Simultaneous isolation of platelet factor 4 and glycoprotein IIb-IIIa complex from rabbit platelets, and characterization of specific chicken antibodies to assay them.
J Immunol Methods
; 284(1-2): 55-72, 2004 Jan.
Article
en En
| MEDLINE
| ID: mdl-14736417
Rabbits are frequently used as models for studying coagulation and platelet disorders. However, few reports on literature have dealt with the purification and characterization of rabbit platelet proteins. Herein a protocol for the simultaneous purification of rabbit platelet factor 4 (PF4) and platelet glycoprotein IIb-IIIa (GPIIb-IIIa, integrin alpha(IIb)beta(3)) is described. Specific antibodies were raised in laying chicken, which were used for assaying PF4 by ELISA, and GPIIb-IIIa by direct immunofluorescence and flow cytometry. Furthermore, the binding of monoclonal antibodies specific for GPIIb-IIIa complex (P2), ligand-induced binding site of GPIIIa (LIBS1) and rabbit P-selectin (12A7), as well as of polyclonal IgY specific for rabbit GPIIb-IIIa, was compared in quiescent and thrombin-activated platelets. Polyclonal anti-rabbit PF4 IgY was a specific and sensitive probe that could be used for assaying PF4 in plasma samples. GPIIb-IIIa expression was increased in thrombin-activated platelets, as evaluated by flow cytometric analysis using P2 and polyclonal antibodies raised in chickens. Rabbit GPIIb-IIIa also exhibited a conformational modification that caused the appearance of ligand-induced binding sites. Increased P-selectin expression, used as a positive control, was also noticeable in thrombin-activated platelets. These data evidence that antibodies raised in laying chickens specific to rabbit PF4 and GPIIb-IIIa, as well as certain monoclonal antibodies specific for human GPIIb-IIIa, may be used for investigating rabbit platelet physiology.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Conejos
/
Plaquetas
/
Inmunoglobulinas
/
Factor Plaquetario 4
/
Complejo GPIIb-IIIa de Glicoproteína Plaquetaria
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
J Immunol Methods
Año:
2004
Tipo del documento:
Article
País de afiliación:
Brasil
Pais de publicación:
Países Bajos