Between September 2013 and August 2014, 43 patients with CHD underwent PCI surgery at Hebei General Hospital (Shijiazhuang, China). In the same time period, 20 healthy volunteers without any history of cardiovascular disease were enrolled in the present study as the control group. The following information was recorded for patients and healthy subjects: i) General clinical data, including gender, age, height, weight, blood pressure, platelet count, fasting plasma glucose level, liver function, kidney function, blood lipid level and plasma fibrinogen content; ii) CHD risk factors, including smoking, drinking, hypertension history and diabetes history; iii) anamnesis, including chronic hepatitis, chronic gastritis and thrombocytopenia; and iv) coronarography and PCI results.

Inclusion criteria for the patients with PCI were as follows: i) Aged between 45 and 80 years; ii) confirmed diagnosis of coronary atherosclerotic heart disease and planned stent implantation (≥70% target lesion stenosis, as indicated by coronarography). Exclusion criteria for patients with PCI were as follows: i) No significant stenosis or percutaneous coronary stent implantation contraindications; ii) severe heart failure, which was defined as left ventricular ejection fraction, ≤30%; iii) severe liver-kidney dysfunction, which was defined as glutamic pyruvic transaminase or glutamic oxaloacetic transaminase, ≥80 µ/l and glomerular filtration rate, ≤125 ml/min; iv) recent history of bleeding or heparin-induced thrombocytopenia; v) cerebrovascular disorder within 3 months; vi) major surgery within 1 month; vii) intake of clopidogrel 1 week prior to hospitalization; viii) life expectancy <12 months; and ix) allergy to aspirin, clopidogrel, heparin, stainless steel or contrast medium. All procedures were approved by the Ethics Committee of Hebei General Hospital. Written informed consent was obtained from all patients or their families.

Peripheral blood samples were supplemented with citrate dextrose, containing 85 mM trisodium citrate, 78 mM citric acid and 111 mM glucose, and centrifuged at 80 × g for 10 min at 22°C to obtain the plasma. Following mixing with 2 mM ethylenediaminetetraacetic acid, the platelet-rich plasma was centrifuged at 1,000 × g for 10 min at 22°C to deposit the platelets. Subsequently, 3 ml beads buffer, containing 0.8% NaCl, 0.02% KCl, 0.144% Na₂HPO₄, 0.024% KH₂PO₄, 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid, was added to resuspend the platelets prior to the addition of 40 µl human CD45 MicroBeads reagent (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Following incubation at room temperature for 45 min with gentle mixing, a MACS^® bead separation system (Miltenyi Biotec GmbH) was used to collect leukocyte-depleted platelets with purity >99.99%.

For bioinformatics analysis, miRWalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/), miRanda (http://www.microrna.org/microrna/home.do) and Targetscan (http://www.targetscan.org/) databases were used to analyze the 3′-UTR region of VASP mRNA. The results of the three databases were collectively used to predict the roles of miR-26a, miR-199 and miR-23a in the regulation of VASP expression (21).

miRNA expression was detected using SYBR Green (Takara Biotechnology Co., Ltd., Dalian, China) RT-qPCR, with U6 gene as an internal reference for relative quantification. cDNA was synthesized from 1 µg total RNA using an M-MLV Reverse Transcription System (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. PCR was performed using a total reaction volume of 25 µl, composed of 12.5 µl SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.), 1 µl PCR forward primer, 1 µl Uni-miR qPCR primer, 2 µl template, and 8.5 µl double distilled H₂O. Primer sequences were as follows: miR-26a, 5′-CGCGCTTCAAGTAATCCAGG-3′; miR-23a, 5′-ATCACATTGCCAGGGATTTCC-3′; miR-199a, 5′-CCCAGTGTTCAGACTACCTGTTC-3′; and U6, 5′-ACGCAAATTCGTGAAGCGTT-3′. PCR amplification conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec, and annealing at 60°C for 20 sec (Veriti Dx Thermal Cycler; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The 2^−ΔΔCq method was used to calculate miRNA relative expression levels (22). Each sample was measured in triplicate.

mRNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. SYBR Green (Takara Biotechnology Co., Ltd.) RT-qPCR was employed to determine the mRNA expression levels of VASP in platelets, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference for quantification analysis. VASP mRNA reaction system was composed of 10 µl SYBR Premix Ex Taq mix, 0.5 µl upstream primer (5-′CAACTCAGGAGGAGCCAGAGG-3′), 0.5 µl downstream primer (5-′TCACCCTCTGTAGGTCCGAG-3′), 1 µl cDNA and 8 µl double distilled H₂O. PCR amplification conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 40 sec and elongation at 72°C for 30 sec, and final extension at 72°C for 1 min. The 2^−ΔΔCq method was used to calculate the relative expression of VASP mRNA (22). Each sample was tested in triplicate.

Platelets, which were purified from 5 ml whole blood, were supplemented with 600 µl precooled radioimmunoprecipitation assay lysis buffer, containing 50 mM Tris-base, 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100 and 1% sodium deoxycholate (Beyotime Institute of Biotechnology, Shanghai, China). Following lysis for 50 min on ice, the mixture was centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was collected in order to determine protein concentration using a bicinchoninic acid protein concentration determination kit (RTP7102; Real-Times Biotechnology Co., Ltd., Beijing, China). Protein samples (50 µg) were subsequently mixed with equal volumes of 2X sodium dodecyl sulfate loading buffer prior to denaturation in a boiling water bath for 5 min. Protein samples (10 µl) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 100 V. Resolved proteins were transferred to polyvinylidene difluoride membranes on ice at 300 mA for 1.5 h, and were subsequently blocked with 50 g/l skimmed milk at room temperature for 1 h. Subsequently, the membranes were incubated with polyclonal rabbit anti-goat VASP (1:5,000; cat. no. ab209093) and polyclonal rabbit anti-goat GAPDH primary antibodies (1:2,000; cat. no. ab37168) (Abcam, Cambridge, MA, USA) at 4°C overnight. Following washing three times with phosphate-buffered saline with Tween 20 (PBST) for 15 min, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1,000; cat. no. ab6721; Abcam) for 1 h at room temperature. Following washing three times with PBST for 15 min, the membrane was developed using an enhanced chemiluminescence detection kit (Sigma-Aldrich, St. Louis, MO, USA). Image Lab software (verson 4.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to acquire and analyze imaging signals. The relative content of VASP protein was expressed as a ratio of VASP/GAPDH.

