Logo Medical Science Monitor

Call: +1.631.470.9640
Mon - Fri 10:00 am - 02:00 pm EST

Contact Us

Logo Medical Science Monitor Logo Medical Science Monitor Logo Medical Science Monitor

17 March 2025: Clinical Research  

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

Masanori Ishiguro1ABCDEF, Yasutaka Kurokawa ORCID logo2ACDEF*, Atsushi Tanooka3B, Takashi Nagamine1D

DOI: 10.12659/MSM.947172

Med Sci Monit 2025; 31:e947172

0 Comments

Abstract

0:00

BACKGROUND: Evaluating the efficacy of antiplatelet therapy is rarely performed in patients with cerebral ischemia, despite the underlying potential recurrence of ischemia and unexpected bleeding. This study aimed to evaluate the role of platelet aggregation tests, which can predict ischemic recurrence and major bleeding.

MATERIAL AND METHODS: We measured the platelet aggregation activity of 768 patients using a standardized turbidimetric technique. Ischemia recurred in 8 of 48 patients who received aspirin alone, and recurred in 68 of 142 patients administered more than 1 antiplatelet agent. Major bleeding occurred in 11 of the same 142 patients. Platelet aggregation was induced by the addition of 4.0 and 20 µmol/L of adenosine diphosphate (ADP). The correlations of the maximal aggregation (MaxAgg), disaggregation rate, and aggregation curve area with the recurrence of ischemia and major bleeding were analyzed.

RESULTS: The MaxAgg in patients without recurrence decreased significantly (74.9%±13.2 to 54.0%±11.3, P<0.001 for 4.0 µmol/L) compared with the values before aspirin treatment. The odds ratio for recurrence (n=68) of total ischemia to non-recurrence (n=74) was 1.057 (1.029-1.086, P<0.001) for 4.0 µmol/L stimulation. The odds ratio for patients with bleeding (n=11) to total ischemia cases (n=142) was 0.828 (0.742-0.924, P=0.001) with 4.0 µmol/L stimulation.

CONCLUSIONS: The recurrence of ischemia was correlated with the lack of decrease in MaxAgg. Major bleeding was correlated with an excessive decrease in MaxAgg.

Keywords: Brain Ischemia, cerebral infarction, Platelet Aggregation, Adenosine Diphosphate, Platelet Aggregation Inhibitors

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.

