RESUMEN
G-proteins play an important role in platelet signal transduction and regulate responses upon activation of G-protein coupled receptors (GPCR). We have previously reported a patient with impaired platelet responses associated with deficiency in platelet Galphaq. To understand the molecular basis for this defect, the cDNA sequence encoding Galphaq (1080 bp) was obtained by reverse-transcription and polymerase chain reaction of platelet RNA; the cDNA sequence showed no mutations in the patient. Platelet Galphaq mRNA levels were decreased by >50% compared to normal subjects; platelet Galphai2 mRNA levels were normal. Neutrophil calcium mobilization and elastase secretion, upon activation with several agonists, and neutrophil Galphaq mRNA and protein levels were normal. These studies demonstrate that the patient has a defect in Galphaq gene expression in platelets but not neutrophils, possibly due to defects in transcriptional regulation or mRNA stability, and suggest a hematopoietic-lineage specific defect.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas/genética , Plaquetas/metabolismo , Trastornos Hemorrágicos/genética , Proteínas de Unión al GTP Heterotriméricas/deficiencia , Neutrófilos/metabolismo , Trastornos de las Plaquetas Sanguíneas/sangre , Señalización del Calcio , Linaje de la Célula , Análisis Mutacional de ADN , ADN Complementario/genética , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Regulación de la Expresión Génica , Trastornos Hemorrágicos/sangre , Proteínas de Unión al GTP Heterotriméricas/biosíntesis , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/fisiología , Humanos , Elastasa de Leucocito/metabolismo , Persona de Mediana Edad , Neutrófilos/enzimología , Especificidad de Órganos , Agregación Plaquetaria , Proteínas Proto-Oncogénicas/biosíntesis , ARN Mensajero/biosíntesis , ARN Mensajero/sangre , Transcripción GenéticaRESUMEN
Defects in glycoprotein (GP)IIb-IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67-year-old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex-specific anti-GPIIb-IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was approximately 50% of normal. An enzyme-linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb-IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb-IIIa, monitored with mAb PAC-1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet-activating factor (PAF); expression of ligand-induced binding sites (LIBS) was < or = 30% of normal. Signal transduction-independent LIBS expression, induced by echistatin, was approximately 60% of normal, suggesting that the integrin present had intact ligand-binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post-translational processing of the complex) and inside-out signalling-dependent activation of the GPIIb-IIIa complex.