Abstract
Background
Different methods are available for quantifying platelet function inhibition. Measuring vasodilator-stimulated phosphoprotein (VASP) phosphorylation is currently the most specific method for assessing the clopidogrel effect. The aim of our study was to compare different tests in view of a clinically applicable bedside test. Further, we examined whether doubling the clopidogrel dose to 150 mg/d in clopidogrel low-responder would lead to a reduction in platelet reactivity.
Methods and results
ADP-, ADP Hs-, and TRAP-induced platelet aggregation were measured by impedance aggregometry in 100 patients with CAD and 18 healthy controls. Moreover, platelet aggregation was assessed by flow cytometrical detection of VASP-phosphorylation and surface P-selectin in a subgroup of 34 patients and in healthy controls. Another 10 patients with CAD, identified as low-responder, were treated with a clopidogrel dose of 150 mg/d. Thereafter, ADP-induced platelet aggregation was assessed by impedance aggregometry. Significant correlations were observed between ADP-induced platelet aggregation assessed by VASP-phosphorylation and by impedance aggregometry. Doubling the dose of clopidogrel to 150 mg/d was associated with a reduction of ADP-induced platelet aggregation in only 60% of the patients.
Conclusions
Impedance aggregometry is a valuable bedside test to assess platelet function inhibition. Doubling the clopidogrel dose is not effective to reduce high on-treatment platelet reactivity in almost half of these patients, pointing to the need of a more powerful platelet inhibitor.
1
Introduction
Clopidogrel, a thienopyridine, belongs to the standard medication regimen in patients with coronary artery disease (CAD) . The P2Y12 receptor antagonist clopidogrel combined with aspirin (ASA) is used as a prophylaxis after stent implantation and reduces the risk of thrombotic vascular occlusion . A large inter-individual and intra-individual variability in reply to clopidogrel were described in various studies . The incidence of so-called low-responder or non-responder fluctuates from 5% to 25% depending on the diagnostic test. Furthermore, the definition of the so-called low-responder or non-responder also varies between different studies .
Patients with high on-treatment platelet reactivity have an increased risk for further cardiovascular events . Up to now, methods for measuring platelet aggregation are laboratory dependent and diversified dependent on the platelet function test used for measuring clopidogrel effectiveness .
A well-known assay for measuring platelet aggregation is the flow cytometrical detection of specific activation markers on platelet surface such as CD62-P upon activation with platelet agonists . P-selectin is stored in the α-granules of platelets. It is expressed on platelet activation by translocation from the α-granules to the platelet surface. Expression of P-selectin is a very sensitive marker for the activation state of platelets. Furthermore, there is an association between the platelet membrane activation markers and the early clinical outcome of elective PTCA .
A very specific method for measuring the effect of clopidogrel is the assessment of the state of vasodilator-stimulated phosphoprotein (VASP) phosphorylation . Clopidogrel reduces the adenosine diphosphate (ADP)-induced VASP-phosphorylation by blocking the P2Y12 receptor (ADP-receptor). The state of VASP-phosphorylation correlates with the inhibition of the P2Y12 receptor and thereby with the inhibition of platelet aggregation . Thus, the quantification of VASP-P in platelets is a good marker for the selective P2Y12-mediated inhibition of platelet function . Another method of measurement platelet aggregation is the impedance aggregometry. Michelson described the advantages and disadvantages of different methods for assessing platelet function such as impedance aggregometry and the measurements for platelet surface P-selectin expression and VASP-phosphorylation. Bouman et al. analyzed the effect of clopidogrel in patients without clopidogrel premedication after a clopidogrel loading dose of 600 mg. The results of the flow cytometric VASP assay, the VerifyNow P2Y12 assay, and the ADP-induced light transmittance aggregometry correlated with the plasma level of the active metabolite of clopidogrel. The authors concluded that the tests mentioned above were suited for the assessment of the clopidogrel effectiveness.
The aim of our study was to compare three different methods such as impedance aggregometry, flow cytometrical detection of VASP-phosphorylation and of platelet surface P-selectin exposure with regard to the evaluation of platelet function inhibition under combined antiplatelet therapy.
Furthermore, we examined whether doubling the clopidogrel dose to 150 mg/d in patients with a reduced responsiveness to the standard dose of clopidogrel (75 mg/d) would lead to a reduction in agonist-induced platelet aggregation as assessed by a suited platelet function test.