Figures

Platelet aggregation test with light transmittance. The aggregation of platelets is judged as the change in light transmittance in platelet-rich plasma (PRP) by stimulation with different concentrations (from 4.0 μmol/L to 20 μmol/L) of adenosine diphosphate (ADP) recorded for 5 minutes. The degree of aggregation increases with the concentration of ADP (left). The y-axis is defined as 0% of transmittance value of PRP and 100% of platelet-poor plasma (PPP). Typical examples of a normal figure (middle) and of hyperaggregation (right) are given.Figure 1. Platelet aggregation test with light transmittance. The aggregation of platelets is judged as the change in light transmittance in platelet-rich plasma (PRP) by stimulation with different concentrations (from 4.0 μmol/L to 20 μmol/L) of adenosine diphosphate (ADP) recorded for 5 minutes. The degree of aggregation increases with the concentration of ADP (left). The y-axis is defined as 0% of transmittance value of PRP and 100% of platelet-poor plasma (PPP). Typical examples of a normal figure (middle) and of hyperaggregation (right) are given. Change in maximal aggregation (MaxAgg) is summarized according to the presence of ischemic recurrence in the patients treated with aspirin. In the control group, MaxAgg remained unchanged (left) by both concentrations of adenosine diphosphate (ADP) stimulation, even between 2 different intervals. MaxAgg decreased significantly in patients without recurrence by stimulation of both concentrations (middle). On the contrary, MaxAgg did not decrease in patients with recurrence (right).Figure 2. Change in maximal aggregation (MaxAgg) is summarized according to the presence of ischemic recurrence in the patients treated with aspirin. In the control group, MaxAgg remained unchanged (left) by both concentrations of adenosine diphosphate (ADP) stimulation, even between 2 different intervals. MaxAgg decreased significantly in patients without recurrence by stimulation of both concentrations (middle). On the contrary, MaxAgg did not decrease in patients with recurrence (right). Change in disaggregation rate (disAggR) in the patients treated with aspirin. In the control group, disAggR remained unchanged (left) by both concentrations of adenosine diphosphate (ADP) stimulation. DisAggR was not changed in the patients on aspirin except by 4.0 μmol/L of ADP stimulation in those without recurrence (top middle).Figure 3. Change in disaggregation rate (disAggR) in the patients treated with aspirin. In the control group, disAggR remained unchanged (left) by both concentrations of adenosine diphosphate (ADP) stimulation. DisAggR was not changed in the patients on aspirin except by 4.0 μmol/L of ADP stimulation in those without recurrence (top middle). Change in aggregation curve area (AgCvAREA) in patients treated with aspirin. In the control group, the AgCvAREA remained unchanged during the period of the study (left). AgCvAREA significantly decreased in patients without recurrence by both concentrations (middle). The AgCvAREA did not decrease in the patients with recurrence after the treatment (right). ADP – adenosine diphosphate.Figure 4. Change in aggregation curve area (AgCvAREA) in patients treated with aspirin. In the control group, the AgCvAREA remained unchanged during the period of the study (left). AgCvAREA significantly decreased in patients without recurrence by both concentrations (middle). The AgCvAREA did not decrease in the patients with recurrence after the treatment (right). ADP – adenosine diphosphate. Odds ratio of maximal aggregation (MaxAgg) for ischemic recurrence. In patients with atherothrombotic infarction and total ischemia, lack of suppression of MaxAgg predisposes to the recurrence of ischemia.Figure 5. Odds ratio of maximal aggregation (MaxAgg) for ischemic recurrence. In patients with atherothrombotic infarction and total ischemia, lack of suppression of MaxAgg predisposes to the recurrence of ischemia. Odds ratio of disaggregation rate (disAggR) for ischemic recurrence. For patients with lacunar infarction, atherothrombotic infarction, and total ischemia, the lack of increase in disAggR predisposes to the recurrence of ischemia.Figure 6. Odds ratio of disaggregation rate (disAggR) for ischemic recurrence. For patients with lacunar infarction, atherothrombotic infarction, and total ischemia, the lack of increase in disAggR predisposes to the recurrence of ischemia. Maximal aggregation (MaxAgg) and disaggregation rate in patients with major bleeding. Major bleeding was correlated only with MaxAgg induced by 4.0 μmol/L of adenosine diphosphate (ADP) stimulation (top). MaxAgg was significantly lower in patients with bleeding compared to patients with ischemia with recurrence and was also significantly lower than in those with ischemia without recurrence (bottom).Figure 7. Maximal aggregation (MaxAgg) and disaggregation rate in patients with major bleeding. Major bleeding was correlated only with MaxAgg induced by 4.0 μmol/L of adenosine diphosphate (ADP) stimulation (top). MaxAgg was significantly lower in patients with bleeding compared to patients with ischemia with recurrence and was also significantly lower than in those with ischemia without recurrence (bottom). Optimal example of the platelet aggregation test stimulated by adenosine diphosphate. Maximal aggregation is sequentially suppressed from left to right. Effectiveness for recurrence suppression is reduced (left), therapeutically effective (middle), and bleeding risk is increased (right).Figure 8. Optimal example of the platelet aggregation test stimulated by adenosine diphosphate. Maximal aggregation is sequentially suppressed from left to right. Effectiveness for recurrence suppression is reduced (left), therapeutically effective (middle), and bleeding risk is increased (right).

