The Role of Multiple Electrode Aggregometry in Detection of Clopidogrel Resistance in Diabetic Patients With Coronary Artery Disease and Prediction of Clinical Outcomes. A Comparative-method, Non Interventional, Single Center Study.

The aim of this study is to evaluate the ability of a new POC device, the multiple electrode aggregometry (Multiplate, Dynabyte, Munich, Germany) to detect clopidogrel resistance and predict the clinical outcome in patients with type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) in comparison with the light transmittance aggregometry (LTA), which is considered the gold standard of platelet function testing [7]. 280 patients with T2DM between 20 and 75 years of age treated with oral and/or parenteral hypoglycaemic therapy for at least 1 month and angiographically established coronary artery disease on the grounds of a previous ST elevation, non-ST elevation acute coronary syndrome or unstable angina treated with clopidogrel 75 mg/day in combination with daily per os aspirin therapy for at least 7 days after documentation of CAD, will be recruited. Patients' enrollment will take place in the Second University Department of Cardiology of the "Attiko" University Hospital in Athens, Greece. Baseline assessment will include recording of demographic data, medical history, cardiovascular risk factors, hematological parameters, number of diseased vessels, number of stented vessels, previous myocardial infarction (MI), previous revascularization, ejection fraction and concomitant medications. Patients with renal (creatinine levels > 2.5 mg/dl) or hepatic (bilirubin level > 2 mg/dL) insufficiency, malignant disease, use of drugs known to affect platelet function, history of bleeding diathesis, platelet count < 100x109/L and a hematocrit < 28% will be excluded. The protocol will be approved by the institutional review board of "Attiko" University Hospital and all patients will give written informed consent before undergoing any study procedure or receiving any study treatment.

Platelet function measurements will be performed on study enrollment. Patients will be identified either as poor or good responders to clopidogrel. The primary end-point will be defined as a composite of death from cardiovascular causes, nonfatal myocardial infarction, stent thrombosis, ischemic stroke, urgent rehospitalization for acute coronary syndrome and/or revascularisation during 1-year follow-up period. Episodes of major bleeding according to PLATO criteria will be recorded during follow-up [8].

The ability of the two methods to detect clopidogrel resistance will be evaluated and the potential correlation between response to antiplatelet treatment and clinical outcome will be assessed. In parallel, patients with similar characteristics, but treated with prasugrel or ticagrelor, will be used as control group of the same sample size.

Platelet Function Analysis Platelet function analysis will be performed in the Laboratory of Haematology and Blood Bank Unit of "Attiko" University Hospital. Blood samples will be collected from an antecubital vein 2 to 4 h after antiplatelet therapy intake. The first 2 to 4 ml of blood will be discarded to avoid spontaneous platelet activation and platelet function tests will be performed within two hours of sampling.

Light transmittance aggregometry (LTA) The whole blood specimen will be collected in 3.8% trisodium citrate and centrifuged at 200 g for 10 minutes to obtain platelet-rich plasma (PRP). The remaining specimen will be re-centrifuged at 2000 g for 15 minutes to obtain platelet-poor plasma (PPP). The platelet count will be adjusted to between 200,000/μl and 300,000/μl with PPP. Aggregation will be performed using a Biodata-PAP-4 aggregometer (Bio/Data Corporation, PA, USA). The 100% line will be set using PPP and a 0% baseline established with PRP before addition of the agonist. The agonist used will be ADP 2.0×10-5 M (Bio/Data Corporation, PA, USA). Test procedure will be performed as previously described [9]. In brief, 0.45 mL PRP is transferred into a cuvette incubated at 37 °C for 3 minutes. Then 0.05 mL of the agonist is added into the PRP and the aggregation pattern is allowed to generate for 6 minutes. Platelet aggregation was determined as the maximal percent change in light transmittance from baseline using PPP as a reference. Cut-off value of > 59% aggregation following 20 microM ADP stimulation will be defined as that indicating of high on-treatment platelet reactivity [10]. Other tested parameters will be late aggregation and the degree of disaggregation.