Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study

Abstract

Background: Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment.

Methods: With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B2 (TXB2)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis.

Results: The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5'-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 = 0.49). In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow cytometry were lower for clopidogrel compared to ASA. Furthermore, adhesion to collagen was lower for ASA and clopidogrel combined compared with either drug alone.

Conclusion: The indirect pharmacodynamic measures of the effects of ASA and clopidogrel might be used together with ADP-induced activation and serum TXB2 for evaluation of anti-platelet treatment. This should be further evaluated in future clinical studies where screening opportunities with the adhesion assay will be optimised towards increased sensitivity to anti-platelet treatment.

Figure 1

Flow chart showing the inclusion of patients and controls. Patients and controls were included consecutively. Blood samples from controls were drawn at two different occasions separated by 2–5.5 months. All patients entering the study received ASA combined with clopidogrel and blood sampling was performed 1.5–6.5 months after initiating the treatment. This was followed by a randomised cross-over enabling all patients to receive monotherapy with both ASA and clopidogrel. The patients received monotherapy for at least 3 weeks and for a maximum of 4.5 months before performing blood sampling. A total of 33 patients and 30 controls entered the study. In the end, 29 patients and 29 controls completed the study.

Figure 2

Effect of platelet inhibiting treatment on serum TXB₂-levels (Factor 13). Serum TXB₂-levels (Factor 13) for patients (n = 29) and healthy controls (n = 29) are presented as mean + SEM. ASA alone or in combination with clopidogrel was significantly different from clopidogrel alone and compared to the mean of the controls (p < 0.001). Also, the difference between controls at visit 1 and visit 2 was significant. ***p < 0.001, ns = not significant.

Figure 3

The influence of ASA and clopidogrel on platelet adhesion. The main figures are representative examples of the variables constituting the respective factors. The insets show the Z-scores for each factor. Also shown in the insets are the comparisons between the control means of visit 1 and 2 and treatment with ASA (A), clopidogrel (C) and the combination of ASA and clopidogrel (A+C). The respective figures show the effect of platelet inhibiting treatment on ADP-induced adhesion (Factor 1, Fig A), ristocetin-induced adhesion to albumin (Factor 6, Fig B), LPA-induced adhesion to albumin (Factor 7, Fig C) and adhesion to collagen (Factor 8, Fig D) for patients (n = 29) and healthy controls (n = 29). All values are presented as mean + SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns = not significant.

Figure 4

The influence of ASA and clopidogrel on platelet activity measured by flow cytometry. The effects of platelet inhibiting treatment on platelet activation detected by flow cytometry induced by ADP (Factor 14, Fig A) and SFLLRN (Factor 15, Fig B) on patients (n = 29). The main figures are representative examples of the variables constituting the respective factors. The insets show the Z-scores for each factor. All values are presented as mean + SEM. ***p < 0.001, ns = not significant.

Figure 5

Correlation between static platelet adhesion and flow cytometry. Correlation between ADP-induced platelet adhesion (Factor 1) and ADP-induced platelet activation as measured by flow cytometry (Factor 14) for patients (n = 29) (r² = 0.49). Data included are from all three separate anti-platelet treatments (ASA and clopidogrel alone as well as ASA and clopidogrel combined).