RESUMO
INTRODUCTION: Reticulated platelet (RP) content is increased in nonvalvular atrial fibrillation (NVAF). The purpose of this study was to determine if platelet content, morphology, and RP proportion are modulated by platelet genes. METHODS AND RESULTS: Expression of six platelet-predominate genes impacting platelet formation and release, platelet count, and RP content was assessed in NVAF patients before and 3-4 months after pulmonary veins isolation (PVI) and compared to normal sinus rhythm (NSR) controls. RNA from isolated platelets was reverse-transcribed assayed against selected genes utilizing real-time qPCR, and expressed as mean cycle threshold (ΔCt) using beta-2-microglobulin as endogenous control. RP content was assessed by flow cytometry. A fourfold lower expression of CFL1 gene coding for nonmuscle cofilin (7.8 ± 0.9 vs. 5.7 ± 1.6, P < 0.001) and twofold lower expression of four other genes were associated with similar platelet counts but fourfold higher (28.7+7.0 vs. 6.7+5.4, P < 0.001) RP content (%) in 97 NVAF cases compared to 51 NSR controls. Three to 4 months after PVI, RP decreased by 28%, while CFL1 gene expression increased over twofold but TUBA4A gene expression decreased almost twofold; NFE2 and MYL6 gene expression remained unchanged. CONCLUSIONS: NVAF is associated with notable downregulation of genes directing platelet production and size but increased RP content. PVI impacts the expression of many of these genes, implying a direct relationship between atrial fibrillation and platelet biogenesis.
Assuntos
Fibrilação Atrial/cirurgia , Plaquetas/metabolismo , Ablação por Cateter , Veias Pulmonares/cirurgia , Potenciais de Ação , Idoso , Fibrilação Atrial/sangue , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/fisiopatologia , Plaquetas/patologia , Estudos de Casos e Controles , Ablação por Cateter/efeitos adversos , Cofilina 1/sangue , Cofilina 1/genética , Feminino , Regulação da Expressão Gênica , Frequência Cardíaca , Humanos , Masculino , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Cadeias Leves de Miosina/sangue , Cadeias Leves de Miosina/genética , Subunidade p45 do Fator de Transcrição NF-E2/sangue , Subunidade p45 do Fator de Transcrição NF-E2/genética , Contagem de Plaquetas , Veias Pulmonares/fisiopatologia , Receptores de Progesterona/sangue , Receptores de Progesterona/genética , Fatores de Tempo , Resultado do Tratamento , Tubulina (Proteína)/sangue , Tubulina (Proteína)/genéticaRESUMO
The processes of megakaryocyte polyploidization and demarcation membrane system (DMS) formation are crucial for platelet production, but the mechanisms controlling these processes are not fully determined. Inhibition of Rho kinase (ROCK) signalling leads to increased polyploidization in umbilical cord blood-derived megakaryocytes. To extend these findings we determined the effect of ROCK inhibition on development of the DMS and on proplatelet formation. The underlying mechanisms were explored by analysing the effect of ROCK inhibition on the expression of MYC and NFE2, which encode two transcription factors critical for megakaryocyte development. ROCK inhibition promoted DMS formation, and increased proplatelet formation and platelet release. Rho kinase inhibition also downregulated MYC and NFE2 expression in mature megakaryocytes, and this down-regulation correlated with increased proplatelet formation. Our findings suggest a model whereby ROCK inhibition drives polyploidization, DMS growth and proplatelet formation late in megakaryocyte maturation through downregulation of MYC and NFE2 expression.