 94.4+/-25.8 mm(3), n=11). In both strains, TA-2 treatment significantly reduced body weight loss and attenuated the hyperthermic response to MCAO. In SHR, treatment with TA-2 significantly reduced brain myeloperoxidase activity. Resting mean arterial blood pressure was unaffected by treatment. Leukocyte counts were elevated in TA-2-treated rats. Fluorescence-activated cell sorting analysis demonstrated the ability of TA-2 to bind to CD3+, CD4+, CD8+, and CD11b+ cells in both naive animals and after MCAO. CONCLUSIONS: These data demonstrate that inhibition of alpha4 integrin can protect the brain against ischemic brain injury and implicate endogenous alpha4 integrin in the pathogenesis of acute brain injury. The mechanism by which alpha4 integrin inhibition offers cerebroprotection is independent of blood pressure modulation and is likely due to inhibition of leukocyte function.

Asunto(s)

Antígenos CD/metabolismo , Infarto Cerebral/prevención & control , Ataque Isquémico Transitorio/metabolismo , Animales , Anticuerpos/farmacología , Antígenos CD/inmunología , Antígenos CD/farmacología , Presión Sanguínea/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/patología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Infarto Cerebral/etiología , Infarto Cerebral/metabolismo , Infarto Cerebral/patología , Modelos Animales de Enfermedad , Citometría de Flujo , Infarto de la Arteria Cerebral Media/complicaciones , Integrina alfa4 , Ataque Isquémico Transitorio/etiología , Ataque Isquémico Transitorio/inmunología , Ataque Isquémico Transitorio/patología , Recuento de Leucocitos , Leucocitos/citología , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Masculino , Peroxidasa/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Reproducibilidad de los Resultados

RESUMEN

Integrins are a family of transmembrane glycoproteins that can interact with components of the extracellular matrix. The alpha4beta1 and alpha4beta7 integrins are heterodimeric leukocyte cell surface molecules critical to their cell and matrix adhesive interactions. Evidence for a central role for the alpha4 integrins in leukocyte pathophysiology in the lung is well documented. In this study, we tested the hypothesis that neutralizing antibody for integrin alpha4 (PS2) may reduce bleomycin (BL)-induced lung fibrosis in vivo. Male C57BL/6 mice were injected intratracheally with saline (SA) or BL (0.08 U/mouse) followed by intraperitoneal injection of SA, isotype control antibody (1E6), or PS2 (100 microg) three times a week. Twenty-one days after the intratracheal instillation, mice were killed for bronchoalveolar lavage (BAL), biochemical, histopathological, and immunohistological analyses. Treatment with PS2 significantly reduced BL-induced increases in lung lipid peroxidation and hydroxyproline content. Lung histopathology also showed reduced fibrotic lesions in the BL-treated lungs by treatment with PS2. BL-treated mouse lungs also showed induction of cells with the myofibroblast phenotype, as indicated by the increased expression of alpha-smooth muscle actin (alphaSMA), whereas treatment with PS2 minimized the BL-induced alphaSMA expression. Furthermore, treatment with PS2 reduced the BL-induced increase in the BAL total cell number, and attenuated the BL-induced increase in the BAL protein level. It is concluded that integrin alpha4 may play an important role in BL-induced pulmonary fibrosis, and the use of anti-alpha4 antibody offers therapeutic antifibrotic potential in vivo.

Asunto(s)

Antígenos CD/inmunología , Antígenos CD/metabolismo , Fibrosis Pulmonar/prevención & control , Actinas/análisis , Animales , Antibacterianos , Anticuerpos/inmunología , Bleomicina , Líquido del Lavado Bronquioalveolar/citología , Diferenciación Celular , Hidroxiprolina/metabolismo , Inmunohistoquímica , Integrina alfa4 , Peroxidación de Lípido , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/enzimología , Fibrosis Pulmonar/patología

