Patients

Between October 2013 and September 2015, all patients admitted with a diagnosis of internal carotid artery stenosis and treated with CAS at the Department of Neurosurgery, Gifu University Hospital were investigated prospectively. Clinical demographic information such as age, sex, previous atherosclerosis-related history, and perioperative medical conditions was recorded. The indication criterion for CAS was stenosis >80% in asymptomatic lesions and/or >50% in symptomatic lesions, in accordance with the North American Symptomatic Carotid Endarterectomy Trial (NASCET) protocol. Patients were treated with antiplatelet therapy comprising some combination of aspirin (100 mg/day), clopidogrel (75 mg/day), and/or cilostazol (200 mg/day) for a minimum of 7 days before the procedure; these agents were selected at the discretion of the attending physicians. Patients continued to receive the same antiplatelet therapy for at least 3 months after CAS. The experimental protocol was approved by the institutional ethics committee and informed consent was obtained from all patients.

Blood sampling and preparation of human platelet-rich plasma

Blood samples were collected before and 4 days after CAS to avoid the effects of heparin or catecholamine, which was used during or after CAS. Blood samples were drawn into a 1/10 volume of 3.2% sodium citrate. Platelet-rich plasma (PRP) was obtained from blood samples by centrifugation at 155g for 12 min at room temperature. Platelet-poor plasma (PPP) was prepared from residual blood by centrifugation at 1400g for 5 min.

ADP and collagen induction of light transmittance platelet aggregation

Aggregation of platelets in citrated PRP was conducted at 37°C in a light transmittance aggregometer (PA-200 Kowa, Tokyo, Japan) with a stirring speed of 800 rpm. ADP (Sigma-Aldrich, St Louis, Missouri, USA) and collagen (Takeda Austria, Linz, Austria) were used to induce aggregation. Platelets were preincubated for 1 min; subsequently, aggregation was monitored for 4 min after the addition of the agonist. The PRP and corresponding PPP transmittance percentages were recorded as 0% and 100%, respectively, and aggregation was expressed as a percentage of the maximum transmittance. Each agonist was tested at three concentrations: ADP 3, 10, and 20 μM and collagen 3, 10, and 20 μg/mL.

The ED₅₀ was defined as the concentration required to induce >50% platelet aggregation transmittance; a low ED₅₀ value indicates high platelet reactivity. We used the ED₅₀ to classify platelet reactivity to ADP stimulation as follows: high reactivity, ED₅₀ ≤3 μM; medium to high reactivity, 3.1–10 μM; medium to low reactivity, 10.1–20 μM; and low reactivity, >20 μM. Similarly, reactivity to collagen stimulation was classified as follows: high reactivity, ED₅₀ ≤3 μg/mL; medium to high reactivity, 3.1–10 μg/mL; medium to low reactivity, 10.1–20 μg/mL; and low reactivity, >20 μg/mL (figure 1). We defined an increase in platelet reactivity as a shift to a higher reactivity category over serial assessments. Patients who exhibited increased platelet reactivity to ADP stimulation, collagen stimulation, or both were categorized as activated.

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Figure 1

Platelet reactivity grade categorization based on effective dose 50% (ED₅₀) values. Representative platelet aggregation curves of each platelet reactivity grade are shown. Blue lines indicate 3 μM ADP and 3 μg/mL collagen, green lines indicate 10 μM ADP and 10 μg/mL collagen, and red lines indicate 20 μM ADP and 20 μg/mL collagen.

MRI analysis

All patients underwent preoperative MRI screening followed by digital subtraction angiography to ascertain the suitability of their lesions for CAS. Previous reports identified carotid artery plaques exhibiting high-intensity signals (HIS) on time-of-flight magnetic resonance angiography (TOF-MRA), as observed on sagittal oblique maximum intensity projection images, as an independent risk factor for ischemic complications in patients subjected to CAS.23 We therefore recorded this sign during plaque evaluation.

In all patients, baseline diffusion-weighted imaging (DWI) was performed after diagnostic angiography and before CAS. A second DWI was performed within 72 hours after CAS, at which time only newly appearing lesions were regarded as ischemic lesions after CAS. MRI findings were evaluated by blinded neuroradiologists.

CAS procedures

All CAS procedures were performed under local anesthesia via the percutaneous transfemoral route. All procedures were performed by a single neurointerventional team. A 100 U/kg heparin bolus was administered immediately before the procedure to increase the activated clotting time to a minimum of 250 s. Two types of embolic protection devices were used: distal balloon protection via a Guardwire (Medtronic AVE, Santa Rosa, California, USA; n=16) or proximal balloon protection via an Optimo (Tokai Medical Products, Aichi, Japan) and Guardwire (n=22). Two types of stents were placed in the stenotic lesions: an open cell stent such as Precise (Johnson & Johnson, Cordis, Minneapolis, Minnesota, USA; n=32) or Protage (Covidien, Mansfield, Massachusetts, USA; n=2), or a Wallstent (Boston Scientific, Natick, Massachusetts, USA; n=4) closed cell stent. Stroke neurologists performed neurological assessments 24 hours after CAS.

Statistical analysis

Values are presented as means±SD. Categorical variables were analyzed using the χ² or Fisher's exact test as appropriate. Continuous variables with normal distributions were analyzed using the Student's t-test or the paired t-test; those with non-normal distributions were analyzed using the Mann-Whitney U test or Wilcoxon signed-rank test as appropriate. Univariate and multivariate logistic regression analyses were performed to determine which factors correlated with increased platelet reactivity after CAS. A p value of <0.05 was considered statistically significant. All statistical analyses were performed using PASW Statistics software, V.18 (SPSS Japan, Tokyo, Japan).