Predictive Value of Platelet Aggregation Tests in Recurrent Cerebral Ischemia and Major Bleeding

Background

Antiplatelet agents are now widely used and are thought to be effective for cerebral ischemia [1–3]. Many different agents are now available on the market, but aspirin is most widely used because of its high cost-benefit value. In clinical practice, these agents are administered without an objective assessment of the in vitro platelet aggregation activity [4] in spite of existing laboratory examinations [5].

Although light transmittance aggregometry (LTA) is one of the most common methods used and is thought to represent the process of platelet aggregation, it requires a fairly difficult laboratory procedure. Additionally, the analysis of a myriad of parameters often impedes judgment of the effectiveness of antiplatelet agents. These are some of the major reasons why LTA is not utilized as a routine examination [6,7].

Furthermore, some researchers claim that there is no objective way to evaluate the efficacy of antiplatelet agents, even by analysis of many cases [8]. Therefore, we aimed to explore parameters of LTA and predict recurrent cerebral ischemia and major bleeding as an adverse event.

Material and Methods

Materials

MATERIALS:

This study was conducted in accordance with the principles of the Helsinki Declaration. The protocol was approved by the Ethics Committee of the Sapporo Medical University (Ethical Number: 21-2-13). Informed consent was obtained verbally with a general written consent form.

All patients (n=768) who experienced symptoms of minor/major stroke or transient ischemic attack (TIA) verified by magnetic resonance imaging (MRI) were included from March 1st, 2003 to October 16th, 2003 (Table 1). Patients who experienced embolic episodes were excluded. We further examined 768 patients by platelet aggregation test at the Asahikawa Neurosurgical Hospital. We utilized the Hema Tracer 801® (MC Medical, Inc., Tokyo, Japan), to measure in vitro platelet aggregation activity, using a standard turbidimetric technique based on changes in optical density.

Among these 768 patients, 589 patients were administered more than 1 antiplatelet agent, and the rest (n=179) received no antiplatelet agent.

ASPIRIN ADMINISTRATION:

The results of platelet aggregation tests, before and after administration of aspirin alone (81 mg/day), were available in 48/589 patients, after excluding patients on concomitant anticoagulation therapy or with surgical procedures (microvascular anastomosis or carotid endarterectomy). In the 5-year follow-up until October 2008, 40/48 patients (mean age: 64.9±14.6; 20 men and 20 women) had no recurrence of ischemia. There were recurring episodes of ischemia in the remaining 8 patients (mean age: 68.3±7.40; 5 men and 3 women).

CONTROL GROUP:

Among 179 patients without any antiplatelet therapy, 24 patients (mean age: 66.0±11.9; 12 men and 12 women) underwent platelet aggregation testing twice, from intervals spanning 1–18 months (mean: 4.6 months). These patients served as our control group.

FACTORS AFFECTING RECURRENCE AND BLEEDING BY LOGISTIC ANALYSIS:

During follow-up, 142 patients receiving antiplatelet agents were analyzed for the prevention of recurrence according to the type of ischemia. One hundred patients were diagnosed with lacunar infarctions at the time of the first ischemic attack. Among them, 55 patients showed no recurrence, while the remaining 45 had recurrent ischemia. Forty-two patients were diagnosed with atherothrombotic infarction. Among them, 19 patients had no recurrence, while the remaining 23 had recurrent ischemia. During the follow-up, 11/589 patients experienced major bleeding episodes such as brain hemorrhage or gastrointestinal bleeding. We excluded patients with nasal bleeding, hematuria, vitreous hemorrhage, or chronic subdural hematoma.

PLATELET AGGREGATION TEST: Platelet-rich plasma (PRP) was prepared by mixing fresh blood from patients with 3.8% sodium citrate in the ratio of 9:1 by volume while resting and fasting. It was then centrifuged at 1000 rpm for 15 minutes. Platelet-poor plasma (PPP), used as a control, was obtained by centrifuging at 3000 rpm for 5 minutes. PRP (0.8 ml) was placed in each cell at a stirring speed of 1000 rpm, and reagents were then added to the PRP in each cell simultaneously [9].

Adenosine diphosphate (ADP) clearly evokes platelet aggregation, which becomes stronger with an increase of ADP concentration (Figure 1; left). Additionally, platelets are known to show primary and secondary aggregation [10,11]. Primary aggregation of platelets is a reversible process due to the lack of intrinsic ADP release. On the contrary, secondary aggregation is an irreversible process showing a firm platelet aggregation with the destruction of platelets releasing intrinsic ADP. These 2 dynamic aggregation processes should be distinguished when the platelet aggregation test is performed in vitro. Therefore, the maximum concentration of ADP showing primary aggregation and the minimum concentration of ADP showing secondary aggregation were set at 4.0 μmol/L and 20 μmol/L of concentration, respectively (Figure 1; left, middle).

Platelet aggregation was measured by the change in light transmittance through PRP during stimulation with the 2 concentrations of ADP (MCM ADP^®: DS Medical Co. Ltd., Tokyo, Japan) and was recorded for 5 minutes. The transmittance values of PRP and PPP were defined as 0% and 100%, respectively.

Aggregation patterns were automatically analyzed using built-in computer software (MC Medical, Inc.). Maximal aggregation (MaxAgg: peak value of the graph), disaggregation rate (disAggR: maximal value-minimal value, divided by maximal value), and aggregation curve area (AgCvAREA: area surrounded by the curve) were calculated for each concentration and the resulting 6 values were analyzed. All assays were performed within 4 hours of sampling.

STATISTICAL ANALYSIS:

Results are expressed as mean±standard deviation. ORIGIN^® (Version 7.5, Origin Lab Corporation: One Roundhouse Plaza, Suite 303, Northampton, MA 01060, USA) and SPSS^® (SPSS Version 25, IBM Japan, Nihombashi-Hakozaki-cho, Tokyo 103-8510, Japan) were used for statistical analysis. A P value of less than 0.01 using one-way ANOVA and the Kruskal-Wallis test was considered statistically significant.

Results

DATA OBTAINED FROM AGGREGOMETER WITH 2 ADP CONCENTRATIONS:

Typical patterns of the platelet aggregation test using the 2 ADP concentrations are shown in Figure 1; middle (normal) and Figure 1; right (hyperaggregation).

FACTORS FOR RECURRENCE AMONG ALL PATIENTS ADMINISTERED ANTIPLATELET AGENTS BY LOGISTIC REGRESSION:

Odds ratios were also calculated using multivariable stepwise logistic regression with backward elimination to elucidate the correlation of obtained factors with lacunar infarction, atherothrombotic infarction, and major bleeding. MaxAgg and disAggR with both concentrations were used, since the AgCvAREA was significantly correlated with MaxAgg, as shown previously. Values are expressed as odds ratio (with 95% confidence interval, P value).

Discussion

LIMITATIONS AND FUTURE CONSIDERATIONS:

Although our research shows that LTA can be a promising tool to evaluate the effectiveness of antiplatelet therapy, there are a few limitations that must be considered. Our research only included patients from a single ethnic group, and results in different regions may vary. To prevent ethnic differences in the interpretation of LTA, multicenter studies may be preferable.

Conclusions

The effectiveness of antiplatelet agents should be assessed by LTA platelet aggregation test. Among the parameters acquired by LTA, only 1 parameter is significant – MaxAgg stimulated by low concentration of ADP – which precisely simulates primary aggregation. The degree of decreasing MaxAgg of the low concentration stimulant indicates the efficacy and complications of antiplatelet treatment.