THE CONCEPT OF DRUG RESISTENCE AS EXEMPLIFIED BY RESISTANCE TO CHEMOTHERAPY
Resistance to chemotherapeutic agents, both antimicrobial and antineoplastic, is a well-known phenomenon in clinical practice. "Drug resistance is the result of microbes changing in ways that reduce or eliminate the effectiveness of drugs, chemicals, or other agents to cure or prevent infections" [1]. This is how the Centers for Disease Control and Prevention defines drug resistance in the context of antibiotic resistance. The same applies to drug resistance in relation to viruses or cancer cells. Therefore, the development of resistance to a given drug implies a change in the pharmacological target. In fact, microbes, viruses, and cancer cells can undergo genetic changes in one or more substrates (enzymes, transmembrane proteins, pumps) that represent drug targets and, as a consequence, drugs targeted at them become less active or inactive.
Three major mechanisms underlie drug resistance:
1. the drug is no longer capable of reaching its therapeutic target, as exemplified by mechanisms impairing the entrance of the drug into the cell or microorganism or by membrane pumps which actively transport the drug outside the eukaryotic or prokaryotic cell (e.g., efflux pumps which actively transport tetracycline and β-lactam antibiotics out of the cell);
2. the drug is no longer active because it can be modified or degraded by microbial enzymes, such as β-lactamase for penicillins and aminoglycoside-modifying enzymes for aminoglycosidic antibiotics;
3. the natural target of the drug is modified, usually through gene mutations, and the affinity of the drug for its target is reduced or the binding site is modified so that the drug can no longer access it (e.g., fluoroquinolone resistance, mutations of penicillin-binding proteins, ribosomal mutations to macrolides and tetracyclines) [2].
The phenomenon of drug resistance is usually triggered by the drug or develops in the presence of the drug. Therefore, drug resistance implies a cause-effect relationship between drug exposure and resistance. This paradigm is summarized by the slogan "Antibiotic therapy: use it and lose it!" [3]. In fact, drug resistance develops as a sort of mechanism of evolutionary "defence" by the target cell (eukaryotic or prokaryotic) against that compound and is often drug-specific (e.g., penicillin resistance does not affect other classes of antibacterial agents). Cross-resistance between different molecules is rarely seen, an exception being the multidrug resistance/P-glycoprotein which is an integral membrane transporter that effluxes a large number of structurally unrelated chemotherapeutic agents in an ATP-dependent fashion. Resistance is also slowly reversible, in that the absence of a given compound over a certain period of time may reconstitute the status quo ante. Therefore, drug resistance appears closely related to the molecular mechanism of action of a given drug, due to the change of at least one of its target ligands.
Drug resistance, at least in the case of antibiotics, is usually quite well-defined in terms of underlying mechanisms. Laboratory detection and identification techniques are available (i.e., antibiogram in vitro testing, molecular identification and antimicrobial susceptibility testing), and the results of these laboratory techniques may impact the clinical therapeutic decision and the outcome. Therefore, true resistance to a given drug has the following features: it implies a change in the usual target of the drug that causes the drug to no longer be effective, and it is identifiable by specific laboratory tests which impact the therapeutic decision.
On the basis of the above-mentioned characteristics of drug resistance, a different situation applies to the more general phenomenon of "treatment failure" and to the substantial variability in the clinical and biological responses to a given drug in different subjects. These phenomena, i.e., treatment failure and interindividual variability in drug response, are quite familiar to practicing physicians, and true drug resistance is likely to account for only a small percentage of treatment failures [1] and for certain classes of drugs only. Under these circumstances, no single laboratory test can diagnose the phenomenon of treatment failure, and the therapeutic algorithm is multifactorial.
Overall, treatment failure can be considered as a "gap" between expected and observed clinical results in response to a given compound. Each single step of the complex process which starts with a diagnostic and therapeutic decision and ends with the observed clinical outcome can be affected by different factors, all of which contribute to the final observed clinical effect in the individual patient (Figure 1).
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Figure 1. Variables that determine the complex relationship between prescribed drug dosage and drug effects on clinical outcome.
(Modified from Rocca B, Patrono C. J Thromb Haemost 2005;3:1597) |
A wrong prescription, poor patient compliance, inadequate patient understanding of the way in which the drug should be taken, altered drug metabolism in vivo due, for instance, to a pre-existing disease affecting drug processing (hepatic and renal disorders), the concomitant administration of other drugs: these are only some of the factors potentially contributing to interindividual variability in responding to the same compound. Antiplatelet drugs make no exception in this context and can display treatment failures, as well as pharmacokinetic and pharmacodynamic variability, similar to all other drugs.
In the following sections we will discuss the evidence supporting the occurrence of interindividual variability in response to antiplatelet drugs as opposed to the existence of a "true" resistance (defined as a laboratory-detectable change in drug target). The determinants of this variability in the individual patient and the clinical, investigational, and practical consequences will be considered.
Before discussing the argument of resistance versus response variability, we will summarize the mechanism of action and the molecular targets of the different classes of antiplatelet drug and describe the most common tests used to explore platelet function and their adequacy as biological indexes of the effects of different antiplatelet drugs. We will then critically review all the various conditions under which "resistance" to antiplatelet drugs has been reported in the literature.
