RESUMO
The pathophysiological mechanisms contributing to the decreased platelet count in immune thrombocytopenia (ITP) are not entirely understood. Here, we investigated the key step of proplatelet formation (PPF) by studying the effect of ITP plasma in thrombopoiesis. Normal cord blood-derived mature megakaryocytes were cultured in the presence of recalcified plasma from ITP patients, and PPF was evaluated by microscopic analysis. Patient samples induced a dose-dependent inhibition in PPF, as well as decreased complexity of proplatelet architecture. Although slightly increased, plasma-induced megakaryocyte apoptosis was not related to PPF impairment. Purified IgG reproduced the inhibitory effect, while platelet-adsorbed plasma induced its reversion, suggesting the involvement of auto-antibodies in the inhibition of thrombopoiesis. Impaired PPF, induced by ITP plasmas bearing anti-GPIIb-IIIa antibodies, was related to their ability to interfere with the normal function of this integrin, as assessed by megakaryocyte PAC-1 binding and ß3 integrin phosphorylation while the presence of anti-glycoprotein Ia-IIa auto-antibodies was associated with loss of normal inhibition of PPF induced by type I collagen. In conclusion, abnormal thrombopoiesis comprising decreased PPF and morphological changes in proplatelet structure are induced by patient samples, unveiling new mechanisms contributing to decreased platelet count in ITP.
Assuntos
Contagem de Plaquetas , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/imunologia , Trombopoese , Adulto , Idoso , Idoso de 80 Anos ou mais , Especificidade de Anticorpos/imunologia , Apoptose/imunologia , Autoanticorpos/imunologia , Plaquetas/citologia , Humanos , Integrinas/imunologia , Megacariócitos/citologia , Megacariócitos/imunologia , Pessoa de Meia-Idade , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Glicoproteínas da Membrana de Plaquetas/imunologia , Trombopoese/imunologia , Adulto JovemRESUMO
Idiopathic thrombocytopenic purpura (ITP) is an autoimmune condition with poorly known etiology, characterized by platelet destruction. Genetic association studies of this disease are scarce, discrepant, and restricted to major histocompatibility complex (MHC) polymorphisms. Hence, a case-control study was conducted with an aim to map the MHC to IPT susceptibility using HLA-B and nine microsatellite loci encompassing MHC class I, II, and III regions. We compared the allelic frequencies in samples of unrelated healthy controls and ITP patients. After correction for multiple tests, only allele MICA*183, also known as A5.1, demonstrated an association, resulting in the identification of a major predisposing region close to STR-MICA. This result may highlight the putative functional role of MICA in the immune response to ITP.
Assuntos
Predisposição Genética para Doença , Antígenos HLA-B/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Púrpura Trombocitopênica Idiopática/genética , Púrpura Trombocitopênica Idiopática/imunologia , Adulto , Autoanticorpos , Brasil , Estudos de Casos e Controles , Feminino , Frequência do Gene , Antígenos HLA-B/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Masculino , Repetições de Microssatélites/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Púrpura Trombocitopênica Idiopática/fisiopatologiaRESUMO
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.
Assuntos
Plaquetas/imunologia , Imunoglobulinas/química , Fator Plaquetário 4/isolamento & purificação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/isolamento & purificação , Coelhos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Galinhas , Cromatografia em Agarose , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Técnica Direta de Fluorescência para Anticorpo , Cobaias , Imunoglobulinas/biossíntese , Imunoglobulinas/imunologia , Masculino , Ativação Plaquetária/imunologia , Fator Plaquetário 4/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Trombina/imunologiaRESUMO
GPIIb/IIIa, the human platelet glycoprotein complex, is the autoantigen most commonly recognized by autoantibodies in autoimmune thrombocytopenic purpura (AITP). Two murine monoclonal antibodies (mAbs), namely Y2/51 and 5B12, directed against gpIIIa and gpIIb/IIIa, respectively, and rabbit anti-human platelet polyclonal antibodies have been used to select AITP-related epitopes from a phage display peptide library expressing random dodecapeptides in the pIII coat protein of M13 phage. The selected phage clones were tested by ELISA for binding to rabbit anti-human platelet antibodies as well as to sera from AITP patients. Seven clones reacted strongly with rabbit anti-human platelet antibodies, and four clones reacted with sera from AITP patients. Some homology between peptide inserts sequences of selected clones and human platelet gpIIIa and gpIb were found.