2
Methods
2.1
Study design
One hundred patients with coronary artery disease (clinical characteristics in Table 1 ) and 18 healthy persons (8 females/10 males) as a control group were enrolled into this study. Patients were classified as follows: 31 with an acute coronary syndrome (ACS) and 69 with a stable coronary artery disease. In all 100 patients, the ADP-, ADP plus prostaglandin (ADP Hs)-, and the thrombin receptor activator peptide (TRAP)-induced platelet aggregation was measured by impedance aggregometry. In a subgroup of 34 patients, the platelet function was additionally analyzed by measuring VASP-phosphorylation and the expression of platelet surface P-selectin. Both measurements were done by flow cytometry. In the control group, agonist-induced platelet reactivity was also examined by the two flow cytometry assays and by impedance aggregometry as mentioned above.
| All patients with coronary artery disease ( n = 100) | Patients with ACS ( n = 31) | Patients with CAD ( n = 69) | P value | |
|---|---|---|---|---|
| Age (years) | 67 (28–86) | 64 (28–86) | 68 (36–83) | n.s. |
| Gender (female/male) | 24/76 | 9/22 | 15/54 | n.s. |
| BMI (kg/m 2 ) | 26.8 (18.3–38.4) | 28.5 (21.7–38.4) | 26.8 (18.3–33.9) | n.s. |
| Cardiovascular risk factors | ||||
| Active smokers | 25 (25%) | 11 (35.48%) | 14 (20.29%) | n.s. |
| Dyslipoproteinemia | 70 (70%) | 21 (67.74%) | 49 (71.01%) | n.s. |
| Systemic hypertension | 71 (71%) | 21 (67.74%) | 50 (72.46%) | n.s. |
| Diabetes mellitus | 26 (26%) | 8 (25.81%) | 18 (26.09%) | n.s. |
| Additional medication | ||||
| Beta-blocker | 93 (93%) | 29 (93.55%) | 64 (92.75%) | n.s. |
| ACE-inhibitor | 81 (81%) | 25 (80.65%) | 56 (81.16%) | n.s. |
| AT1-inhibitor | 14 (14%) | 4 (12.90%) | 10 (14.49%) | n.s. |
| Statins | 97 (97%) | 31 (100%) | 66 (95.65%) | n.s. |
| Diuretics | 52 (52%) | 17 (54.84%) | 35 (50.72%) | n.s. |
All 100 patients received a loading dose of 600 mg clopidogrel during the cardiac catheter examination or directly before going to the catheter lab followed by a daily therapy with clopidogrel (75 mg/d) and aspirin (100 mg/d).
Patients with a current clopidogrel therapy of 75 mg/d were treated with a loading dose of clopidogrel 300 mg before or during cardiac catheter examination. The measurements were done within 24 h after the administration of the loading dose. In the control group, no medications were given.
Another 10 CAD patients were enrolled in this study because they suffered from thrombotic events or recurrent angina despite clopidogrel therapy (75 mg/d).
All these 10 patients exhibited high on-treated platelet reactivity. In these patients, the clopidogrel dose was increased to 150 mg/d to reduce the platelet hyperreactivity.
High on-treatment platelet reactivity was defined as a post-treatment platelet aggregation above the 75th percentile in ADP-induced platelet aggregation measured by impedance aggregometry.
Sibbing et al. defined a cut-off point at the upper quintile (416 AU⁎min) as clopidogrel low responsiveness. Matetzky et al. divided patients into four quartiles according to the percentage reduction of ADP-induced platelet aggregation at day 6 compared to the baseline activity of each patient. Patients with a high on-treatment platelet aggregation above the 4th quartile were classified as clopidogrel low-responder. Therefore, we choose the 75th percentile as a threshold value for clopidogrel low responsiveness.
All patients with a high on-treatment platelet reactivity received 150 mg/d clopidogrel not less than 30 days. ADP- and ADP Hs-induced platelet aggregation was assessed by impedance aggregometry between 4 and 15 days.
Two patients obtained 150 mg/d clopidogrel for 15 days before testing platelet function, three patients for 8 days, one patient for 7 days, two patients for 6 days, and two patients for 4 days only.
Measurements of platelet function were performed early in the latter two patients, because these patients were not available at a later stage due to the need of rehabilitation procedures.
The institutional review board of the hospital approved the study and written informed consent was obtained from each subject. All procedures were performed in accordance with the Declaration of Helsinki.
2.2
Platelet surface P-selectin measured by flow cytometry
Venous blood was drawn from each patient to flow cytometrically assess agonist-induced P-selectin exposure on the platelet surface. After isolation of platelets from blood, the platelets were stimulated with 20 μM ADP or 10 μM TRAP (CALBIOCHEM®/SIGMA®) or were left unstimulated for 5 min at ambient temperature. After fixation with 2% paraformaldehyde, the platelets were incubated with IgG-FITC conjugated anti-mouse antibody and were analyzed by flow cytometry. Evaluation was done with Cell Quest® software. Data are given as the median immunofluorescence intensity (MFI) after subtraction of the unspecific mouse IgG binding. The median of the curve is given as activity units (AU).
