Author + information
- Steven R. Steinhubl, MD⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Steven R. Steinhubl, Cardiovascular Wellness, Geisinger Health System, 100 North Academy Avenue, MC 27-70, Danville, Pennsylvania 17822
Real knowledge is to know the extent of one's ignorance.
Having the ability to measure whether and how much a drug is able to influence platelet function ex vivo has led to some remarkable contributions to cardiovascular medicine over the last several decades. For example, the nearly 40-year journey for aspirin to go from a hypothesized to a proven therapy for ischemic heart disease may have never occurred if not for a series of mechanistic investigations of its effects on platelet function (2). The entire thienopyridine class of antiplatelet agents may have been forever relegated to the trash heap of failed pharmaceutical entities if not for the fortuitous finding that it prevented adenosine diphosphate (ADP)-induced platelet aggregation after it initially failed for its proposed use as an anti-inflammatory agent (3). And the parenteral platelet glycoprotein (GP) IIb/IIIa inhibitors are a drug class that was specifically designed around their ability to potently inhibit platelet aggregation. But even with these notable successes, historically, platelet function testing has proven to be a somewhat unreliable and confusing “crystal ball”—inconsistent in its ability to predict what, if any, antithrombotic efficacy an agent may have—and still of no proven clinical use in guiding individual therapy. Nonetheless, because our ability to adequately tame the platelet is so critical for the successful treatment and prevention of arterial thrombosis, and because, as of yet, we have no viable testing alternative, ex vivo platelet function assessments have and will continue to play a substantial role in clinical research and drug development.
To the busy clinician, the constant barrage of academic research and industry marketing focusing solely on platelet function testing results and the achievement of “optimal” platelet inhibition might imply a level of understanding greater than what truly exists. Further improving our real knowledge of assessing platelet function and how to interpret its results requires a deeper appreciation and understanding of the 3 basic issues summarized in the following text.
There are hundreds of variations and methods to test platelet function; all generate different results
The origins of platelet function testing date back over 100 years to the earliest description of bleeding times, and the first ex vivo platelet function test was described in 1929 (2). Since then, every manner of platelet disintegration, adhesion, stickiness, aggregation, agglutination, and activation has been quantitated in some form as a measure of platelet activity. The test sample for these various tests is whole blood or platelet-rich plasma and has in some cases been stored overnight, spun in glass jars, poured over glass beads, mixed with fibrinogen-coated beads, sucked through capillary tubes or pin holes in collagen discs, stirred, or otherwise “assaulted” in a number of ways. Most of the time, the blood or plasma is mixed with some kind of an antithrombotic agent at varying amounts; often it is citrate, but sometimes heparin, hirudin, or a number of other compounds. Next, a platelet agonist is usually added such as ADP, collagen, thromboxane, thrombin, and many others, with the concentration chosen somewhat arbitrarily and with up to a 100-fold variation among laboratories. What is critical to appreciate is that every one of these variations, both big and small, can have a profound impact on measured platelet function. As a simple example, GP IIb/IIIa inhibitors can be said to inhibit platelet aggregation anywhere from only 30% to nearly 100%, depending on the agonist and its concentration (4).
Even if we focus on just 3 somewhat similar and common methods of platelet function testing, surprising differences are found. Light transmittance aggregometry (LTA), the VerifyNow P2Y12 assay (Accumetrics, San Diego, California), and the Multiplate device (Roche, Basel, Switzerland) are all based on the same general premise of adding an agonist to a sample obtained through venipuncture and measuring changes to the sample over time due to platelet “aggregation.” The blood samples are drawn into citrated tubes for the VerifyNow P2Y12 assay and commonly for LTA, but hirudin is recommended for the Multiplate. For LTA, the blood is next centrifuged to yield platelet-rich plasma, whereas whole blood is used in both the VerifyNow P2Y12 assay and Multiplate, but for the latter, the blood is diluted 2:1 with saline. The platelet agonist, for example, ADP, is next added. For the Multiplate and VerifyNow P2Y12 assay, the concentration is set, but in the VerifyNow P2Y12 assay, prostaglandin E1 is added to the ADP. With LTA, the concentration is operator dependent, with concentrations commonly varying from 0.1 to 20 μmol/l. Following the mixing of agonist and sample, an aggregation curve is then generated based on changes over time in measured light passing through the sample for LTA and VerifyNow P2Y12 assay, or changes in electrical impedance for Multiplate. The most striking difference among the 3 testing platforms is that each measures something completely different on these generated curves, and then reports that measure as platelet function. In the Multiplate, the area under the curve is reported. For VerifyNow P2Y12 assay, it is the slope of the initial portion of the curve. And for LTA, it is operator dependent, but it is most typically the maximal height of the curve, although the final height, and more rarely the slope, have also been reported.
What all of this variability tells us is that our search for “optimal” platelet inhibition will need to come with a very detailed roadmap that is specific for the type of test, the appropriate sample preparation, and the “correct” agonist(s) and concentration(s).
The ability to inhibit platelet function ex vivo does not guarantee clinical antithrombotic efficacy
One of the landmark studies of modern cardiovascular medicine was the Canadian multicenter trial, proving that aspirin was significantly more effective than placebo in decreasing cardiac events, including cardiac death, in patients with unstable angina (5). What is often forgotten about this trial is that it included randomization to another antiplatelet agent, sulfinpyrazone. Although it is likely you have never prescribed sulfinpyrazone, at that time, it was considered by some to be “… one of the most effective platelet inhibitors currently available …” based on ex vivo platelet function testing (6). Clinically, however, it provided no antithrombotic benefit, and in the final analysis of the study, sulfinpyrazone-treated patients were grouped with the placebo arm.
