RESUMO
To clarify the mechanism underlying a high risk of thrombotic complications in diabetic patients, we investigated the relationship between HSP27 phosphorylation and the platelet activation induced by adenosine diphosphate (ADP) in diabetic patients. Platelet-rich plasma was prepared from the blood of type 2 diabetes mellitus (DM) patients. By measuring the dose response of platelet aggregation to ADP, an individual ED50 was determined. Based on the normal range identified in non-DM controls, the subjects were divided into a hyper-aggregate (Group 1) and a normo- or hypo-aggregate group (Group 2). The protein phosphorylation was analyzed by western blotting. The release of PDGF-AB and sCD40 ligand (sCD40L) was measured by ELISA. In both groups, ADP induced HSP27 phosphorylation at Ser-78 and Ser-82. The phosphorylation at Ser-78 and the release of both PDGF-AB and sCD40L induced by a low dose of ADP (1 µM) in Group 1 were significantly higher than these values in Group 2. There was a significant relationship between the ADP-induced HSP27 phosphorylation level at Ser-78 and the ADP ED50 value of platelet aggregation. The ADP (1 µM)-induced phosphorylation of HSP at Ser-78 observed in the platelets from Group 1 was inhibited by PD98059 or SB203580. The use of aspirin ameliorated the accelerated microaggregation of platelets in Group 1, and the low-dose ADP-induced phosphorylation of HSP27 at Ser-78 was no longer observed. These results strongly suggest that the phosphorylation of HSP27 at Ser-78 is correlated with the acceleration of platelet aggregation induced by ADP in type 2 DM patients.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Proteínas de Choque Térmico HSP27/metabolismo , Agregação Plaquetária , Processamento de Proteína Pós-Traducional , Difosfato de Adenosina/farmacologia , Difosfato de Adenosina/fisiologia , Idoso , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Ligante de CD40/metabolismo , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Flavonoides/farmacologia , Proteínas de Choque Térmico , Humanos , Imidazóis/farmacologia , Masculino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Chaperonas Moleculares , Fosforilação , Piridinas/farmacologia , Serina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
AIMS: We developed a novel method for diagnosing platelet hyper-aggregation in patients with type 2 diabetes mellitus (DM). MAIN METHODS: By measuring the dose response of platelet aggregation to collagen, an individual ED(50) was determined. Based on the normal range identified in non-DM controls (mean+/-two SEM=0.460+/-0.082 microg/ml, n=47), type 2 DM patients were divided into high ED(50) (ED(50)>0.542 microg/ml; n=32: group I) or low ED(50) groups (ED(50)<0.378 microg/ml; n=32; group II). In these patients, collagen-induced levels of phospho-p38 MAPK and phospho-p44/p42 MAPK were measured using Western blots and enzyme-linked immunosorbent assays (ELISA). KEY FINDINGS: In group II, the collagen (0.3 and 1 microg/ml)-induced levels of both phospho-p38 MAPK and phospho-p44/p42 MAPK measured by western blot analysis were found to be significantly higher than those in group I. The individual ED(50) was found to be significantly correlated with the collagen-induced levels of phospho-p38 MAPK and phospho-p44/p42 MAPK. This correlation was also observed when ELISA was used to measure phospho-p38 MAPK levels in a different population of DM patients (n=90). SIGNIFICANCE: These results strongly suggest that the phosphorylation levels of collagen-induced p38 MAPK and p44/p42 MAPK represent the hyperaggregability of platelets and that the quantification of phospho-p38 MAPK can be a new and useful diagnostic biomarker of platelet hyper-aggregation in DM patients.