Understanding the determinants of the response to each antiplatelet drug may help in optimizing and individualizing antiplatelet strategies and improving their risk/benefit profile.
CLASSIFICATION OF ANTIPLATELET DRUGS BASED ON THEIR MECHANISM OF ACTION
Biochemical aspects of platelet function related to the mechanism of action of antiplatelet drugs
Platelets are vital components of physiological hemostasis and key participants in thrombosis by virtue of their capacity to adhere to injured blood vessels and to accumulate at sites of injury [4]. Although platelet adhesion and activation can be viewed as a physiological repair response to the sudden fissuring or rupture of an atherosclerotic plaque, uncontrolled progression of such a process, through a series of self-sustaining amplification loops, may lead to intraluminal thrombus formation, vascular occlusion, and transient ischemia or infarction (Figure 2). Currently available antiplatelet drugs interfere with some of the steps involved in the activation process, including adhesion, release, and/or aggregation [4], and have a measurable impact on the risk of arterial thrombosis that cannot be dissociated from an increased risk of bleeding [5].
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Figure 2. Hemostatic platelet plug forming in a cut arteriole (electron micrograph x650).
(Reproduced from Born G, Patrono C. Br J Pharmacol 2006;147:S241, with permission) |
Platelets form by fragmentation of the cytoplasm of bone marrow megakaryocytes and have a maximum circulating life span of about 10 days in man [6]. Approximately 1011 platelets are produced each day under physiological circumstances. This level of production can increase up to tenfold at times of increased need [6]. Several lines of evidence indicate that platelet reactivity may vary as a function of platelet age, similar to what is observed in erythropoiesis, where reticulocytes, i.e., young erythrocytes, appear much more active and reactive than their senescent counterparts [7,8]. This phenomenon might confer heterogeneity in terms of hemostatic capacity of younger versus older platelets. Younger platelets express detectable amounts of RNA that allows their laboratory identification as "reticulated platelets," are likely to have increased size, as shown by higher mean platelet volume in immune thrombocytopenia versus aplastic anemia [9], and are endowed with a different repertoire of proteins that resembles more closely that of parent megakaryocytes. Consistent with this hypothesis, younger platelets express more adhesive proteins [8] as well as the inducible isoform of cyclooxygenase (COX-2) and the membrane type of prostaglandin (PG)E-synthase, and this phenomenon is markedly amplified in clinical conditions associated with accelerated platelet regeneration [10]. The different reactivity of young versus old platelets might have a clinical counterpart in the different degree of bleeding diathesis observed in thrombocytopenias of different origins, i.e., thrombocytopenia due to megakaryocyte dysfunction (prevalence of old platelets, such as in myelodysplastic syndromes) versus thrombocytopenia due to excess of peripheral platelet destruction and compensatory increased platelet output from megakaryocytes (e.g., immune-mediated forms, recovery on marrow transplantation). Therefore, both the increased percentage of reticulated platelets and enhanced platelet response to agonists observed in immune thrombocytopenic purpura might contribute to the maintenance of a better hemostasis, despite low platelet counts, as compared to aplastic anemia [11,12].
Platelets are anucleated blood cells, but they can provide a circulating source of chemokines, cytokines, and growth factors that are pre-formed and packaged in their storage granules (Figure 3).
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Figure 3. Electron microscopic image of a resting human platelet sectioned in the equatorial (upper panel) and cross-section (lower panel) planes. The typical discoid shape is visible on the cross-section, and dense granules are visible on both sections.
(Mag x33,000; from Rocca B, unpublished data, 1999) |
Besides releasing pre-formed substances, activated platelets are metabolically active and capable of synthesizing several mediators which sustain and amplify their hemostatic response. For instance, biologically active metabolites derived from arachidonic acid (AA), such as thromboxane (TX) A2, are synthesized upon activation (Figure 4).
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Figure 4. Arachidonic acid metabolism through cyclooxygenases in platelets and mechanism of action of aspirin. Bold arrows indicate preferential metabolic routes. |
Furthermore, activated platelets have the capacity of translating constitutive mRNAs into proteins, including interleukin-1b [13]. At variance with the rapid production of prostanoids, protein translation requires several hours [13]. This property may confer to platelets previously unrecognized roles in inflammation and vascular injury, beyond their fast, hemostatic response. Thus, antiplatelet drugs may be expected to impact on platelet-derived protein signals for inflammatory and/or proliferative responses that may contribute to atherothrombosis.
Activation and aggregation of platelets will be discussed in the following paragraphs. In addition to intra- and extraplatelet activatory signals, negative modulation of platelet adhesion and aggregation is exerted by several physiological mechanisms, including endothelium-derived prostacyclin (PGI2), nitric oxide, CD39/ecto-ADPase, and platelet endothelial cell adhesion molecule-1 (PECAM-1). Some drugs may interfere with these regulatory pathways, as exemplified by the dose-dependent inhibition of PGI2 production by aspirin and other COX-inhibitors [5].
