Impact of Adenosine Diphosphate and Calcium Chelation on Platelet Aggregation Testing in Patients Receiving Clopidogrel Therapy

To the Editor:

Large clinical trials have established the role of clopidogrel in acute coronary syndrome and percutaneous coronary intervention (1–3). Light transmission aggregometry (LTA) is the conventional method for measuring ex vivo platelet aggregation in patients receiving clopidogrel. Most studies have used adenosine diphosphate (ADP) as the agonist to trigger platelet aggregation. However, there is currently no consensus regarding the optimal concentration of ADP to use, thus making comparisons between studies difficult. Furthermore, LTA is routinely performed in blood samples anticoagulated with citrate. Because extracellular Ca²⁺plays a role in platelet aggregation (4), the calcium chelation induced by citrate may have an effect on the measurement of the platelet inhibitory activity of clopidogrel. Therefore, the aims of this study are: 1) to determine the optimal concentration of ADP required to consistently detect maximum platelet aggregation, and 2) to evaluate whether the Ca²⁺chelation induced by citrate overestimates platelet inhibition in patients on clopidogrel therapy.

The study population consisted of 34 patients with coronary artery disease receiving aspirin and clopidogrel therapy. Therefore, no baseline blood samples were obtained before the patients received clopidogrel and aspirin. To allow for an individual’s full response to clopidogrel, we included only patients who had been receiving maintenance 75 mg therapy for >5 days or had received a 300 mg or 600 mg loading dose at least 4 h before samples were taken. Patients were excluded if they met any of the following criteria: platelet count <150,000 or >450,000, hemoglobin <8 g/dl, or use of glycoprotein IIb/IIIa inhibitors or warfarin in the past seven days. The Baylor College of Medicine Institutional Review Board approved the study.

Blood samples were obtained by polypropylene syringes and collected from the arterial sheaths. Blood from each patient was immediately transferred into a tube containing 3.8% trisodium citrate and another tube containing the non-CA²⁺chelating anticoagulant PPACK (D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone) and then inverted gently. Platelet aggregation was assessed via standard LTA. Platelet aggregation was assessed using the PACKS-4 (Helena Laboratories, Beaumont, Texas) four-channel light transmission aggregometer. Maximum extent of aggregation was calculated after the addition of different concentrations of ADP (1, 5, 10, and 20 μM). Platelet aggregation was determined as the percent change in light transmission using platelet-poor plasma as the reference. No platelet count adjustments were made.

Data are expressed as mean ± SD. Comparisons between citrate and PPACK at all four concentrations of ADP were analyzed by paired ttest. Comparisons among the four different concentrations of ADP were accomplished using one-way analysis of variance. A p value of <0.05 was considered statistically significant.

Patient characteristics are displayed in Table 1.Platelet aggregation in the citrate anticoagulated samples was 34.6 ± 18.9% at 20 μmol/l, 31.3 ± 15.4% at 10 μmol/l, 23.3 ± 13.9% at 5 μmol/l, and 5.6 ± 5.5% at 1 μmol/l ADP. In the PPACK anticoagulated tubes, platelet aggregation was 45.4 ± 21.1% at 20 μmol/l, 40.6 ± 19.2% at 10 μmol/l, 32.8 ± 15.2% at 5 μmol/l, and 12.3 ± 9.1% at 1 μmol/l ADP (Fig. 1).There was no significant difference in percent of platelet aggregation between 20 μmol/l and 10 μmol/l ADP in either the citrate (p = 0.4) or PPACK (p = 0.3) samples. Significantly greater platelet aggregation was seen in the PPACK samples as compared to citrate at all ADP concentrations (p = 0.03 at 20 μmol/l, p = 0.03 at 10 μmol/l, p < 0.01 at 5 μmol/l, p < 0.001 at 1 μmol/l). All comparisons of platelet aggregation with different concentrations of ADP in either the citrate group or the PPACK group (20 vs. 5 μmol/l, 20 vs. 1 μmol/l, 10 vs. 5 μmol/l, 10 vs. 1 μmol/l, and 5 vs. 1 μmol/l) were significantly different except for those between 10 μmol/l and 20 μmol/l in both groups.

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Figure 1

Percent platelet aggregation in patients on clopidogrel using different concentrations of adenosine diphosphate (ADP) and either citrate (open bars)or PPACK (solid bars)as anticoagulant. *p < 0.05.

Even though ADP is the agonist most widely used to initiate platelet aggregation for patients receiving clopidogrel, there is no current standard regarding the optimal concentration of ADP or the anticoagulant to use. Therefore, it is difficult to make valid comparisons of patients’ responses among different studies. This study demonstrates that there is no statistical difference between the aggregatory response at 10 μmol/l and 20 μmol/l ADP; therefore, it would be reasonable to use 10 μmol/l as the standard concentration of ADP for testing.

To our knowledge, this study is the first to compare the differential effects of citrate versus a non-Ca²⁺chelating anticoagulant (PPACK) on platelet aggregation in patients receiving dual antiplatelet therapy of clopidogrel and aspirin. The results demonstrate that citrate anticoagulated plasma undergoes markedly less platelet aggregation, which was significant at all concentrations of ADP tested. This is noteworthy because studies have suggested that there is a relationship between the degree of platelet inhibition and major adverse clinical events (5).

• American College of Cardiology Foundation