RESUMEN

Eosinophil accumulation has been associated with the pathogenesis of numerous allergic inflammatory disorders. Despite the great interest in this response, many aspects of eosinophil accumulation remain unknown. This is particularly true with respect to tissue-specific mechanisms that may regulate the accumulation of eosinophils in different organs. This study addressed this issue by investigating and comparing the roles of alpha(4)-integrins and vascular cell adhesion molecule 1 (VCAM-1) adhesion pathways in interleukin 4 (IL-4)-induced eosinophil accumulation in 2 different rat models of inflammation, namely pleural and cutaneous inflammation. Similar to our previous findings in studies in rat skin, locally administered IL-4 induced a time- and dose-dependent accumulation of eosinophils in rat pleural cavities, a response that was associated with generation of the chemokine eotaxin. The IL-4-induced eosinophil accumulation in skin and pleural cavities was totally inhibited by an antirat alpha(4)-integrins monoclonal antibody (mAb) (TA-2). In contrast, whereas an antirat VCAM-1 mAb (5F10) totally blocked the response in skin, IL-4-induced eosinophil accumulation in rat pleural cavities was not affected by VCAM-1 blockade. A radiolabeled mAb technique demonstrated that endothelial-cell VCAM-1 expression was induced in response to IL-4 in both skin and pleural membrane. The results indicate that although endothelial-cell VCAM-1 is present in skin and pleura, a functional role for it in IL-4-induced eosinophil accumulation was evident only in skin. These findings suggest the existence of tissue-specific adhesive mechanisms in regulating leukocyte migration in vivo and demonstrate a dissociation between VCAM-1 expression and eosinophil accumulation.

Asunto(s)

Quimiocinas CC , Eosinófilos/efectos de los fármacos , Interleucina-4/farmacología , Molécula 1 de Adhesión Celular Vascular/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/efectos de los fármacos , Antígenos CD/inmunología , Antígenos CD/fisiología , Calcimicina/farmacología , Moléculas de Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Quimiocina CCL11 , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio/química , Endotelio/citología , Eosinófilos/química , Eosinófilos/citología , Inflamación/patología , Inflamación/fisiopatología , Integrina alfa4 , Interleucina-4/fisiología , Ligandos , Masculino , Modelos Animales , Pleura/química , Pleura/patología , Ratas , Ratas Sprague-Dawley , Piel/patología , Factores de Tiempo , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/inmunología

RESUMEN

Integrin alpha(4)beta(1) on the surface of T lymphocytes interacts with vascular cell adhesion molecule-1 (VCAM-1) and fibronectin during migration of lymphocytes from the blood to sites of inflammation. Migrating lymphocytes actively modify their environment through a number of mechanisms including proteolysis of the extracellular matrix by matrix metalloproteinases (MMP) synthesized by the cells. In this study, expression of MMP upon alpha(4)beta(1)-mediated adhesion of leukocytes to two major ligands, the IIICS-1 domain of fibronectin and VCAM-1, has been examined. Adhesion of T lymphoblastoid Jurkat cells to the CS-1 peptide induced expression of mRNA for two MMPs, gelatinase A (MMP-2) and gelatinase B (MMP-9). As evaluated by relative RT-PCR and Northern blot analyses, the level of mRNA was upregulated about 4- to 5-fold for both MMPs compared to control cells maintained in suspension. With time, both enzymes were detected in conditioned media and inside the cells, and their identities were verified by Western blotting and gelatin zymography. Adhesion of Jurkat cells to the second major alpha(4)beta(1) ligand, VCAM-1, upregulated mRNA for MMP-2 (3.5-fold) and failed to induce expression of mRNA for MMP-9. Accordingly, only MMP-2 protein was detected in conditioned media of cells adherent to VCAM-1. Occupancy of alpha(4)beta(1) on the surface of suspended cells with soluble CS-1 peptide or VCAM-1 did not upregulate synthesis and release of MMPs. A similar pattern of induction of MMPs after adhesion to CS-1 and VCAM-1 was observed in T lymphocytes isolated from human blood. These results demonstrate that adhesion of T lymphocytes through alpha(4)beta(1) to different ligands, which bind to similar or overlapping sites in the integrin, induces intracellular events leading to distinct patterns of MMPs biosynthesis.