Glycoprotein IIb/IIIa
The final effector of most platelet-activating signals is the glycoprotein (GP) αIIb-β3, also known as GPIIb/IIIa. This is the most abundant of the platelet integrins and binds fibrinogen with the highest affinity but can also bind other arginine-glycine-aspartic acid (RGD)-containing molecules such as fibronectin, von Willebrand factor, and vitronectin [14]. This integrin is required for platelet aggregation, clot retraction, and platelet spreading over vascular matrices but also plays an important role in platelet transmembrane signalling [15]. On quiescent platelets, this receptor exhibits minimal binding affinity for von Willebrand factor and plasma fibrinogen [16]. In an activated state, "inside-out" signal transduction mechanisms trigger a conformational change in the receptor to a high-affinity ligand-binding state that is competent to bind adhesive proteins and form a platelet plug. After ligand binding, "outside-in" signal transduction mechanisms mediate integrin-cytoskeleton interactions [17]. These have been shown to be required for postligand-occupancy events, such as cell spreading and formation of focal adhesion sites. The conformational status of GPIIb/IIIa is regulated not only by platelet agonists, such as thrombin or adenosine diphosphate, but also by antagonists such as PGI2 and nitric oxide.
The crucial role of GPIIb/IIIa in mediating platelet aggregation is demonstrated by the severe mucosal bleeding tendency that characterizes subjects affected by Glanzmann's thrombasthenia. This is an autosomal recessive disorder characterized by deficient or dysfunctional GPIIb/IIIa complexes [18]. The hallmark of the disease is impaired platelet aggregation stemming from defective fibrinogen binding to GPIIb/IIIa. Also, IIIa-null mice show clinical and laboratory thrombasthenic features similar to those seen in the human disease, including mucosal hemorrhage, poor clot retraction, and reduced platelet aggregation [19]. Because of its crucial role in platelet function, this integrin is an important target in cardiovascular medicine (see below). Furthermore, the Pl(A2) polymorphism of the IIIa subunit has been reported to be associated with an increased risk of coronary thrombosis [20].
Platelet arachidonic acid metabolism
In response to a variety of pro-aggregatory stimuli such as thrombin, adenosine diphosphate (ADP), or collagen, platelets synthesize a variety of eicosanoids that are biologically active metabolites derived from AA. On activation, intraplatelet Ca2+ increases, rapidly activating cytosolic phospholipase (PL)A2, which mobilizes AA from the membrane phospholipid bilayer. AA is then converted to PGH2 by PGH-synthase-1 or -2, also referred to as COX-1 or -2, and to 12-(S)-hydroxyeicosatetraenoic acid (HETE) by 12-lipoxygenase. Downstream isomerases and reductases, differentially expressed in a cell-specific fashion, catalyze the conversion of the PGH2 intermediate to biologically active end-products, including PGE2, PGF2α, PGD2, PGI2, and TXA2, known collectively as prostanoids (Figure 4) [21]. TXA2 is the most abundant prostanoid from activated platelets, and its biosynthesis is almost completely COX-1-dependent under normal circumstances. PGE2 is much less abundant (approximately 30- to 40-fold less than TXA2) and is largely COX-1-dependent. Under conditions of increased platelet turnover, the contribution of COX-2 to the biosynthesis of both PGE2 and TXA2 increases: up to 40% of PGE2 and up to 20% of TXA2 released on activation in vitro are inhibited by COX-2 blockade [10]. Therefore, platelets possess COX-1, COX-2, TX-synthase, and both isoforms of PGE-synthases, with the inducible, membrane-associated PGE synthase being coexpressed with COX-2 in younger platelets [10].
COX isozymes are integral membrane proteins that catalyze a two-step reaction, first cyclizing AA to form PGG2 and then reducing the 15-hydroperoxy group of PGG2 to form PGH2. They share approximately 60% homology within a given species and exhibit remarkable structural homology at the atomic level [21]. Nevertheless, they are encoded by different genes, located on distinct chromosomes, and, most importantly, may be coupled to different prostanoid synthases, even within the same cell type [22]. COX-1 is the prevalent isoform expressed by mature platelets, while COX-2 expression has been described in the youngest platelet population and is derived from the parent megakaryocytes [10,23-25]. Therefore, COX-2 expression is physiologically present only in a small fraction (<_525_-825_29_ of="" the="" circulating="" platelet="" pool="">24]. However, COX-2-expressing platelets increase substantially in clinical settings associated with high platelet regeneration [10].
Thus, platelet COX-1 activity is largely predominant and mainly coupled to TX-synthase, although a smaller but consistent amount of COX-1-derived PGE2 is also released during platelet activation. At variance with COX-1, COX-2-dependent TXA2 synthesis from thrombin-stimulated platelets seems negligible under physiological circumstances [10,26]. COX-2 activity appears mainly coupled with PGE-synthase(s), although under conditions of high platelet turnover a detectable albeit small amount of TXA2 is COX-2-derived [10].
