Simultaneous isolation of platelet factor 4 and glycoprotein IIb-IIIa complex from rabbit platelets, and characterization of specific chicken antibodies to assay them.
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.
Santoro
Marcelo Larami
ML
Laboratory of Pathophysiology, Institute Butantan, Av. Vital Brazil, 1500, 05503-900, São Paulo-SP, Brazil. santoro@butantan.gov.br
Barbaro
Kátia Cristina
KC
Flores da Rocha
Tânia Rúbia
TR
Soares Torquato
Ricardo José
RJ
Hirata
Izaura Yoshico
IY
Sano-Martins
Ida Sigueko
IS
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
D000911:Antibodies, Monoclonal; C016512:IgY; D007136:Immunoglobulins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010978:Platelet Factor 4; D013917:Thrombin