Asunto(s)

Proteínas Portadoras/fisiología , Adhesión Celular/fisiología , Fibronectinas/fisiología , Integrinas/fisiología , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Oligopéptidos/fisiología , Receptores Mensajeros de Linfocitos/fisiología , Linfocitos T/citología , Linfocitos T/fisiología , Molécula 1 de Adhesión Celular Vascular/fisiología , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Inducción Enzimática , Fibronectinas/química , Fibronectinas/genética , Expresión Génica , Humanos , Integrina alfa4beta1 , Células Jurkat , Cinética , Ligandos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solubilidad

RESUMEN

The integrin alpha9beta1 is expressed on epithelial cells, smooth muscle cells, skeletal muscle, and neutrophils and recognizes at least three distinct ligands: vascular cell adhesion molecule 1 (VCAM-1), tenascin-C, and osteopontin. The alpha9 subunit is structurally similar to the integrin alpha4 subunit, and alpha9beta1 and alpha4beta1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of alpha4 integrins to ligands, would also be a functional inhibitor of alpha9beta1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of alpha9beta1-mediated adhesion to VCAM-1 and of neutrophil migration across tumor necrosis factor-activated endothelial cells. A peptide containing a novel MLDG motif shared by both of these disintegrins also inhibited alpha9beta1- and alpha4beta1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of alpha9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other alpha9beta1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SVVYGLR, which we have previously shown inhibit binding of alpha9beta1-expressing cells to tenascin-C and osteopontin, respectively, had no effect on adhesion to VCAM-1. These data suggest that there are structurally distinct requirements for interactions of the alpha9beta1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.

Asunto(s)

Desintegrinas/farmacología , Integrinas/metabolismo , Sialoglicoproteínas/metabolismo , Tenascina/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Venenos de Víboras/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Dimerización , Desintegrinas/química , Desintegrinas/aislamiento & purificación , Desintegrinas/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Humanos , Integrinas/antagonistas & inhibidores , Integrinas/genética , Datos de Secuencia Molecular , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Osteopontina , Fragmentos de Péptidos/farmacología , Alineación de Secuencia , Análisis de Secuencia de Proteína , Especificidad por Sustrato , Venenos de Víboras/química , Venenos de Víboras/aislamiento & purificación , Venenos de Víboras/metabolismo

RESUMEN

Leukocyte recruitment to target tissue is initiated by weak rolling attachments to vessel wall ligands followed by firm integrin-dependent arrest triggered by endothelial chemokines. We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium. Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, alpha4beta1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1. Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date. Although VLA-4 affinity is not altered upon chemokine signaling, subsecond VLA-4 clustering at the leukocyte-substrate contact zone results in enhanced leukocyte avidity to VCAM-1. Endothelial chemokines thus regulate all steps in adhesive cascades that control leukocyte recruitment at specific vascular beds.

Asunto(s)

Adhesión Celular/fisiología , Endotelio Vascular/metabolismo , Integrinas/metabolismo , Leucocitos/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Anticuerpos Monoclonales , Movimiento Celular/fisiología , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Endotelio Vascular/citología , Citometría de Flujo , Humanos , Integrina alfa4beta1 , Microscopía Confocal , Microscopía por Video , Transducción de Señal , Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/genética

RESUMEN

We have isolated and characterized EMS16, a potent and selective inhibitor of the alpha2beta1 integrin, from Echis multisquamatus venom. It belongs to the family of C-lectin type of proteins (CLPs), and its amino acid sequence is homologous with other members of this protein family occurring in snake venoms. EMS16 (M(r) approximately 33K) is a heterodimer composed of two distinct subunits linked by S-S bonds. K562 cells transfected with alpha2 integrin selectively adhere to immobilized EMS16, but not to two other snake venom-derived CLPs, echicetin and alboaggregin B. EMS16 inhibits adhesion of alpha2beta1-expressing cells to immobilized collagen I at picomolar concentrations, and the platelet/collagen I interaction in solution at nanomolar concentrations. EMS16 inhibits binding of isolated, recombinant I domain of alpha2 integrin to collagen in an ELISA assay, but not the interaction of isolated I domain of alpha1 integrin with collagen IV. Studies with monoclonal antibodies suggested that EMS16 binds to the alpha2 subunit of the integrin. EMS16 inhibits collagen-induced platelet aggregation, but has no effect on aggregation induced by other agonists such as ADP, thromboxane analogue (U46619), TRAP, or convulxin. EMS16 also inhibits collagen-induced, but not convulxin-induced, platelet cytosolic Ca(2+) mobilization. In addition, EMS16 inhibits HUVEC migration in collagen I gel. In conclusion, we report a new, potent viper venom-derived inhibitor of alpha2beta1 integrin, which does not belong to the disintegrin family.