TXA2 is synthesized and released in response to a variety of platelet agonists and provides a signal that amplifies platelet activation by inducing further platelet recruitment and irreversible aggregation. TXA2 exerts its biological actions by binding a specific transmembrane receptor, TP. One gene and two alternative splicing isoforms of TP have been identified in humans: the a isoform is a heptahelical transmembrane G-protein-coupled receptor, preferentially expressed on the platelet membrane. It couples Gq, inhibiting adenylyl cyclase activity, thus leading to intraplatelet activatory signals [27]. The TPb isoform has been cloned from an umbilical endothelial cell library and its presence and function in platelets remain controversial. Furthermore, on the basis of ligand/binding studies, two different types of PGH2/TXA2 receptors have been pharmacologically defined on human platelets, characterized by high and low affinity states [28]. Whether they correspond to the two cloned isoforms of TP is presently uncertain.
AA can also be metabolized by the platelet-type 12-lipoxygenases (P-12-LO) and converted to 12-(S)-HETE. Mice lacking the P-12-LO gene showed increased sensitivity to ADP-induced aggregation, implying an inhibitory modulation exerted by 12-HETE on platelet activation [29]. A human P-12-LO has been cloned [30], although its physiological role is less clearly defined compared to mice.
Besides undergoing enzymatic metabolism, AA can also be nonenzymatically converted to isomeric eicosanoid species called "isoeicosanoids" [31], through a process of free radical-catalyzed lipid peroxidation [32]. Among this family of compounds, the most extensively studied are the F2 isoprostanes, isomers of PGF2α, and, in particular, 8-iso-PGF2α. Nanomolar concentrations of the latter cause vasoconstriction and activate platelets, causing shape change and potentiating the platelet response to subthreshold concentrations of other agonists [32]. Pharmacological studies in vitro or using genetically manipulated mice, either lacking or overexpressing the TP gene, strongly suggest that 8-iso-PGF2α acts as a TP agonist [33], although some data suggest that other, currently unknown, receptors may bind 8-iso-PGF2α in platelets [34]. Importantly, F2 isoprostanes can be measured both in plasma and urine [32] and therefore provide a noninvasive index of lipid peroxidation in vivo.
Platelet P2Y receptors
Platelet activation by nucleotides such as ADP plays a crucial role in thrombus formation. Nucleotides can be released in extracellular fluids by different sources: cell necrosis, efflux through membrane channels, or exocytosis of secretory granules, as in the case of platelets that store ADP in their dense granules. Once in the extracellular milieu, these molecules are rapidly degraded by various ectonucleotidases [35].
Nucleotides interact with two large families of purinergic (P) receptors: the ionotropic P2X and the G protein-coupled P2Y receptors. Two types of P2Y and one type of P2X receptors are expressed by human platelets. Among the eight different subtypes of P2Y receptors cloned so far, the P2Y1 and P2Y12 types are present on platelets, acting as receptors for ADP. The importance of these receptors in both physiological and pathological platelet function is largely derived from human disorders, mouse models, and pharmacological interventions. P2Y1- and P2Y12-null mice show a similar phenotype characterized by impaired platelet aggregation in response to ADP, increased bleeding time, and low susceptibility to experimental thrombosis [36,37]. Human subjects with defective P2Y12 receptors have been reported to have a moderate to severe bleeding diathesis [38].
The P2Y1 receptor is mainly Gq-coupled, can trigger shape change, and is responsible for a weak and transient aggregation of human platelets, mediated through calcium release. The P2Y1 receptor is also coupled to G12/G13-linked Rho-kinase activation in human platelets [39].
The P2Y12 receptor is Gi-coupled and is responsible for adenylyl cyclase inhibition. It accounts for the completion and amplification of the platelet response to ADP and other agonists, including TXA2, thrombin, and collagen. Furthermore, P2Y12 activation potentiates platelet secretion and stabilizes platelet aggregates [39]. Polymorphisms of the P2Y12 gene have been reported to be associated with increased risk of ischemic diseases [40].
In addition to the presence of P2Y1 and P2Y12 receptors, platelets express the P2X1 receptor as well. This receptor is a ligand-gated cation channel responsible for fast calcium entry and is bound by ATP, but not ADP. This receptor, on ATP ligation, induces a transient platelet shape change and is involved in platelet activation by collagen under high shear conditions. Studies in null mice indicate that the P2X1 receptor might contribute to thrombus formation within small arteries [41].
The P2Y12 receptor is a well-established target of antiplatelet drugs, such as ticlopidine and clopidogrel. Studies in P2Y1 and P2X1 knockout mice and experimental thrombosis models using selective P2Y1 and P2X1 antagonists have shown that these receptors could also be potential targets for new antithrombotic drugs.
Irreversible and reversible inhibitors of platelet COX-1
Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs worldwide. These drugs act through the inhibition of COX activity and consequently prevent the formation of prostanoids, though with different mechanisms and with variable COX-isozyme selectivity [5].
Irreversible inhibitors: Aspirin
The best characterized mechanism of action of aspirin is related to permanent inactivation of the COX activity of COX-1 and COX-2 [42,43] (Figure 4). By diffusing through cell membranes, aspirin enters the COX-channel, a narrow hydrophobic channel connecting the cell membrane to the catalytic pocket of the enzyme. Aspirin first binds to an arginine-120 residue, a common docking site for all NSAIDs; then it selectively acetylates a serine residue, serine-529 in human COX-1 and serine-516 in human COX-2. These acetylated serine residues are located in the narrowest section of the channel, thereby preventing access of arachidonic acid to the COX catalytic site of the enzyme. However, aspirin acetylates human COX-1 and COX-2 with different affinities and is approximately 50- to 100-fold more potent in inhibiting platelet COX-1 than monocyte COX-2 [44]. This may account, at least in part, for the different dose requirements of the analgesic and anti-inflammatory versus antiplatelet effects of the drug.