2.3
State of VASP-phosphorylation measured by flow cytometry
Citrated blood was collected for the assessment of VASP-phosphorylation. The platelet VASP/P2Y12-Kit, manufactured by Biocytex, France, distributed by American diagnostic®, was used according to manufactures instructions.
VASP is an intracellular platelet protein, which is activated through phosphorylation . Prostaglandin E 1 (PGE 1 ) binds on platelet surface and leads to conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) and then via protein kinase A to the phosphorylation of VASP . ADP binds to its receptor P2Y12 and thereby inhibits by binding the prostaglandin E 1 -induced signaling.
The platelet reactivity index (PRI) was calculated via revised edition of mean fluorescence intensities (MFIc): PRI = [(MFIc PGE1 − MFIc (PGE1 + ADP) )/MFIc PGE1 ] · 100.
2.4
Impedance aggregometry
The Multiplate® instrument (Dynabyte medical) is a platelet analyzer with five channels for parallel measurement platelet function in whole blood. Each measuring cell has two sensor wires for internal quality inspection. The principle of measuring platelet function is the impedance aggregometry. Activated platelets adhere and aggregate on sensor wires and thus change the electric resistance, leading to differences in the impedance .
For this assay, hirudin–blood was incubated with NaCl in a measurement chamber. Each sample was tested with the three agonists: TRAP, ADP, or ADP HS. After adding these agonists to the diluted blood, the measurement was started immediately. Values are given as the area under the aggregation curve (AUC [AU⁎min]) (10 AU⁎min = 1 U).
2.5
Statistical analysis
Statistical analysis was performed with SPSS (SPSS 12.0 for Windows) and GraphPad Prism 4. With the Kolmogorov–Smirnov test, data were tested for normal distribution. Data are given as median, the 25th and 75th percentile. Not normally distributed data were compared with the Mann–Whitney test. For the assessment of correlations, the Pearson correlation for parametric data and the Spearman’s rank correlation coefficient for non-parametric data were used. A P value of < .05 (two-sided test) was considered as statistically significant.
2
Methods
2.1
Study design
One hundred patients with coronary artery disease (clinical characteristics in Table 1 ) and 18 healthy persons (8 females/10 males) as a control group were enrolled into this study. Patients were classified as follows: 31 with an acute coronary syndrome (ACS) and 69 with a stable coronary artery disease. In all 100 patients, the ADP-, ADP plus prostaglandin (ADP Hs)-, and the thrombin receptor activator peptide (TRAP)-induced platelet aggregation was measured by impedance aggregometry. In a subgroup of 34 patients, the platelet function was additionally analyzed by measuring VASP-phosphorylation and the expression of platelet surface P-selectin. Both measurements were done by flow cytometry. In the control group, agonist-induced platelet reactivity was also examined by the two flow cytometry assays and by impedance aggregometry as mentioned above.
| All patients with coronary artery disease ( n = 100) | Patients with ACS ( n = 31) | Patients with CAD ( n = 69) | P value | |
|---|---|---|---|---|
| Age (years) | 67 (28–86) | 64 (28–86) | 68 (36–83) | n.s. |
| Gender (female/male) | 24/76 | 9/22 | 15/54 | n.s. |
| BMI (kg/m 2 ) | 26.8 (18.3–38.4) | 28.5 (21.7–38.4) | 26.8 (18.3–33.9) | n.s. |
| Cardiovascular risk factors | ||||
| Active smokers | 25 (25%) | 11 (35.48%) | 14 (20.29%) | n.s. |
| Dyslipoproteinemia | 70 (70%) | 21 (67.74%) | 49 (71.01%) | n.s. |
| Systemic hypertension | 71 (71%) | 21 (67.74%) | 50 (72.46%) | n.s. |
| Diabetes mellitus | 26 (26%) | 8 (25.81%) | 18 (26.09%) | n.s. |
| Additional medication | ||||
| Beta-blocker | 93 (93%) | 29 (93.55%) | 64 (92.75%) | n.s. |
| ACE-inhibitor | 81 (81%) | 25 (80.65%) | 56 (81.16%) | n.s. |
| AT1-inhibitor | 14 (14%) | 4 (12.90%) | 10 (14.49%) | n.s. |
| Statins | 97 (97%) | 31 (100%) | 66 (95.65%) | n.s. |
| Diuretics | 52 (52%) | 17 (54.84%) | 35 (50.72%) | n.s. |
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