Asunto(s)

Integrinas/antagonistas & inhibidores , Lectinas Tipo C , Lectinas/farmacología , Venenos de Víboras/química , Secuencia de Aminoácidos , Señalización del Calcio/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Dimerización , Disulfuros , Endotelio Vascular/efectos de los fármacos , Humanos , Integrinas/genética , Integrinas/metabolismo , Lectinas/clasificación , Datos de Secuencia Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Unión Proteica , Receptores de Colágeno , Análisis de Secuencia de Proteína

RESUMEN

The leukocyte integrin very late antigen-4 (alpha(4)beta(1), CD49d/CD29) is an adhesion receptor that plays an important role in allergic inflammation and contributes to antigen-induced late responses (LAR) and airway hyperresponsiveness (AHR). In this study, we show that single doses of a new small-molecule, tight-binding inhibitor of alpha(4), BIO-1211, whether given by aerosol or intravenously, either before or 1.5 h after antigen challenge blocks allergen- induced LAR and post-antigen-induced AHR in allergic sheep. Multiple treatments with doses of BIO-1211 that were ineffective when given singly, were protective. BIO-1211 also provided dose-dependent inhibition of the early airway response (EAR) to antigen. In conjunction with the functional protection against the antigen-induced LAR and AHR, sheep treated with BIO-1211 before challenge showed significantly reduced: (1) numbers of eosinophils in bronchoalveolar lavage (BAL), (2) BAL levels of the inflammatory marker tissue kallikrein, and (3) numbers of inflammatory cells (lymphocytes, eosinophils, metachromatic staining cells, and neutrophils) in bronchial biopsies obtained after challenge when compared with corresponding biopsies after vehicle treatment. More importantly, we show for the first time that an inhibitor of alpha(4) was able to reverse post-antigen-induced AHR, thereby decreasing the time of recovery from the normal period of > 9 d to 3 d. Our results show that effective inhibition of antigen-induced airway responses can be achieved with single doses of a potent small-molecule inhibitor of alpha(4) and that such agents may be used therapeutically, as well as prophylactically, to alleviate allergen- induced inflammatory events. These data provide further support and extend the evidence for the role of alpha(4) integrins in the pathophysiologic events that follow airway antigen challenge.

Asunto(s)

Asma/fisiopatología , Integrina beta1/fisiología , Integrinas/antagonistas & inhibidores , Integrinas/fisiología , Oligopéptidos/farmacología , Receptores Mensajeros de Linfocitos/fisiología , Receptores de Antígeno muy Tardío/fisiología , Animales , Asma/tratamiento farmacológico , Hiperreactividad Bronquial/tratamiento farmacológico , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Carbacol/administración & dosificación , Eosinófilos/citología , Integrina alfa4beta1 , Calicreínas/análisis , Oligopéptidos/administración & dosificación , Oligopéptidos/uso terapéutico , Ovinos

RESUMEN

BACKGROUND: The alpha4beta1 (or very late antigen-4 [VLA-4]) integrin is thought to play a role in inflammatory processes, mediating mononuclear leukocyte infiltration. The adventitial response to balloon injury is an important determinant of neointimal formation and arterial remodelling. OBJECTIVES: To determine whether the monoclonal antibody hHP1/2 directed against the human alpha4-integrin subunit decreases neoadventitial formation and subsequent luminal narrowing following balloon injury. DESIGN: Randomized, double-blind, placebo controlled study. SETTING: Tertiary care, Canadian university hospital vascular biology laboratory. ANIMALS AND METHODS: In 16 pigs, two coronary arteries were injured with an oversized balloon, while a third coronary artery was designated as an uninjured control vessel. One hour before balloon injury, 5 mg/kg of hHP1/2 was administered to eight animals, while another eight animals received an infusion of a saline placebo. Animals were killed three and 14 days following balloon injury. MAIN RESULTS: Administration of hHP1/2 resulted in an immediate decrease in circulating monocyte and lymphocyte counts. These parameters returned to normal within three days. There was a decrease in neoadventitial formation 14 days after arterial injury in pigs treated with hHP1/2 compared with controls (2.26+/-0.77 versus 3.42+/-1.01 mm, respectively, P=0.04). There was a loss of lumen area between days 3 (4.33+/-1.09 mm2) and 14 (3.09+/-0.38 mm2, P=0.02) after balloon injury in pigs treated with saline, but not in the pigs treated with hHP1/2. CONCLUSIONS: Administration of an antibody to the alpha4-integrin subunit is associated with less neoadventitial formation and less lumenal narrowing after balloon injury. This novel therapy may play an important role in modulating arterial remodelling and thereby may reduce restenosis following percutaneous coronary interventions in humans.