The unique pharmacological features of aspirin, such as its short plasma half-life (15-20 min) and its efficiency in permanently inactivating COX-1 at low concentrations, as well as the unique biological features of anucleated platelets, such as their lack of substantial new protein synthesis, result in complete and persistent blockade of platelet COX-1-dependent TXA2 biosynthesis on once daily administration of low-dose aspirin in vivo [45]. Single oral doses of aspirin ranging from 5- to 100-mg dose-dependently reduce platelet TXB2 production in healthy subjects, as measured by the whole blood assay ex vivo (see below) [45]. Furthermore, the inhibitory effect of doses of aspirin <100 mg="" is="" cumulative="" on="" repeated="" daily="" _dosing2c_="" reaching="" a="" plateau="" within="" 3="" to="" 10="" _days2c_="" depending="" the="" dose="" and="" platelet="" turnover="">45,46] (Figure 5). Moreover, on aspirin withdrawal, the kinetics of TXA2 synthesis recovery is consistent with the rate of platelet turnover, reflecting the entrance into the peripheral circulation of new platelets bearing nonacetylated COX-1 enzyme [45]. Thus, aspirin pharmacodynamics is completely dissociated from its pharmacokinetics.
 |
Figure 5. Inhibitory effect of doses of aspirin <100 _mg2c_="" as="" single="" dose="" or="" daily="">
(Reproduced from Patrono C et al. Circulation 1985;72:1177, with permission) |
The consistency of dose requirements and saturability of the effects of aspirin in acetylating platelet COX-1, inhibiting TXA2 production, and preventing atherothrombotic complications constitute the best evidence that aspirin prevents thrombosis through inhibition of platelet TXA2 biosynthesis [42,43]. In fact, a very large database of randomized clinical trials now offers the most compelling evidence that prevention of myocardial infarction and ischemic stroke by aspirin is largely due to permanent inactivation of platelet COX-1 [43]. These studies, which tested the efficacy and safety of the drug when given at daily doses ranging from as low as 30 mg to as high as 1500 mg, have established two important facts. First, the antithrombotic effect of aspirin is saturable at doses in the range of 75 to 100 mg, as would be expected from human studies of platelet COX-1 inactivation [45,46]. Second, despite a half-life of approximately 20 minutes in the human circulation, the antithrombotic effect of aspirin is observed with a dosing interval of 24 hours, reflecting the permanent nature of platelet COX-1 inactivation and the duration of TXA2 suppression following oral dosing in man. Other mechanisms of action that have been suggested as contributing to the antithrombotic effect of aspirin, such as an anti-inflammatory effect of the drug, are not compatible with these unique properties [42,43].
Although the search for the lowest effective dose of aspirin for platelet inhibition was largely driven by the explicit concern of concomitant inhibition of vascular PGI2 production, it is still uncertain whether dose-dependent suppression of the latter attenuates the antithrombotic effect of aspirin in clinical syndromes of vascular occlusion [43]. The biochemical selectivity of low-dose aspirin arises from both pharmacokinetic determinants, such as the acetylation of platelet COX-1 that occurs in portal blood (prior to first-pass metabolism), and pharmacodynamic determinants, such as the limited sensitivity of endothelial COX-2 to the drug [43]. Aspirin is an effective antithrombotic agent in a wide range of daily doses [5]. Whether dose-dependent inhibition by aspirin of a mediator of thromboresistance, such as PGI2, may be responsible for a somewhat attenuated efficacy at high daily doses [47,48] remains to be demonstrated convincingly.
Aspirin's unique features in inhibiting platelet COX-1-its ability to inactivate the enzyme permanently through a short-lived active moiety-are ideally suited to its role as an antiplatelet drug, because they severely limit the extent and duration of extraplatelet effects of the drug, including the inhibition of PGI2. Moreover, the cumulative nature of platelet COX-1 acetylation by repeated low doses of aspirin [45,46] explains the clinical efficacy of doses as low as 30 to 50 mg daily [5], the predictable high-grade inhibition of platelet TXA2 biosynthesis, and the persistence of the drug's effect. These features, in turn, may limit the consequences of less-than-ideal compliance in a real world setting.
Permanent inactivation of platelet COX-1 by aspirin may lead to the prevention of thrombosis as well as to excess bleeding. At least two distinct COX-1-dependent mechanisms contribute to the increased risk of upper gastrointestinal (GI) bleeding associated with aspirin exposure: inhibition of TXA2-mediated platelet function and impairment of PGE2-mediated cytoprotection in the GI mucosa [5]. Whereas the former effect is dose-independent, at least for daily doses in excess of 30 mg, the latter effect is clearly dose-dependent. Inhibition of platelet function is largely responsible for the twofold increase in the risk of upper GI bleeding associated with daily doses of aspirin in the range of 75 to 100 mg, in as much as a similar relative risk is associated with other antiplatelet agents that do not act on COX and therefore do not affect PGE2-mediated cytoprotection [43]. Inhibition of COX-1-dependent cytoprotection amplifies the risk of bleeding/perforation by causing new mucosal lesions or aggravating existing ones and is associated with a relative risk of 4 to 6 at the higher, analgesic or anti-inflammatory doses of aspirin. Assessing the net effect of aspirin requires an estimation of the absolute risk of the individual patient for thrombotic and hemorrhagic complications [43].
