Methods

Study population

We collected data on 801 patients with type 2 diabetes suffering from acute ischemic cardio-cerebral vascular events, including acute coronary syndrome (ACS) and acute ischemic stroke (AIS), at the Guangdong Provincial People’s Hospital and Guangdong Geriatrics Institute during the past four years from the hospital information system(seen in Figure. 1). All enrolled patients must be within one week from onset to admission, and were treated with dual antiplatelet therapy (DAPT, including clopidogrel (Plavix) and aspirin).

Exclusion criteria:

patients with atrial fibrillation, cerebral embolism, hemorrhagic stroke, thrombocytopenia, thrombocytosis, platelet dysfunction, type 1 diabetes, coagulation dysfunction, severe liver disease, hematopoietic dysfunction, chronic kidney disease (≥stage 3), pregnancy, or malignant tumors and patients treated with anticoagulant agents or thrombolytic or novel antiplatelet drugs within one week before admission. Patients with partial clinical data or laboratory results were also excluded.

Data collection and organization:

Data collection:

Staff employed in the statistics office of our hospital conducted a data search based on important fields, such as diagnosis, laboratory results, and primary clinical medications.

Data arrangement:

Data such as diagnosis, laboratory results, and clinical medications were integrated into the same data file based on ”patient identification number & hospitalization date” by Excel for Windows; thus, a medical database was established.

Data elimination:

To avoid selective bias and confounding factors, we chose the data from patients treated with a single hypoglycemic drug. Data with a small sample, such as patients treated with short-acting insulin, dipeptidyl peptidse-4 inhibitors, repaglinide, or thiazolidinedione, were eliminated. Moreover, patients with insulin duration <7 days were excluded, and for core data such as the maximum amplitude of adenosine diphosphate-induced platelet-fibrin clots (MAADP), the outliers were eliminated, the criteria were -3 ≤ z ≤ 3.
These details are shown in Figure. 1.

Data supplementation:

We reviewed medical documents and supplied some demographic data (height, weight, etc.), lifestyle data (smoking, drinking, etc.), and data on past medical history and drugs taken before admission. For those who did not have a detailed record, we contacted the patient by phone.

Data verification:

All core data were carefully verified by 2 staff members.

Antiplatelet therapy regimen:

According to clinical record data, all patients included presented two groups, Conventional DAPT (clopidogrel 75 mg daily, aspirin 100 mg daily) should be carried out at least 7 days before admission. For those who did not take any antiplatelet drugs before admission or only took one antiplatelet drug, they received intensive DAPT (clopidogrel 300 mg daily, aspirin 300 mg daily) after admission. The clopidogrel taken by patients was a reference listed drug, not a generic drug.

CYP2C19 Genotyping

As stated in the published article, 14 CYP2C19 was determined in our hospital by a genotyping kit (DNA Microarray, BaiO Inc., Shanghai, China).CYP2C19*2 mutations include 681GA and 681AA, while CYP2C19*3 mutations include 636GA and 636AA.

Thromboelastography (TEG)

Thrombelastograms (TEGs) reflects not only thrombin generation kinetics and fibrin­network formation during clot generation but also platelet function.15 MAADP refers to the maximum fibrin clot strength induced by adenosine diphosphate(ADP), and is an important indicator in TEG. The quantitative assessment of MAADP measured by TEG can assess the patient’s response to oral antiplatelet therapy and can be related to the prognosis of cardiovascular and cerebrovascular events,15-17 and with the increase of MAADP, the risk of thrombosis increased or the antiplatelet effect was poor.18 In the study, we adopted MAADP measured by TEG as a main indicator to evaluate patients’ effect on antiplatelet treatment.
Blood samples that were collected from patients at least 6 h after they received intensive treatment or 7 days after they received regular antiplatelet therapy were sent for analysis in vacutainer tubes containing 3.2% trisodium citrate. The vacutainer tubes were filled to capacity and inverted 3–5 times to ensure complete mixing of the anticoagulant. Thrombelastography® uses four channels to detect the effects of antiplatelet therapy acting via the arachidonic acid and ADP pathways. A detailed description of this method has been outlined previously.19 The TEG® 5000 Thrombelastograph® Hemostasis Analyzer System (Haemoscope Corporation, Niles, USA) and automated analytical software were used to measure the physical properties.

Statistical Analysis

Statistical analysis was performed using IBM SPSS Statistics 23 software. The Kolmogorov-Smirnov test (KS test) was used to perform normal distribution analysis for all measurement data. Data that were fit for analyses were transferred to the normal distribution. Continuity variables are expressed as the mean ± the standard deviation (SD). Each candidate risk factor was analyzed by univariate linear regression analysis, and P < 0.1 was defined as the screening criterion. Then, stratified regression analysis was carried out to gradually adjust for the demographic data, genetic factors, lifestyle, biochemical indicators, antiplatelet regimen, etc., and to analyze the effects of hypoglycemic treatment on MAADP tested by TEG. p < 0.05 was defined as statistically significant.
The present study was approved by the Human Ethics Committee of Guangdong Provincial People’s Hospital and Guangdong Academy of Medical Sciences (No. GDREC2017280H) and adhered to the tenets of the Declaration of Helsinki.