Asunto(s)

Angioplastia Coronaria con Balón , Anticuerpos Monoclonales/farmacología , Vasos Coronarios/lesiones , Integrinas/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Túnica Íntima/lesiones , Animales , Vasos Coronarios/inmunología , Vasos Coronarios/patología , Displasia Fibromuscular/inmunología , Displasia Fibromuscular/patología , Displasia Fibromuscular/prevención & control , Humanos , Integrina alfa4beta1 , Integrinas/fisiología , Recuento de Linfocitos , Receptores Mensajeros de Linfocitos/fisiología , Porcinos , Túnica Íntima/inmunología , Túnica Íntima/patología

RESUMEN

Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAb's against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.

Asunto(s)

Artritis/prevención & control , Adhesión Celular/fisiología , Colágeno/metabolismo , Dermatitis Alérgica por Contacto/prevención & control , Hipersensibilidad Tardía/prevención & control , Integrinas/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Artritis/inmunología , Artritis/patología , Colágeno/toxicidad , Dermatitis Alérgica por Contacto/inmunología , Dermatitis Alérgica por Contacto/patología , Dermatitis Irritante/inmunología , Dermatitis Irritante/patología , Dermatitis Irritante/prevención & control , Edema/etiología , Edema/prevención & control , Femenino , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/patología , Integrina alfa1beta1 , Integrinas/inmunología , Leucocitos/patología , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores de Colágeno

RESUMEN

Alpha5beta1, a major fibronectin receptor, is a widely distributed integrin that is essential for cell growth and organ development. Here, we describe a novel heterodimeric disintegrin named EMF10, isolated from the Eristocophis macmahoni venom, that is an extremely potent and selective inhibitor of alpha5beta1. EMF10 inhibited adhesion of cells expressing alpha5beta1 to fibronectin (IC(50) = 1-4 nM) and caused expression of a ligand-induced binding site (LIBS) on the beta1 subunit of alpha5beta1 integrin. It partially inhibited adhesion of cells expressing alphaIIbbeta3, alphavbeta3, and alpha4beta1 to appropriate ligands only at concentration higher than 500 nM. Guinea pig megakaryocytes expressing alpha5beta1 adhered to immobilized EMF10 and showed extensive spreading and cytoskeletal mobilization. As determined by electrospray mass spectrometry, EMF10 is composed of two species with molecular masses of 14 575 and 14 949 Da, respectively. EMF10 is a heterodimer containing two subunits: EMF10A (Mr 7544 Da) and EMF10B (Mr 7405 and 7032 Da) linked covalently by S-S bonds. Subunit B showed heterogeneity and may be present as EMF10B1 (Mr 7032) and EMF10B2 (Mr 7405). In putative hairpin loops, EMF10A and EMF10B contained CKKGRGDNLNDYC and CWPAMGDWNDDYC motifs, respectively. The reduced and alkylated subunit B of EMF10 inhibited adhesion of K562 cells to fibronectin in a dose-dependent, saturable manner with IC(50) of 3 microM. The synthetic, cyclic CKKGRGDNLNDYC and CWPAMGDWNDDYC peptides expressed their inhibitory activity in the same system with IC(50) of 100 microM. We propose that alpha5beta1 recognition of EMF10 is associated with the MGDW motif located in a putative hairpin loop of the B subunit and that the expression of activity may also depend on the RGDN motif in the subunit A and on the C-termini of both subunits.

Asunto(s)

Desintegrinas/química , Receptores de Fibronectina/antagonistas & inhibidores , Venenos de Víboras/química , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Células CHO , Adhesión Celular/efectos de los fármacos , Cricetinae , Citoesqueleto/metabolismo , Citoesqueleto/fisiología , Dimerización , Desintegrinas/aislamiento & purificación , Desintegrinas/metabolismo , Desintegrinas/fisiología , Fibronectinas/metabolismo , Cobayas , Humanos , Células Jurkat , Células K562 , Ligandos , Megacariocitos/metabolismo , Megacariocitos/fisiología , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Receptores de Fibronectina/biosíntesis , Receptores de Fibronectina/metabolismo , Relación Estructura-Actividad , Venenos de Víboras/aislamiento & purificación , Venenos de Víboras/metabolismo , Venenos de Víboras/farmacología