The efficacy and safety of aspirin has been evaluated in patients covering the whole spectrum of atherothrombosis, from apparently healthy low-risk individuals to patients presenting with an acute myocardial infarction or an acute ischemic stroke. Among patients with occlusive vascular disease, both individual studies [5] and a meta-analysis of trials of antiplatelet therapy [48] have shown that low-dose aspirin reduces the risk of a serious vascular event by approximately one-quarter. This represents a composite of one-third reduction in nonfatal myocardial infarction, one quarter reduction in nonfatal stroke, and one-sixth reduction in death from a vascular or unknown cause [48]. Since each of these proportional reductions applies similarly to all categories of patients with vascular disease, the absolute benefits of aspirin in the individual patient can be estimated by applying a one-third reduction to her/his absolute risk of nonfatal myocardial infarction, a one-quarter reduction to the risk of nonfatal stroke, and a one-sixth reduction to the risk of vascular death [48]. Thus, among a wide range of patients with vascular disease, in whom the annual risk of a serious vascular event ranges from 4% to 8%, aspirin typically prevents at least 10 to 20 fatal and nonfatal vascular events for every 1000 patients treated for 1 year (number needed to treat: 50 to 100) [48]. No appreciable gender- or age-related differences in the effects of aspirin were found in these analyses.
Observational studies and a meta-analysis of trials [48] among high-risk patients have demonstrated that long-term therapy with low-dose aspirin is associated with around a twofold increased risk of major extracranial (mostly, upper GI) bleeding, and this proportional excess hazard appears similar regardless of the variable underlying cardiovascular risk of the patient. In middle-aged patients, this corresponds to an estimated absolute excess of approximately 1 to 2 major bleeding complications per 1000 patients treated with low-dose aspirin for 1 year (number needed to harm: 500 to 1000) [43,48]. Therefore, for most high-risk patients using low-dose aspirin, the number avoiding a serious vascular event clearly outweighs the number experiencing a major bleeding, unless there is some particular reason for an increased susceptibility to bleeding, such as advanced age, history of prior ulcer, or concomitant treatment with other drugs interfering with primary hemostasis. Such a favorable harm/benefit balance for low-dose aspirin in high-risk patients has resulted in consistent level 1 recommendations by both North American [5] and European [49] consensus documents and in regulatory approval by the Food and Drug Administration and European health authorities for practically all vascular indications except peripheral arterial disease.
Thus, aspirin is recommended in all clinical conditions in which antiplatelet prophylaxis has a favorable risk/benefit profile. In consideration of dose-dependent GI toxicity and its potential impact on compliance, physicians are encouraged to use the lowest dose of aspirin that has been shown to be effective in each clinical setting [5]. The available evidence supports daily doses of aspirin in the range of 75 to 100 mg for the long-term prevention of serious vascular events in high-risk patients [43]. In clinical settings where an immediate antithrombotic effect is required (such as in acute coronary syndromes or in acute ischemic stroke), a loading dose of 160 to 200 mg should be given at diagnosis in order to ensure rapid and complete inhibition of thromboxane-dependent platelet aggregation [5].
Although the benefits of low-dose aspirin are clear among patients with vascular disease, the balance of benefit and harm of the same preventive strategy is substantially uncertain in low-risk individuals with no clinically apparent vascular disease. The decision to prescribe lowdose aspirin in a person with no history of vascular disease must rely on the individual judgment that the likely benefits of aspirin will exceed any risks of bleeding. On the basis of the available evidence from six primary prevention trials, low-dose aspirin therapy for 4 to 10 years prevents nonfatal myocardial infarction by one quarter, but it has no clear protective effect against ischemic stroke or vascular death [43]. Therefore, assessing the benefits and risks of lowdose aspirin requires balancing any absolute reduction in nonfatal myocardial infarction (1 to 3 per 1000 treated for 1 year) against an increased risk of major GI bleeding (1 to 2 per 1000) and hemorrhagic stroke (0.1 to 0.2 per 1000) [43]. The recently reported Women's Health Study [50] found no evidence of a protective effect of aspirin 100 mg on alternate days against myocardial infarction but a marginally significant 17% reduction in the risk of stroke (relative risk, 0.83; 95% CI, 0.69 to 0.99; P=.04). It should be noted that stroke represented a secondary end-point of the study and that the overall effects of 10-year prophylaxis with aspirin on the primary end-point (a combination of nonfatal myocardial infarction, nonfatal stroke, and death from cardiovascular causes) failed to reach statistical significance (relative risk, 0.91; 95% CI, 0.80 to 1.03; P=.13). Despite the large sample size of approximately 40,000 initially healthy women and the long duration of follow-up, the Women's Health Study was probably underpowered to detect a moderate treatment effect because of the lower than expected rate of vascular events (only 0.3% per year in the control group) and lower than expected relative risk reduction associated with aspirin (9% vs 25%) [50]. Thus, its results should be viewed within the context of all randomized evidence from aspirin trials [43].
