Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters











Database
Language
Publication year range
1.
Arterioscler Thromb Vasc Biol ; 40(3): 624-637, 2020 03.
Article in English | MEDLINE | ID: mdl-31969014

ABSTRACT

OBJECTIVE: The αIIbß3 antagonist antiplatelet drug abciximab is the chimeric antigen-binding fragment comprising the variable regions of murine monoclonal antibody 7E3 and the constant domains of human IgG1 and light chain κ. Previous mutagenesis studies suggested that abciximab binds to the ß3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent ß1-α1 helix. These studies could not, however, assess whether 7E3 or abciximab prevents fibrinogen binding by steric interference, disruption of either the αIIbß3-binding pocket for fibrinogen or the ß3 SDL (which is not part of the binding pocket but affects fibrinogen binding), or some combination of these effects. To address this gap, we used cryo-electron microscopy to determine the structure of the αIIbß3-abciximab complex at 2.8 Å resolution. Approach and Results: The interacting surface of abciximab is comprised of residues from all 3 complementarity-determining regions of both the light and heavy chains, with high representation of aromatic residues. Binding is primarily to the ß3 SDL and neighboring residues, the ß1-α1 helix, and ß3 residues Ser211, Val212 and Met335. Unexpectedly, the structure also indicated several interactions with αIIb. As judged by the cryo-electron microscopy model, molecular-dynamics simulations, and mutagenesis, the binding of abciximab does not appear to rely on the interaction with the αIIb residues and does not result in disruption of the fibrinogen-binding pocket; it does, however, compress and reduce the flexibility of the SDL. CONCLUSIONS: We deduce that abciximab prevents ligand binding by steric interference, with a potential contribution via displacement of the SDL and limitation of the flexibility of the SDL residues.


Subject(s)
Abciximab/ultrastructure , Cryoelectron Microscopy , Integrin alpha2/ultrastructure , Integrin beta3/ultrastructure , Platelet Aggregation Inhibitors , Abciximab/metabolism , Binding Sites , Binding, Competitive , HEK293 Cells , Humans , Integrin alpha2/genetics , Integrin alpha2/metabolism , Integrin beta3/genetics , Integrin beta3/metabolism , Ligands , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Mutation , Platelet Aggregation Inhibitors/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins/ultrastructure , Structure-Activity Relationship
2.
Histol Histopathol ; 19(3): 735-42, 2004 07.
Article in English | MEDLINE | ID: mdl-15168335

ABSTRACT

Aortic explants were obtained from mouse fetuses and cultured in collagen gels. Immuno-fluorescence microscopy, antibodies (anti alpha1, alpha2 and alpha3 integrin subunits) were used. Fibroblastic cells migrated from the aortic explant after one day of cultivation. The migrating cells located in the peripheral part of the aortic explant were positive for alpha1 and alpha2 integrin subunit antibodies. Immuno-fluorescence-positive staining for the alpha3 integrin subunit antibody was clearly seen in the migrating cells located near the aortic explant and surrounding tube-like structures. In an immuno-electron microscope study performed by pre-embedding immuno labeling, gold particles associated with the alpha3 integrin subunit were found to reside on the membranes of the cells surrounding the capillary-like tubes. Two synthetic peptides, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and KDGEA (Lys-Asp-Gly-Glu-Ala), were added to the growth medium to study their effects on cell migration. KDGEA, a compound containing the recognition sequence for alpha2beta1 integrin, decreased cell migration, while GRGDSP exhibited no effect. The migration of fibroblastic cells is an important phenomenon for tube formation. The present study suggested that the alpha1 and alpha2 integrin subunits are both involved in the cell migration, and more specifically, that the alpha2 integrin subunit participates in cell migration through the KDGEA sequence. The alpha3 integrin subunit played a role in tube formation.


Subject(s)
Integrin alpha1/metabolism , Integrin alpha2/metabolism , Integrin alpha3/metabolism , Neovascularization, Physiologic , Protein Subunits/metabolism , Animals , Aorta/cytology , Cell Movement/drug effects , Collagen Type I/metabolism , Culture Media , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Fluorescent Antibody Technique, Indirect , Gels , Immunohistochemistry , Integrin alpha1/immunology , Integrin alpha1/ultrastructure , Integrin alpha2/immunology , Integrin alpha2/ultrastructure , Integrin alpha3/immunology , Integrin alpha3/ultrastructure , Magnesium/metabolism , Mice , Microscopy, Confocal , Microscopy, Immunoelectron , Oligopeptides/pharmacology , Organ Culture Techniques , Time Factors
SELECTION OF CITATIONS
SEARCH DETAIL