phosphorylation before and after the introduction of clopidogrel
5.1.1 Introduction
From 2003-2005, several research groups published data supporting a variation in response to clopidogrel (69,154,156,207). The majority of this data used optical platelet aggregation in response to ADP as the principle means of assessing the variability. Flow cytometric
measurement of fibrinogen activation in response to ADP was also recognised as a marker of response to clopidogrel (147). However, 2 novel commercial assays emerged during this period; VerifyNow P2Y12 and flow cytometric measurement of VASP-phosphorylation (193,208). As discussed in Chapter 2, these novel assays presented some advantages over more traditional methods. VerifyNow P2Y12 is a rapid, easy to use cartridge-based assay that has the potential to be used as a bedside test in the clinical environment by staff with minimal training. VASP-PRI is a more time consuming, laboratory-based assay which measures the concentration of an intraplatelet phosphoprotein that is specifically affected by stimulation of the platelet P2Y12 receptor, the target of clopidogrel. It also has the advantage of producing stable results for at least 24 hours after sampling. Thus, VerifyNow P2Y12 appealed on the basis of potential clinical utility and VASP-PRI on the basis of specificity and flexibility in the management of laboratory resources. I therefore sought to investigate the change in these 2 assay results following the introduction of clopidogrel in a pilot study of patient attending for coronary angiography and intervention.
It has been suggested that coronary intervention itself may stimulate platelet activation and there is data demonstrating that the assessed response to clopidogrel may vary over the days following PCI (67,147). However, most patients undergoing intervention either electively or following an ACS (excluding ST elevation myocardial infarction) are discharged the day following this procedure. In light of this, I also investigated how the assessed response to clopidogrel using these assays changes on the day following intervention. As a reference assay, I measured platelet fibrinogen binding following stimulation with ADP using flow cytometry.
Page | 140 5.1.2 Patient selection
Patients with suspected CAD attending for angiography with a view to PCI were recruited prior to the introduction of clopidogrel. Exclusion criteria previously described were applied.
All patients had been taking aspirin 75mg/day for at least 5 days and were naive to clopidogrel at the time of recruitment. The remaining selection criteria are those detailed in Section 3.1.
Following recruitment, patients had a baseline sample collected for the assessment of platelet activation in response to ADP according to the three assays; VerifyNow P2Y12, VASP-PRI and platelet fibrinogen binding. Patients were then introduced on to clopidogrel prior to their PCI procedure. A second sample was taken following the introduction of clopidogrel
immediately prior to coronary angiography. As a reflection of the available data outlined in Chapters 2 and 3, and in keeping with the local practice in our centre, a patient was deemed to have been fully introduced onto clopidogrel if they had received a loading dose of 600mg > 2 hours previously, a 300mg loading dose >24hrs previously or had received 75mg/day of clopidogrel for at least 5 days. In those patients that underwent PCI, a third sample was collected to reassess the response to clopidogrel by all three assays 12-24 hours following the procedure.
5.1.3 Sample collection and processing
All samples were collected from a large calibre vein by RG as previously described (Section 3.2). An initial 3.5ml 3.2% sodium citrate tube was collected and then discarded followed by 2 further 3.5ml 3.2% sodium citrate tubes and a 2ml 3.2% sodium citrate tube supplied by Accumetrics. The retained sample tubes were mixed by gently inverting 5 times.
Details of sample processing are given in Chapter 3. In summary, prior to sample acquisition, flow cytometry tubes were prepared for the assessment of fibrinogen binding to platelets. A 3.8ml citrated whole blood tube was utilised within 10 minutes of sampling for these assays.
Samples were run in duplicate to assess reproducibility. After 30 minutes, the 2ml citrated
Page | 141 tube was used to test clopidogrel response using the VerifyNow P2Y12 system. A single
citrated tube was set aside for the assessment of VASP-PRI within 24 hours.
5.1.4 Statistical Methods
This was an exploratory study to investigate the utility of these assays for the assessment of clopidogrel response. The distribution of assay results was summarized using the median, interquartile range and range for each assay. Assay results from the 3 sample time points were compared using paired t-tests. The 3 different assay results at each time point were also compared using Pearson correlation coefficients with an assumption of normality.
Page | 142 n=41
Demographic Age (yrs)
Mean±SD 64.7 ± 10.6
Male (%) 25 (61)
BMI (kg/m2)
Mean±SD 26.7 ± 6.0
Ethnicity
Caucasian (%) 41 (100)
Medical History
Hypertension (%) 24 (59)
Diabetes (%) 6 (15)
Family History (%) 16 (39)
Dyslipidaemia (%) 24 (59)
Previous TIA/CVA (%) 5 (12)
Previous MI (%) 12 (29)
Previous PCI (%) 7 (17)
Previous CABG (%) 4 (10)
Smoking Status
Current (%) 8 (20)
Medication
Statin (%) 31 (76)
Beta Blocker (%) 17 (66)
ACE Inhibitor (%) 12 (29)
ARB (%) 1 (2)
Calcium Channel Blocker (%) 16 (39)
Nitrates (%) 8 (20)
K+ Channel Activators (%) 11 (27) Proton Pump inhibitor (%) 7 (17) Table 11: The demographics of patients included in Study 5.1
Page | 143 5.1.5 Results
41 patients were recruited to the study. The demographics of these patients are shown in Table 11. A summary of results for the three assays used to assess platelet activation in response to ADP are provided in Table 12. In 2 patients, I was unable to obtain a sample prior to coronary angiography. Of the 39 patients sampled prior to coronary angiography, 28 went on to have coronary intervention and thus had a further sample taken 12-24hours following this procedure for the assessment of clopidogrel response. Of these patients, 2 received intravenous GPIIbIIIa inhibitor (1 abciximab, 1 tirofiban) during the procedure and hence their post-PCI results were excluded. Duplicate assays were undertaken for the measurement of fibrinogen platelet
binding. The correlation between these duplicate samples was excellent (r2 = 0.94) thus the average value was calculated.