Within the last decade, the best examples of a platelet inhibition–antithrombotic efficacy disconnect is exemplified by the string of failed oral GP IIb/IIIa antagonists. All 4 agents evaluated in large phase III trials significantly and substantially inhibited platelet aggregation greater than their controls over a 24-h period. Although it was not surprising that all of these agents increased bleeding, what was unanticipated and still not well understood was that their use did not even lead to a trend in improved antithrombotic efficacy, and instead caused a significant increase in mortality and increased myocardial infarction (MI) rates among acute coronary syndrome patients (7).
Most recently, 2 promising antiplatelet drugs, vorapaxar and cangrelor, failed to show significant clinical antithrombotic efficacy in large-scale phase III trials, despite achieving greater levels of platelet inhibition based on separate ex vivo testing (8–10).
The message from these agents seems clear: the ability to inhibit platelet aggregation ex vivo is an unreliable surrogate for clinical antithrombotic efficacy, especially between drug classes and when considering the impact on the population. Therefore, any attempt to identify an “optimal” level of platelet inhibition will need to be drug specific and is unlikely to be additive between classes (unlike targeting a specific blood pressure and achieving that level with several agents).
Measured platelet function may have more to do with the plasma than the platelets
No matter how platelet function is tested in the clinical setting, a lot more than just platelets are being tested. Whether whole blood or platelet-rich plasma is used, the test sample contains literally thousands of different molecular entities, many of which are known to influence platelet function, including clotting factors, inflammatory cytokines, and microparticles. The potential impact of these constituents is best demonstrated through a simple but very informative study carried out by Gawaz et al. (11) in the mid-1990s. Using washed platelets from healthy volunteers, plasma was added from either acute MI patients or from control subjects with stable angina. Despite the platelets being identical, platelet aggregation was markedly greater when combined with the plasma from MI patients versus that of controls. Similar results were found for measures of platelet activation and platelet–endothelial cell adhesion.
These findings form a basis for potentially explaining why high platelet reactivity has been consistently associated with worse clinical outcomes, yet some prospective trials altering antiplatelet function based on ex vivo–measured platelet reactivity have not translated into improved clinical outcomes. A better appreciation for the fact that platelets, and therefore platelet antagonists, play only a partial role in defining measured platelet reactivity suggests that identifying “optimal” platelet inhibition may not be achievable until all of the nonplatelet factors influencing measured platelet function are fully understood.
So in light of the above caveats, how are we to interpret the 2 well-done and interesting platelet function–based studies in this issue of JACC: Cardiovascular Interventions? In the study by Ahn et al. (12), on-treatment platelet reactivity was determined in over 1,200 post-PCI patients using the VerifyNow P2Y12 assay, and the association with locally defined high platelet reactivity and 1-year outcomes were described. Similar to prior reports, they found high platelet reactivity to be associated with worse outcomes, although this was statistically significant only in the acute MI population. Based on the information described earlier, that plasma from acute MI patients leads to significant differences in platelet function relative to patients with stable coronary disease (11), it seems logical that the identification of the most appropriate cut-point for high platelet reactivity should be based on the patient's clinical acuity. Doing so would likely improve its prognostic value, although what to do with that information still needs to be defined.
Valgimigli et al. (13) addressed an interesting question with a very well-done and detailed study in which various antiplatelet regimens were tested, with the endpoint based on 20 μmol/l ADP-induced final aggregation. The premise of the study was that “suboptimal” platelet inhibition with a thienopyridine can be identified and then treated with a GP IIb/IIIa antagonist until “optimal” platelet inhibition is achieved by the thienopyridine. As logical as that seems, it is difficult to reconcile that hypothesis with the clinical results of trials such as BRAVE 3 (Bavarian Reperfusion Alternatives Evaluation-3) and EARLY ACS (Early Glycoprotein IIb/IIIa Inhibition in Non–ST-Segment Elevation Acute Coronary Syndrome), in which the vast majority of control-arm patients would have to be assumed to have “suboptimal” platelet inhibition relative to the GP IIb/IIIa treatment arm, yet that difference in platelet inhibition did not lead to differences in clinical outcomes (14,15). How or whether mixing and matching of P2Y12 antagonists with GP IIb/IIIa inhibitors will reduce ischemic event rates requires outcomes data and should not be based on platelet function results.
Antiplatelet therapies are some of our most potent interventions in the treatment and prevention of the complications of atherosclerotic disease. Yet, they remain the only routinely utilized drug class in which patients are treated with a 1-size-fits-all approach, and that flies in the face of everything known about genetic and environmental influences and their impact on interindividual variability. But the desire to individualize therapy needs to be tempered by the knowledge that there is a long way to go and much still to learn in our search for the illusive “optimal” platelet inhibition.
Dr. Steinhubl has reported that he has no relationships relevant to the contents of this paper to disclose.
↵⁎ Editorials published in JACC: Cardiovascular Interventions reflect the views of the authors and do not necessarily represent the views of JACC: Cardiovascular Interventions or the American College of Cardiology.
- American College of Cardiology Foundation
- ↵(1938) The Analects of Confucius Translated by Arthur Waley (The Macmillan Company, New York).
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- There are hundreds of variations and methods to test platelet function; all generate different results
- The ability to inhibit platelet function ex vivo does not guarantee clinical antithrombotic efficacy
- Measured platelet function may have more to do with the plasma than the platelets
- Current studies