RESUMO
To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from beta3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet-fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Antígenos/imunologia , Plaquetas/imunologia , Hibridomas/imunologia , Glicoproteínas de Membrana , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos CD/genética , Antígenos CD/imunologia , Plaquetas/citologia , Plaquetas/metabolismo , Fusão Celular , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Fibrinogênio/imunologia , Hibridomas/citologia , Integrina beta1/imunologia , Integrina beta3 , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Peso Molecular , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/genética , Tetraspanina 29 , TrombinaRESUMO
To investigate the role of the glycosylation of the platelet receptor glycoprotein Ib (GPIb, CD 42b), platelets and purified GPIb were deglycosylated by neuraminidase, O- and N-glycosidases. N-deglycosylation and neuraminic-acid cleavage had little effect on ristocetin and botrocetin-induced platelet agglutination. However, O-deglycosylation reduced the response by approximately 50%, and total deglycosylation (the combination of all three glycosidases) fully abolished the response to ristocetin. Interestingly, binding of von Willebrand Factor (vWF) to purified GPIb in the presence of ristocetin and botrocetin in a standardized microtiter plate assay was not altered by partial or even by total deglycosylation. Electron microscopy indicated that the normally stretched approximately 50 nm long molecule was approximately 32 nm after N-deglycosylation, approximately 20 nm after O-deglycosylation, and reduced in a approximately 15 nm long collapse by total deglycosylation. These results suggest that deglycosylation has major structural impacts on GPIb, strongly impairing platelet-vWF interactions; however, vWF binding to isolated GPIb remains unaffected.
Assuntos
Plaquetas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/ultraestrutura , Sítios de Ligação , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Sequência de Carboidratos , Venenos de Crotalídeos/farmacologia , Glicosídeo Hidrolases/farmacologia , Glicosilação , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Neuraminidase/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Ristocetina/farmacologia , Fator de von Willebrand/metabolismoRESUMO
Complement component C6 is known to contain two factor I modules in tandem at its C-terminus. To localize the disulfide bridges in those domains, native C6 was cleaved with trypsin, followed by subtilisin. The resulting digests were separated by reversed-phase HPLC, and all of the potential cystine-containing fragments were detected by a fluorescence assay and amino acid composition analyses. Final identification of the disulfide bonds was achieved by Edman degradation of the corresponding peptides. From the data gained a 1-3, 2-9, 4-7, 5-10, 6-8 pattern was determined (Cys752-Cys802, Cys763-Cys780, Cys765-Cys816, Cys772-Cys795, Cys841-Cys852, Cys846-Cys898, Cys859-Cys876, Cys861-Cys911, Cys867-Cys891). These findings are compared with the strongly related complement components C7 and factor I.
Assuntos
Complemento C6/química , Cisteína , Cistina , Fibrinogênio/química , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Dissulfetos , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Subtilisinas , TripsinaRESUMO
C9 is the most abundant protein of the membrane attack complex of complement. By means of limited proteolysis, different chromatographic techniques, a thiol-specific fluorescence assay, amino acid analysis, and Edman degradation 9 out of 12 disulfide bridges are definitely assigned (Cys22-Cys57, Cys33-Cys36, Cys67-Cys73, Cys121-Cys160, Cys233- Cys234, Cys359-Cys384, Cys489-Cys505, Cys492-Cys507, Cys509-Cys518). Weaker evidence permits to reduce the number of possible configurations for the remaining 3 cystines (Cys80-Cys91, Cys86-Cys104, Cys98-Cys113, or Cys80-Cys91, Cys86-Cys113, Cys98-Cys104). These findings are discussed in comparison with the strongly related components C6, C7, C8alpha, and C8beta.