Pre Clopidogrel (n=41)
Pre-PCI (n=39)
Post-PCI (n=26)
Med. IQR Range Med. IQR Range Med. IQR Range Fibrinogen
binding (% +ve) 94 6 82-99 78 18 51-99 76 26 30-97 VerifyNow P2Y12
%inhibition 10 12 0-15 40 44 1-96 52 43 1-95
VASP-PRI 84 10 63-99 62 31 9-93 61 26 13-88
Table 12: Variation in clopidogrel response before clopidogrel, before PCI and after PCI assessed using 3 different assays.
Page | 144 VASP-PRI VerifyNow P2Y12
(%inhibition) Fibrinogen
binding (%+ve)
Pearson Correlation 0.65 -0.76
p (2-tailed) <0.001 <0..001
VASP-PRI Pearson Correlation - -0.76
p (2-tailed) - <0.001
Table 13: Correlation coefficients between the assays used to measure clopidogrel response
Significant correlations were seen between all 3 assays at all three time points, with the strongest between VerifyNow P2Y12 and VASP-PRI (r = 0.768, Table 13). The weakest correlation was between VASP-PRI and fibrinogen binding (r = 0.65, Figure 37) It should be noted that the correlation coefficients between VerifyNow P2Y12 and the other assays are negative given that this assay presents results as %inhibition of ADP response.
Page | 145 Figure 37: Correlation between fibrinogen binding and VASP-PRI for all assays
(baseline, pre-PCI and post-PCI)
Figure 38: VASP-PRI results for patients from study 5.2
Base = before clopidogrel; Pre PCI = after clopidogrel, before coronary intervention;
Post PCI = after coronary intervention
There was clearly a significant change in all three assays following the introduction of clopidogrel. There was a trend towards greater platelet inhibition to ADP following PCI.
Comparing the mean values between these data sets using paired sample t-tests suggested, for this relatively small cohort of 26 patients, the differences did not reach statistical significance (Verify Now P2Y12, mean dif. +4.4%, p = 0.13; VASP-PRI, mean dif -3.8%, p = 0.10;
fibrinogen binding, mean dif. -3.0%, p = 0.181).
5.1.6 Discussion
Although a relatively small number of patients, this pilot study produced important results. In a population representative of the West of Scotland patient attending for coronary angiography for suspected coronary disease, taking regular aspirin therapy, I was able to demonstrate using
Page | 146 three different assays that there was a measurable reduction in platelet response to ADP
following the introduction of clopidogrel. I also showed that, in keeping with work from others, the extent of platelet inhibition in response to clopidogrel varied considerably. In addition, testing platelet response the day following coronary intervention suggested a trend towards increased platelet inhibition by clopidogrel at this time point. This is at odds with work from Gurbel et al (147).
The optimal time point to measure clopidogrel response remains uncertain. However, the introduction of GPIIbIIIa inhibitors at the time of PCI may have significant effects on the assay results, particularly VerifyNow P2Y12 and flow cytometric measurement of fibrinogen platelet binding, both of which are affected by GPIIbIIIa receptor inhibitors. There is some evidence that VASP-PRI is less affected by GPIIbIIIa inhibitors (209). In view of this, the most practical time point to measure clopidogrel response is in preloaded patients prior to coronary angiography/intervention.
It also remains uncertain which assay is superior for measuring clopidogrel response. The results of this study demonstrated that although all three methods confirm a wide variation in clopidogrel response, correlations between the assays were only moderate.
I found the measurement of fibrinogen binding to platelets to be particularly cumbersome.
The assay tubes required preparation immediately before blood sampling and patients could only be tested when there were the available staff and resources. In addition, this assay had already been reported elsewhere and in our cohort it showed the weakest correlation with the other clopidogrel response assays. The strongest correlation was between VerifyNow P2Y12 and VASP-PRI. VerifyNow P2Y12 offered a simple and rapid assessment of clopidogrel response requiring minimal training. Although time consuming, VASP-PRI appealed due to the direct relationship with stimulation of the P2Y12 receptor by ADP. In addition, the stability of this assay for 24-48 offered considerable logistical advantages. As a consequence, these assays became the focus of my further investigation of clopidogrel response.
Ultimately, the primary concern was whether these assay results have any bearing on clinical outcome. Again, a much larger dataset was required. Study 5.2 describes the assessment of response to clopidogrel in the cohort of patients recruited for Study 4.2.