RESUMEN

The aim of the present study was to investigate the role of the adhesion pathway alpha4 integrins/vascular cell adhesion molecule type 1 (VCAM-1) in rapid eosinophil accumulation induced by the chemoattractants PAF and LTB4. For this purpose we have used an in vivo model of local 111In-eosinophil accumulation to quantify eosinophil accumulation induced by intradermal administration of platelet-activating factor (PAF) and leukotriene B4 (LTB4) in rats. Initial experiments carried out over 4 hr demonstrated that intravenous administration of an anti-VCAM-1 monoclonal antibody (mAb; 5F10) or an anti-alpha4 integrin mAb (TA2) caused a significant reduction in PAF- or LTB4-induced 111In-labelled eosinophil accumulation. Time-course experiments demonstrated that the anti-VCAM-1 mAb was effective at suppressing early phases of the 111In-labelled eosinophil accumulation induced by PAF and LTB4 (e.g. within the first 60 min). In contrast, 111In-labelled eosinophil accumulation induced by these chemoattractants was unaffected by the local administration of the transcriptional inhibitor actinomycin D, suggesting a role for basally expressed VCAM-1. Indeed, basal expression of VCAM-1 in rat skin sites was demonstrated by the localization of intravenously administered radiolabelled mAb. The localization of the radiolabelled antibody was not altered in skin sites injected with PAF or LTB4. Finally, the inhibitory effects seen with the anti-VCAM-1 mAb were enhanced when the antibody was co-injected into rats with an anti-intercellular adhesion molecule-1 (ICAM-1) mAb (1A29). The combination of these two mAb also caused a significant inhibition of PAF-induced oedema, as quantified by the local accumulation of 125I-labelled human serum albumin. The results indicate a role for alpha4 integrins/VCAM-1 and ICAM-1, in PAF- and LTB4-induced eosinophil accumulation in vivo and suggest that basally expressed VCAM-1 may have a functional role in rapid accumulation of eosinophils induced by chemoattractants.

Asunto(s)

Quimiotaxis de Leucocito/inmunología , Eosinófilos/inmunología , Piel/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Dactinomicina/farmacología , Edema/inmunología , Técnicas para Inmunoenzimas , Integrina alfa4 , Integrinas/inmunología , Leucotrieno B4/inmunología , Masculino , Factor de Activación Plaquetaria/inmunología , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Sprague-Dawley , Enfermedades de la Piel/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

Eotaxin is a potent eosinophil-specific CC-chemokine, which has been shown to play a role in the selective induction of eosinophil accumulation in a number of allergic models of inflammation. Many aspects of the mechanism by which eotaxin induces eosinophil accumulation in vivo remain unresolved. In the present study, we investigated the direct effect of synthetic human eotaxin on leucocyte/endothelial cell interactions within rat mesenteric venules, as quantified by intravital microscopy. Topical eotaxin (30 pmol) induced rapid firm adhesion and extravasation of leucocytes within the rat mesentery, the extravasated leucocytes all being eosinophils, as determined by histological analysis. Whilst eotaxin was unable to stimulate the interaction of rat eosinophils with vascular cell adhesion molecule-1 (VCAM-1) under static conditions in vitro, eotaxin-induced responses in vivo were significantly suppressed by anti-alpha4 integrin and anti-VCAM-1 monoclonal antibodies (mAbs). The anti-alpha4 integrin mAb, HP2/1 (3.5 mg/kg), inhibited the eotaxin-induced firm adhesion and extravasation, 60 min postapplication of the chemokine, by 89% and 84%, respectively. In the same set of experiments, the anti-VCAM-1 mAb, 5F10 (3.5 mg/kg), inhibited leucocyte adhesion and extravasation by 61% and 63%, respectively. These results demonstrate that eotaxin-induced migration of eosinophils through rat mesenteric venules in vivo is dependent on an alpha4 integrin/VCAM-1 adhesion pathway, the significance of which may only be evident under flow conditions and/or following the ligation of other adhesion molecules expressed on eosinophils.