It has been suggested that low-dose aspirin may be appropriate for individuals whose estimated annual risk of a coronary event, based on a risk prediction algorithm, exceeds a particular threshold. Various guidelines have adopted this approach using risk thresholds for coronary events ranging from 0.6% to 1.5% per year. In particular, the suggestion that aspirin therapy is safe and worthwhile at coronary event risks ≥1.5% per year is potentially attractive [51]. However, there is inadequate information from randomized clinical trials assessing the efficacy and safety of low-dose aspirin in asymptomatic subjects at an estimated annual risk of 1.5% to 3.0%, and more evidence is clearly needed [43].
Reversible COX-1 inhibitors
A variety of nonselective NSAIDs can inhibit TXA2-dependent platelet function through competitive, reversible inhibition of COX-1. When used at conventional anti-inflammatory dosage, these drugs generally inhibit platelet COX-1 activity by 70% to 90% [5,43]. However, such degrees of inhibition are insufficient to block platelet aggregation adequately in vivo because of the substantial biosynthetic capacity of human platelets to produce TXA2 [5] (Figure 6).
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Figure 6. Maximal capacity of human platelets to synthesize thromboxane (TX) B2 (left panel), rate of TXB2 production in healthy subjects (middle panel), and nonlinear relationship between the inhibition of platelet cyclooxygenase activity and TXB2 biosynthesis in vivo (right panel). Left panel depicts the level of TXB2 production stimulated by endogenous thrombin during whole-blood clotting at 37°C. The middle panel shows the metabolic fate of TXA2 in vivo and the calculated rate of its production in healthy subjects on the basis of TXB2 infusions and measurements of its major urinary metabolite. The right panel depicts the nonlinear relationship between the inhibition of serum TXB2 measured ex vivo and the reduction in the excretion of thromboxane metabolites measured in vivo.
(Reproduced with permission from Patrono C, García Rodríguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med 2005;353:2373-2383. Copyright © 2005 Massachusetts Medical Society. All rights reserved) |
The only reversible COX-1 inhibitors that have been examined for antithrombotic efficacy in relatively small randomized clinical trials are sulfinpyrazone, flurbiprofen, indobufen, and triflusal [5]. None of these reversible COX-1 inhibitors is approved as an antiplatelet drug in the United States, though they are available in a few European countries. Moreover, the randomized clinical trials comparing indobufen to aspirin and triflusal to aspirin largely lack adequate statistical power to test biologically plausible differences in efficacy, and they were not designed to establish therapeutic equivalence [5]. In fact, population-based observational studies have failed to detect an association between nonaspirin NSAID use and risk of MI, though whether individual pharmacokinetic/pharmacodynamic features may explain conflicting results on naproxen is subject to debate [5,52]. Unfortunately, such case-control studies involve inherent biases that may be difficult or impossible to identify and adjust for, so they cannot reliably detect moderate treatment effects.
P2Y12 antagonists: thienopyridines and related compounds
Thienopyridines
Ticlopidine and clopidogrel are structurally related thienopyridines with platelet inhibitory properties. Both drugs selectively inhibit ADP-induced platelet aggregation, with no direct effects on the metabolism of arachidonic acid [5]. Ticlopidine and clopidogrel also can inhibit platelet aggregation induced by collagen and thrombin, but these inhibitory effects are abolished by increasing the agonist concentration and, therefore, likely reflect blockade of ADP-mediated amplification of the response to other agonists [5].
Neither ticlopidine nor clopidogrel affect ADP-induced platelet aggregation when added in vitro up to 500 mM, thus suggesting that in vivo hepatic transformation to an active metabolite(s) is necessary for their antiplatelet effects. A short-lived, active metabolite of clopidogrel has been characterized [53]. Clopidogrel and, probably, ticlopidine induce irreversible alterations of the platelet ADP receptor P2Y12, mediating inhibition of stimulated adenylyl cyclase activity by ADP (Figure 7) [54]. Inhibition of platelet function by clopidogrel is associated with a selective reduction in ADP-binding sites, with no consistent change in the binding affinity. Permanent modification of an ADP receptor by thienopyridines is consistent with time-dependent, cumulative inhibition of ADP-induced platelet aggregation on repeated daily dosing and with slow recovery of platelet function on drug withdrawal [5].