Flow cytometry was used to measure the phosphorylation levels of VASP following stimulation with vasodilators, and to calculate the PRI. At least 6 h prior to surgery, patients were administered 300 mg clopidogrel, and clopidogrel was maintained at 75 mg daily following surgery. Seven days after clopidogrel administration, fasting blood samples (4 ml) were collected from patients and tested within 48 h according to the manufacturer's protocol (PLTVASP/P2Y12 kit; Diagnostica Stago S.A.S., Paris, France). Following count analysis using FACSCalibur (BD Biosciences, San Jose, CA, USA), the corrected mean fluorescence intensity (MFI) values (MFIc) of tubes T1 and T2 were determined. MFIc was calculated from MFI values obtained from MAb anti-VASP-P (tubes T1 and T2) following subtraction of the negative controls (tube T3): MFIc (PGE1)=MFIc (T1)=MFI (T1) - MFI (T3); MFIc (PGE1 + ADP)=MFIc (T2)=MFI (T2) - MFI (T3). PRI was calculated using the following function: PRI=[MFIc (PGE1) - MFIc (PGE1 + ADP)]/MFIc (PGE1) × 100. At least one normal sample was tested as a control for each patient sample. Low response to clopidogrel was defined as PRI ≥50% on day 7 following administration, whereas high response was defined as PRI <50% (23).

Student's t-tests were performed using SPSS 16.0 for Windows (SPSS, Inc., Chicago, IL, USA). All data were expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Clinical data were collected from 43 patients and 20 healthy subjects (Table I). The data demonstrated that patients with low clopidogrel response were not significantly different from those with high clopidogrel response, according to all clinical data and postoperative cardiovascular physiological and biochemical indicators. In addition, healthy subjects were not significantly different from patients with low or high clopidogrel response in gender, age, smoking, body mass index or platelet count. However, healthy subjects did not present with type II diabetes, hyperlipidemia or hypertension. Although still within the normal range, patients with low or high clopidogrel response exhibited significantly increased fibrinogen content, as compared with the healthy subjects (P<0.05). These results indicated that PCI may not significantly alter CHD risk factors, such as type II diabetes, hypertension and age, in patients with CHD.

To calculate PRI, flow cytometry was performed to measure the phosphorylation levels of VASP. Analysis of the results demonstrated that the number of patients with low clopidogrel response (PRI ≥50%) was 17 (39.53%); whereas the number of patients with high clopidogrel response (PRI <50%) was 26 (60.47%). In addition, the average PRI values of patients with low response or normal response to clopidogrel were significantly higher, as compared with the healthy subjects (P<0.05). Notably, the average PRI of patients with low clopidogrel response was significantly increased, as compared with the high clopidogrel response group (P<0.05) (Fig. 1). These results suggested that platelet activity is increased in patients with low or high clopidogrel response, as compared with healthy subjects, on day 7 following clopidogrel treatment.

Western blotting was performed to measure VASP protein expression in platelets. The results demonstrated that platelet VASP protein expression did not significantly differ between patients with high clopidogrel response and healthy subjects, whereas platelet VASP protein expression was significantly higher in patients with low clopidogrel response, as compared with healthy subjects and patients with high clopidogrel response (P<0.05) (Fig. 2). These results suggested that platelet VASP protein expression may be inhibited by clopidogrel in patients with high clopidogrel response, whereas VASP protein expression may be elevated in patients with low clopidogrel response, who may have high platelet reactivity.

RT-qPCR was used to determine VASP mRNA expression levels in platelets. The results demonstrated that there was no significant difference between the platelet VASP mRNA expression levels of patients with high clopidogrel response and healthy subjects. However, patients with low clopidogrel response exhibited significantly increased platelet VASP mRNA expression levels, as compared with healthy subjects and patients with high clopidogrel response (P<0.05) (Fig. 3). These results indicated that VASP gene transcription is significantly increased in patients with low clopidogrel response, as compared with healthy subjects and patients with high clopidogrel response, which exhibit similar low expression levels.

RT-qPCR was performed to determine whether miR-26a, miR-199 or miR-23a in platelets can target VASP mRNA. The results demonstrated that relative expression of platelet miR-26a in patients with low clopidogrel response was significantly increased, as compared with healthy subjects or patients with high clopidogrel response (P<0.05; Fig. 4A); however, this phenomenon was not observed for either miR-199 or miR-23a (Fig. 4B and C). These results suggested that the expression of miR-26a in platelets may be associated with high platelet reactivity, whereas miR-199 and miR-23a are not.

To investigate whether serum miR-26a, miR-199 or miR-23a participate in clopidogrel resistance, their respective expression levels were measured using RT-qPCR. The results demonstrated that the serum expression levels of miR-26a, miR-199 and miR-23a did not differ among the three groups (Fig. 5A–C). These results indicated that serum miR-26a, miR-199 and miR-23a are not involved in clopidogrel resistance.