References

1. The SALT Collaborative Group, Swedish Aspirin Low-Dose Trial (SALT) of 75 MG aspirin as secondary prophylaxis after cerebrovascular ischemic events: Lancet, 1991; 338; 1345-49

2. van Gijn J, Algra A, Kappelle JDutch TIA Trial Study Group, A comparison of two doses of aspirin (30 mg vs. 283 mg a day) in patients after a transient ischemic attack or minor ischemic stroke: N Engl J Med, 1991; 325; 1261-66

3. Diener HC, Cunha L, Forbes CEuropean Stroke Prevention Study, 2. Dipyridamole and acetylsalicylic acid in the secondary prevention of stroke: Neurol Sci, 1996; 143; 1-13

4. Pongrácz E1, Measurement of platelet aggregation during antiplatelet therapy in ischemic stroke: Clin Hemorheol Microcirc, 2004; 30; 237-42

5. Sudo T, Ito H, Ozeki Y, Estimation of anti-platelet drugs on human platelet aggregation with a novel whole blood aggregometer by a screen filtration pressure method: Br J Pharmacol, 2001; 133; 1396-404

6. Cha JK, Jeon HW, Kang MJ, ADP-induced platelet aggregation in acute ischemic stroke patients on aspirin therapy: Eur J Neurol, 2008; 15; 1304-8

7. Wadowski PP, Eichelberger B, Kopp CW, Disaggregation following agonist-induced platelet activation in patients on dual antiplatelet therapy: J Cardiovasc Transl Res, 2017; 10; 359-57

8. Hilkens NA, Algra A, Diener HC, Balancing benefits and risks of long-term antiplatelet therapy in noncardioembolic transient ischemic attack or stroke: Stroke, 2021; 52; 3258-65

9. Gurbel PA, Gesheff M, Bliden KP, Chapter 7 Light transmission aggregometry: Antiplatelet therapy in cardiovascular disease, 2014; 51-57, Wiley Online Library

10. Brandt JT, Platelet response to agonists: Clinical laboratory medicine, 2002; 1011-13, Philadelphia, LWW

11. Rodgers GM, Chapter 46 Diagnostic approach to the bleeding disorders, Part 5 Disorders of hemostasis and coagulation: Wintrobe’s Clinical Hematology, 2023, Philadelphia, LWW

12. Lim ST, Platelet function testing in transient ischaemic attack and ischaemic stroke: A comprehensive systematic review of the literature: Platelets, 2015; 26; 402-12

13. Hilkens NA, Algra A, Greving JP, Prediction models for intracranial hemorrhage or major bleeding in patients on antiplatelet therapy: A systematic review and external validation study: J Thromb Haemost, 2016; 14; 167-74

14. Liao JK, Secondary prevention of stroke and transient ischemic attack: Is more platelet inhibition the answer?: Circulation, 2007; 115; 1615-21

15. Hvas A-M, Favaloro EJ, Platelet function analyzed by light transmission aggregometry: Hemostasis and thrombosis methods and protocols Totowa, 2017; 321-31, Humana Press

