title: Simultaneous isolation of platelet factor 4 and glycoprotein IIb-IIIa complex from rabbit platelets, and characterization of specific chicken antibodies to assay them.
abstract: 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.
authors.affiliation: Laboratory of Pathophysiology, Institute Butantan, Av. Vital Brazil, 1500, 05503-900, São Paulo-SP, Brazil. santoro@butantan.gov.br
authors.forename: Marcelo Larami
authors.initials: ML
authors.lastname: Santoro
authors.forename: Kátia Cristina
authors.initials: KC
authors.lastname: Barbaro
authors.forename: Tânia Rúbia
authors.initials: TR
authors.lastname: Flores da Rocha
authors.forename: Ricardo José
authors.initials: RJ
authors.lastname: Soares Torquato
authors.forename: Izaura Yoshico
authors.initials: IY
authors.lastname: Hirata
authors.forename: Ida Sigueko
authors.initials: IS
authors.lastname: Sano-Martins
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001792:Blood Platelets / Q000276:immunology*; D002645:Chickens; D002847:Chromatography, Agarose; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D019085:Fluorescent Antibody Technique, Direct; D006168:Guinea Pigs; D007136:Immunoglobulins / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D008297:Male; D015539:Platelet Activation / Q000276:immunology; D010978:Platelet Factor 4 / Q000276:immunology / Q000302:isolation & purification*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits / Q000276:immunology*; D013917:Thrombin / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; C016512:IgY; D007136:Immunoglobulins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010978:Platelet Factor 4; D013917:Thrombin