Platelet P66Shc/NO Signaling Pathway Mediates Clopidogrel Resistance In CHD Patients With DM

Objective: Coronary heart disease(CHD)is one of the diseases with the highest morbidity and mortality in the world.Clopidogrel combined with aspirin has been proven as a powerful solution of treatment of acute coronary syndromes and has been recommended in many treatment guidelines.However,despite intensified dual antiplatelet therapy,about 30% patients still don’t reach the same degree of benefit from the given drug,which is called clopidogrel response variability or clopidogrel resistance.Diabetes mellitus(DM)is a major risk factor for clopidogrel resistance in CHD patients.In this study,we investigated the incidence of clopidogrel resistance and its correlation with recurrent cardiovascular events in CHD patients with DM,and we further explored the regulatory effect of platelet p66Shc/NO signaling pathway on clopidogrel resistance.It was expected to understand the mechanism and develop the novel antiplatelet therapeutic drug target in CHD patients with DM.Methods: Ⅰ Clinical research: 257 patients with coronary heart disease in the Department of Cardiology,Zhongda Hospital,Southeast University,from October 2018 to October 2019,were enrolled in this study.According to whether they have type 2 diabetes mellitus(T2DM)or not,patients are divided into two groups: coronary heart disease without type 2 diabetes(CHD-T2 DM group)and coronary heart disease with type 2 diabetes(CHD+T2DM group).Patients were given clopidogrel load dose before coronary angiography,and blood samples were collected at 0 h and 12 h(10-14 h)after clopidogrel load dose,respectively.Light transmittance aggregometry(LTA)was used to detect the platelet aggregation function.The maximum platelet aggregation rate(MAR)before and after taking clopidogrel was detected,and the inhibition of platelet aggregation(IPA)was calculated,to classify of clopidogrel effectiveness.The patients were followed up for 6 months to investigate the occurrence of major adverse cardiovascular events(MACE)of cardiovascular death,myocardial infarction and stroke.Ⅱ Basic Research: A filter method based on platelet-rich plasma was established to isolate and purify platelets.BCA protein concentration and RNA concentration were determined to verify the content of the obtained platelet samples.q RT-PCR and agarose gel electrophoresis were further used to verify the purity of platelet samples.According to whether clopidogrel resistance,patients were further divided into four groups,namely: the CHD-T2DM-CR group,the CHD-T2DM+CR group,the CHD+T2DM-CR group and the CHD+T2DM+CR group.The expression levels of NO,p66 Shc and other indicators in platelets of each group were detected by NOS activity detection kit,q RT-PCR,Western Blot and other methods.Results: 1)The basal platelet aggregation rate in the CHD+T2DM group was significantly higher than that in the CHD-T2 DM group(P < 0.001);After taking clopidogrel,the incidence of clopidogrel resistance and low response in the CHD+T2DM group was higher than that in the CHD-T2 DM group(P < 0.05);The incidence of MACE in the CHD+T2DM group was also significantly higher than that in the CHD-T2 DM group(P < 0.05);2)The plasma NO and platelet NO levels in the CHD+T2DM+CR group were significantly lower than those in the CHD-T2DM-CR group(P < 0.05);The m RNA and protein levels of platelet p66 Shc in the CHD+T2DM+CR group were significantly higher than those in the CHD-T2DM-CR group(P < 0.05).Platelet p66Shc/NO signaling pathway may be involved in mediating clopidogrel resistance in CHD patients with T2 DM.Conclusions: CHD patients with T2 DM have a higher basal platelet aggregation rate.The incidence of clopidogrel resistance and low response after taking clopidogrel was significantly higher than that of CHD patients without T2 DM,and it was related to the incidence of MACE.Through further analysis,it was found that the NO levels in the serum and platelets of patients in the CHD+T2DM+CR group were significantly lower than in the CHD-T2DM-CR group,and the expression level of platelet p66 Shc was also significantly higher than in the CHD-T2DM-CR group.The signal pathway may be involved in mediating clopidogrel resistance in CHD patients with T2 DM.