Figures

Figure 1. Platelet aggregation test with light transmittance. The aggregation of platelets is judged as the change in light transmittance in platelet-rich plasma (PRP) by stimulation with different concentrations (from 4.0 μmol/L to 20 μmol/L) of adenosine diphosphate (ADP) recorded for 5 minutes. The degree of aggregation increases with the concentration of ADP (left). The y-axis is defined as 0% of transmittance value of PRP and 100% of platelet-poor plasma (PPP). Typical examples of a normal figure (middle) and of hyperaggregation (right) are given.Figure 2. Change in maximal aggregation (MaxAgg) is summarized according to the presence of ischemic recurrence in the patients treated with aspirin. In the control group, MaxAgg remained unchanged (left) by both concentrations of adenosine diphosphate (ADP) stimulation, even between 2 different intervals. MaxAgg decreased significantly in patients without recurrence by stimulation of both concentrations (middle). On the contrary, MaxAgg did not decrease in patients with recurrence (right).Figure 3. Change in disaggregation rate (disAggR) in the patients treated with aspirin. In the control group, disAggR remained unchanged (left) by both concentrations of adenosine diphosphate (ADP) stimulation. DisAggR was not changed in the patients on aspirin except by 4.0 μmol/L of ADP stimulation in those without recurrence (top middle).Figure 4. Change in aggregation curve area (AgCvAREA) in patients treated with aspirin. In the control group, the AgCvAREA remained unchanged during the period of the study (left). AgCvAREA significantly decreased in patients without recurrence by both concentrations (middle). The AgCvAREA did not decrease in the patients with recurrence after the treatment (right). ADP – adenosine diphosphate.Figure 5. Odds ratio of maximal aggregation (MaxAgg) for ischemic recurrence. In patients with atherothrombotic infarction and total ischemia, lack of suppression of MaxAgg predisposes to the recurrence of ischemia.Figure 6. Odds ratio of disaggregation rate (disAggR) for ischemic recurrence. For patients with lacunar infarction, atherothrombotic infarction, and total ischemia, the lack of increase in disAggR predisposes to the recurrence of ischemia.Figure 7. Maximal aggregation (MaxAgg) and disaggregation rate in patients with major bleeding. Major bleeding was correlated only with MaxAgg induced by 4.0 μmol/L of adenosine diphosphate (ADP) stimulation (top). MaxAgg was significantly lower in patients with bleeding compared to patients with ischemia with recurrence and was also significantly lower than in those with ischemia without recurrence (bottom).Figure 8. Optimal example of the platelet aggregation test stimulated by adenosine diphosphate. Maximal aggregation is sequentially suppressed from left to right. Effectiveness for recurrence suppression is reduced (left), therapeutically effective (middle), and bleeding risk is increased (right).

In Press

Clinical Research  

Buccal Acupuncture Reduces the Dose of Sufentanil Needed in Laparoscopic Gynecological Surgery

Med Sci Monit In Press; DOI: 10.12659/MSM.947088  

0:00

Review article  

Global Guidelines and Trends in HPV Vaccination for Cervical Cancer Prevention

Med Sci Monit In Press; DOI: 10.12659/MSM.947173  

0:00

Clinical Research  

Serum Prolidase and Ischemia-Modified Albumin Levels in Neural Tube Defects: A Comparative Study of Myelome...

Med Sci Monit In Press; DOI: 10.12659/MSM.947873  

0:00

Clinical Research  

Impact of Depression, Fatigue, and Pain on Quality of Life in Slovak Multiple Sclerosis Patients

Med Sci Monit In Press; DOI: 10.12659/MSM.947630  

Most Viewed Current Articles

17 Jan 2024 : Review article   7,932,897

Vaccination Guidelines for Pregnant Women: Addressing COVID-19 and the Omicron Variant

DOI :10.12659/MSM.942799

Med Sci Monit 2024; 30:e942799

0:00

16 May 2023 : Clinical Research   702,919

Electrophysiological Testing for an Auditory Processing Disorder and Reading Performance in 54 School Stude...

DOI :10.12659/MSM.940387

Med Sci Monit 2023; 29:e940387

0:00

01 Mar 2024 : Editorial   29,882

Editorial: First Regulatory Approvals for CRISPR-Cas9 Therapeutic Gene Editing for Sickle Cell Disease and ...

DOI :10.12659/MSM.944204

Med Sci Monit 2024; 30:e944204

0:00

28 Jan 2024 : Review article   23,844

A Review of IgA Vasculitis (Henoch-Schönlein Purpura) Past, Present, and Future

DOI :10.12659/MSM.943912

Med Sci Monit 2024; 30:e943912

0:00

Your Privacy

We use cookies to ensure the functionality of our website, to personalize content and advertising, to provide social media features, and to analyze our traffic. If you allow us to do so, we also inform our social media, advertising and analysis partners about your use of our website, You can decise for yourself which categories you you want to deny or allow. Please note that based on your settings not all functionalities of the site are available. View our privacy policy.

Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750