Asunto(s)

Moléculas de Adhesión Celular/fisiología , Quimiocinas CC , Factores Quimiotácticos Eosinófilos/farmacología , Citocinas/farmacología , Venas Mesentéricas/inmunología , Administración Tópica , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígenos CD/fisiología , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/inmunología , Movimiento Celular , Quimiocina CCL11 , Eosinófilos/efectos de los fármacos , Eosinófilos/fisiología , Humanos , Integrina alfa4 , Leucocitos/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Estadísticas no Paramétricas , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/fisiología

RESUMEN

We have used the highly specific alpha4beta1 inhibitor 4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study alpha4beta1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of alpha4beta1. Observed KD values for BIO1211 binding ranged from a value of 20-40 nM in 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 mM Mn2+ to 18 pM when binding was measured after co-activation by 2 mM Mn2+ plus 10 microgram/ml of the integrin-activating monoclonal antibody TS2/16. The large range in KD values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 x 10(-4) s-1 to >140 x 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 x 10(6) M-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 alpha4beta1 that 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 alpha4beta1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin.

Asunto(s)

Integrinas/metabolismo , Oligopéptidos/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Cationes Bivalentes , Humanos , Integrina alfa4beta1 , Integrinas/antagonistas & inhibidores , Células Jurkat , Cinética , Ligandos , Unión Proteica , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

The migration of leukocytes into glomeruli in crescentic glomerulonephritis is fundamental to pathogenesis, and offers important therapeutic opportunities. We addressed the importance of VCAM-1, and its leukocyte ligand very late antigen-4 (VLA-4), in such leukocyte migration. In a rat model of nephrotoxic nephritis, glomerular expression of VCAM-1, studied by immunohistochemistry, was up-regulated by day 6 of nephritis. To quantify kidney endothelial VCAM-1 expression, a differential radiolabeled mAb technique was used, which demonstrated that protein expression was not up-regulated by day 2 of nephritis, but rose threefold between days 2 and 5, and remained elevated until at least day 28. An in vivo study was then performed, using blocking mAbs to either VCAM-1 or VLA-4, starting mAb treatment on the day prior to disease induction, and continuing until animals were sacrificed at day 7. mAbs to VLA-4 significantly attenuated renal injury (albuminuria, glomerular fibrinoid necrosis, and crescent formation), but mAbs to VCAM-1 had no significant effect. Surprisingly, the number of leukocytes within glomeruli was unaffected by anti-VLA-4 mAb therapy, despite the reduction in renal injury. Paradoxically, classical markers of macrophage activation were increased in the anti-VLA-4- and anti-VCAM-1-treated animals. This study demonstrates that kidney endothelial VCAM-1, in contrast to ICAM-1, is not up-regulated by day 2 of nephrotoxic nephritis, and plays little part in early leukocyte influx into glomeruli. However, VLA-4 is an important mediator of glomerular injury, operating after transendothelial leukocyte migration, and presumably binding to alternate ligands within the kidney.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Endotelio Vascular/metabolismo , Glomerulonefritis/inmunología , Glomerulonefritis/metabolismo , Integrinas/inmunología , Glomérulos Renales/patología , Receptores Mensajeros de Linfocitos/inmunología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Albuminuria/inmunología , Albuminuria/patología , Albuminuria/terapia , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Apoptosis/inmunología , Movimiento Celular/inmunología , Endotelio Vascular/inmunología , Glomerulonefritis/patología , Glomerulonefritis/terapia , Inmunohistoquímica , Integrina alfa4beta1 , Integrinas/fisiología , Radioisótopos de Yodo/metabolismo , Glomérulos Renales/inmunología , Glomérulos Renales/metabolismo , Leucocitos/patología , Masculino , Ratas , Ratas Endogámicas WKY , Receptores Mensajeros de Linfocitos/fisiología , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/fisiología

RESUMEN

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing alpha4beta1 and alpha4beta7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6-30 nM, adhesion of K562 cells (alpha5beta1) to fibronectin with IC50 = 150 nM, and adhesion of alphaIIbbeta3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alphavbeta3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 microM), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mM), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5-10 mM) in contrast to RGDS peptide (IC50 = 3 mM), inhibiting completely at 10 mM.

Asunto(s)

Antígenos CD/efectos de los fármacos , Desintegrinas/farmacología , Oligopéptidos/farmacología , Venenos de Víboras/química , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Dimerización , Desintegrinas/química , Humanos , Integrina alfa4 , Integrina alfa5 , Células K562 , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido