EDITORIAL COMMENT
Will Measuring Vasodilator-Stimulated Phosphoprotein Phosphorylation Help Us Optimize the Loading Dose of Clopidogrel?*
Neal S. Kleiman, MD, FACC*
Methodist DeBakey Heart Center, Houston, Texas; and Weill Cornell Medical College, New York, New York.
* Reprint requests and correspondence: Dr. Neal Kleiman, 6565 Fannin, MS F-1035, Houston, Texas 77030. (Email: nkleiman{at}tmhs.org).
Thienopyridines have robust effects on the ischemic manifestations of coronary artery disease. Randomized clinical trials have shown that treatment with thienopyridines dramatically lowers the rates of intracoronary stent thrombosis (1); reduces the rate of cardiovascular death, stroke, or reinfarction in patients with non–ST-segment elevation acute coronary syndromes (2); and reduces the likelihood of death after treatment for ST-segment elevation myocardial infarction (3). Yet, for the past 5 years we have known that the biological response to clopidogrel, the best-tolerated and most widely used thienopyridine currently available, is heterogeneous (4). Patients in whom platelet reactivity remains persistently elevated after treatment with clopidogrel may be at elevated risk for serious ischemic events, such as thrombosis of an intracoronary stent or myocardial infarction.
Although the heterogeneity of the response to clopidogrel is fairly well characterized, it is poorly understood. A variety of explanations have invoked genetic variation of its target receptor, variability in the hepatic conversion of clopidogrel to its active metabolite, and drug–drug interactions. It is also unclear to what extent increasing the dose of clopidogrel used as an initial load can increase the biological effect of the drug on platelets. Use of an initial loading dose of clopidogrel, as compared with a maintenance dose only, shortens the time required to achieve maximum inhibition of platelet aggregation from about 24 to 6 h; increasing the loading dose further to 600 mg shortens this period to about 2 h. Whether global implementation of a 900-mg loading dose shortens this time further and leads to a more profound antithrombotic effect is controversial.
In this issue of the Journal, Bonello et al. (5) studied a novel variant of personalized medicine to determine whether a more profound antiplatelet effect could be achieved by further augmenting the loading dose in selected patients (i.e., those in whom the antiplatelet effect of clopidogrel is minimal). Understanding this approach requires understanding the action of clopidogrel on the platelet. Clopidogrel is converted (as are the other thienopyridines) by members of the hepatic cytochrome P450 family from a prodrug to an active metabolite that has only recently been identified (4,6). The prodrug antagonizes the action of adenosine diphosphate (ADP) (released by other activated platelets and by damaged cells) on P2Y12, 1 of the 3 purinergic receptors on the human platelet. Platelet aggregation and activation responses to ADP are initiated by another receptor, P2Y1, but cannot be completed without activation of P2Y12. The ADP-induced activation of P2Y12 causes, through a Gi protein-based intracellular signaling pathway, a decrease in adenyl cyclase resulting in lower levels of intracellular cyclic adenosine monophosphate (cAMP). Cyclic AMP is a necessary co-factor for phosphorylation of a protein known as vasodilator-stimulated phosphoprotein (VASP) (7). Vasodilator-stimulated phosphoprotein is important in the platelet for regulation of the actin cytoskeleton and for conversion of glycoprotein IIb/IIIa to its active conformation, thus permitting platelets to aggregate; VASP exists in both phosphorylated and dephosphorylated states. The phosphorylated form is inactive. Inhibition of cAMP activity after activation of P2Y12 by ADP leads to an increase in VASP dephosphorylation, whereas blockade of P2Y12 by the active metabolite of a thienopyridine leads to an increase in phosphorylated VASP. Thus, the ratio of phosphorylated to dephosphorylated VASP reports the degree of P2Y12 blockade (i.e., activity of the active thienopyridine metabolite that has bound P2Y12) (8). Whereas measurement of VASP phosphorylation originally required Western blotting techniques, flow cytometry now allows this measurement to be performed with considerably greater ease and precision (9,10). This technique has the additional advantage that samples remain stable for about 24 h after they are collected. Artifacts of platelet activation after sample collection (commonly seen in platelet aggregation studies) are thus avoided. Most investigators who study this field have adopted a measure known as the platelet reactivity index (PRI), which compares maximally stimulated VASP phosphorylation with the amount of phosphorylation present after ADP stimulation of the platelet. When P2Y12 is blocked, the index decreases (Fig. 1). In volunteer subjects given clopidogrel, close correlation between light transmittance aggregometry and PRI has been reported (r = 0.66 to 0.9, depending on the ADP concentration used) (11), whereas in patients undergoing a percutaneous coronary intervention (PCI), coarser correlation has been reported between the PRI and light transmittance aggregometry (p = 0.47) and aggregation measured using a point of care assay, the VerifyNow device (Accumetrics, San Diego, California) (p = 0.52) (12). The index shows very little variance among healthy volunteers and decreases linearly when varying concentrations of a competitive P2Y12 antagonist are added to ex vivo blood samples (10).
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Figure 1 Pathways Activated Following Stimulation of P2Y12 by ADP
The pathway resulting in phosphorylation and dephosphorylation of VASP is surrounded by the broken line. ADP = adenosine diphosphate; Akt = serine threonine kinase; cAMP = cyclic adenosine monophosphate; cGMP = cyclic guanosine monophosphate; GP = glycoprotein; NO = nitric oxide; PIP3 = phosphatidylinositol (3,4,5) triphosphate; VASP = vasodilator-stimulated phosphoprotein. Figure illustration by Rob Flewell.
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Frere et al. (13) reported that PRI predicted ischemic outcomes in patients with acute coronary syndromes, whereas Bonello et al. (14) previously showed that a PRI <50 was associated with a reduced rate of ischemic events after PCI in patients who had received 300 mg of clopidogrel. In the current study (5), they calculated the PRI in patients loaded with a 600-mg dose of clopidogrel 24 h before intracoronary stent implantation. Patients with a high (>50) PRI were randomized to receive either serial doses of clopidogrel until the PRI decreased to <50, or to undergo PCI without additional clopidogrel doses. Consistent with previous reports using the same assay, about one-half of the patients studied had an elevated PRI after a single 600-mg dose of clopidogrel. Serial dosing of clopidogrel resulted in a reduction of the PRI to <50 in 86% of patients randomized to guided therapy. One month after PCI, major adverse cardiac events (composite cardiovascular death, stent thrombosis, recurrent acute coronary syndromes, or revascularization) occurred in 10% of patients in the control group and no patients receiving PRI-guided therapy.
These findings are supported by those of a recent randomized trial of prasugrel, a third-generation thienopyridine with effects on platelet aggregation that are more homogeneous than those of clopidogrel (15). Prasugrel was associated with a 19% reduction in a composite of death or myocardial infarction compared with clopidogrel in a population with acute coronary syndromes (16). They are also consistent with a growing body of observational studies suggesting that patients with greater residual platelet aggregation after clopidogrel dosing are at higher risk for ischemic events compared with patients in whom platelet aggregation is reduced.
Does this mean that the PRI is likely to become established as a biomarker of thienopyridine activity and should be used to titrate the dosing of clopidogrel and other P2Y12 antagonists? It is useful to reflect on what is meant by a biomarker. In 2005, Fleming (17) pointed out 3 characteristics requisite for the evaluation of a biomarker. First, correlation of the potential marker with a clinically accepted end point is a necessary but not sufficient condition. Second, the proposed biomarker should fully capture the effect of a particular treatment on the end point. Implicit in this requirement is that the biomarker be a necessary part of a biological step in the mechanism of a treatment effect. Third, reproducibility of the effect measured by the biomarker should be present in a meta-analysis of clinical trials of different therapies within a class of treatments. A desirable correlate of this characteristic is evaluation within a clinical trial that shows the effect of an intervention administered based on a measurement of the biomarker.
How does the PRI fare with regard to these 3 requisites? First, correlation of the PRI with clinical end points has thus far been performed in a relatively small number of patients undergoing PCI, although given the current level of interest in this marker, it is likely that more correlations will be performed before very long. Similarly, as new antagonists of P2Y12 undergo large clinical trials, it is likely that we will see multiple analyses of the PRI within this drug class, thus fulfilling the third requisite. Fulfilling the second requisite, capturing the full effect of P2Y12 antagonist therapy, is less certain. Clearly, VASP phosphorylation is more specific to the P2Y12-initiated pathway of platelet activation than are either measurement of platelet aggregation or of P-selectin expression. However, activation of P2Y12 by ADP initiates other signaling pathways involving molecules such as phosphoinositide-3-kinase (18) and Rap1b (19) that activate and stabilize glycoprotein IIb/IIIa independent of VASP phosphorylation. Furthermore, the antibody 16C2 used for VASP measurement reports phosphorylation at a serine residue that is also phosphorylated by cyclic guanosine monophosphate-dependent kinases that are not regulated by P2Y12 (20).
The use of a flow cytometric assay also introduces logistic and economic concerns. Although well regarded as a tool for differentiating cell types, flow cytometry is expensive, requires the availability of experienced personnel, and can hardly be considered a bedside tool. As any researcher who has fought for time on a flow cytometer can attest, results do not become available quickly. The median time between administration of the first clopidogrel dose and performing the first VASP assay was 24 h in the Bonello et al. (5) study, and 33% of patients required 3 assays and subsequent clopidogrel doses to achieve a PRI <50. These considerations are difficult to disregard, particularly in the U.S., where only a small minority of patients receive a clopidogrel loading dose 2 h or more before placement of a stent. Such an approach must also be viewed in light of competing technologies, some of which, like bedside platelet aggregation technologies, lend themselves more easily to point-of-care application. Currently, a large randomized trial is poised to test whether a bedside aggregation device can be used to achieve similar results in the chronic phase of clopidogrel therapy.
Although widespread measurement of the PRI before PCI may be impractical, the current study provides an early view into the potential utility of directing therapy based on a pathway-specific assay of the biological activity of an antiplatelet drug, and suggests that once a target level of inhibition is appropriately validated, an individualized approach to dosing may ultimately be preferable to a shotgun approach to dose selection.
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Footnotes
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Dr. Kleiman has received research grants from Sanofi-Aventis Inc., Eli Lilly Inc., and Schering-Plough Inc.; received gifts of laboratory supplies from Accumetrics; and is an advisor to Thrombovision.
* Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology. 
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References
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