After single oral doses of clopidogrel, ADP-induced platelet aggregation was inhibited in a dose-dependent fashion in healthy volunteers, with an apparent ceiling effect (i.e., 40% inhibition) at 400 mg. Inhibited platelet aggregation was detectable 2 hours after oral dosing of 400 mg, and it remained relatively stable up to 48 hours. With repeated daily dosing of 50 to 100 mg in healthy volunteers, ADP-induced platelet aggregation was inhibited from the second day of treatment (25%-30% inhibition) and reached a steady state (50%-60% inhibition) after 4 to 7 days. Such a ceiling effect is comparable to that achieved with ticlopidine (500 mg daily). Ticlopidine, however, is characterized by a slower onset of the antiplatelet effect as compared with clopidogrel.
 |
Figure 7. Scheme of the two platelet P2Y receptors and downstream intraplatelet pathways.
|
Thus, the active metabolite of clopidogrel has a pharmacodynamic pattern quite similar to that of aspirin in causing cumulative platelet inhibition on repeated daily low-dose administration [5]. However, only incomplete inhibition of ADP-induced aggregation is achieved with clopidogrel. As with aspirin, platelet function returned to normal 7 days after the last dose. Both the cumulative nature of the inhibitory effects of daily dosing and the slow recovery rate of platelet function on drug withdrawal are consistent with the active moieties of aspirin (i.e., acetylsalicylic acid) and clopidogrel (i.e., active metabolite) causing a permanent defect in a platelet protein that cannot be repaired during the 24-hour dosing interval and can be replaced only through platelet turnover [5]. This also justifies the once-daily regimen of both drugs despite their short half-life in the circulation. Bleeding times measured in the same multiple-dose study of clopidogrel described earlier showed a comparable prolongation (by 1.5-2.0-fold over controls) at 50 to 100 mg daily or ticlopidine at 500 mg daily [5].
Clopidogrel has undergone a rather fast clinical development, with limited phase II studies and a single large
phase III trial (i.e., CAPRIE) to test its efficacy and safety at 75 mg daily compared with aspirin at 325 mg daily in 19,185 high-risk patients [55]. Clopidogrel was slightly more effective than aspirin, and there was some suggestion from a marginally significant heterogeneity test that clopidogrel may be particularly effective at preventing vascular events in patients with symptomatic peripheral arterial disease. This interesting and unexpected finding suggests that the pathophysiologic importance of TXA2 and ADP may vary in different clinical settings. In the CAPRIE trial, the frequency of severe rash was higher with clopidogrel than with aspirin (absolute excess approximately 1-2 per 1000), as was the frequency of diarrhea, thus reproducing the characteristic side effects of ticlopidine. No excess neutropenia, however, was associated with clopidogrel, but the frequency of this serious complication was extremely low (0.05%) in this trial [55]. The CURE trial [56] has demonstrated the efficacy and safety of adding clopidogrel (a loading dose of 300 mg followed by 75 mg daily) to aspirin in the long-term management of patients with acute coronary syndromes without ST-segment elevation. Moreover, the combination of aspirin and clopidogrel has become standard treatment for 1 month after coronary stent implantation [57]. The CREDO trial [58] has demonstrated that following percutaneous coronary interventions, long-term (1-year) clopidogrel therapy significantly reduces the risk of adverse ischemic events. The recently reported COMMIT trial [59] in 45,852 patients admitted to Chinese hospitals within 24 hours of suspected acute myocardial infarction demonstrated that adding a 2-week course of clopidogrel therapy (75 mg daily) on top of conventional treatments (including aspirin, 162 mg daily) produced a highly significant 9% proportional reduction in death, reinfarction, or stroke, corresponding to 9 fewer events per 1000 patients. These effects of clopidogrel appeared consistent across a wide range of patients and independent of other treatments being used. At variance with the CURE trial [56], in which the additional benefit of clopidogrel was associated with enhanced risk of bleeding, no statistically significant excess risk (considering all fatal, transfused, or cerebral bleeds) was noted in the COMMIT trial, either overall or in patients aged older than 70 years [59].
In the recently reported CHARISMA trial [60], among 15,603 patients with established coronary, cerebral, or peripheral arterial disease or with multiple risk factors for atherothrombosis, the addition of clopidogrel to aspirin did not result in a statistically significant lowering of the risk of experiencing a major vascular event during a 28-month follow-up.
Other P2Y12 antagonists
A new class of direct P2Y12 antagonists (e.g., AR-C69931MX) and a novel thienopyridine (prasugrel) [61] are currently being developed that appear to block this ADP receptor more effectively than clopidogrel and to have rapid onset and long duration of action.
Dipyridamole
Dipyridamole is a pyrimidinopyrimidine derivative with vasodilator and antiplatelet properties [5]. The precise mechanism of platelet inhibition by dipyridamole is not known, although several have been proposed. For example, dipyridamole is thought to act by inhibiting the uptake of adenosine into erythrocytes and endothelial cells. This results in increased plasma adenosine levels, which means that there is more available for binding to the adenosine receptor on the platelet [62]. Adenosine activates the release of adenylate cyclase, which converts cyclic adenosine triphosphate (cATP) to cyclic adenosine monophosphate (cAMP). Dipyridamole also blocks the enzyme cyclic guanine monophosphate (cGMP) phosphodiesterase, thereby inhibiting the breakdown of cGMP. Therefore, dipyridamole also acts by inhibiting cyclic nucleotide phosphodiesterases [63], thus increasing intraplatelet levels of cyclic nucleotides which have antiaggregatory effects. The third mechanism of action for dipyridamole is probably by potentiation of the antiaggregating effects of endothelium-derived relaxing factor [64].
The addition of dipyridamole to aspirin has not been shown clearly to produce additional reductions in serious
vascular events in an overview of 25 trials among approximately 10,000 high-risk patients [48], although one trial
suggested that there may be a worthwhile further reduction in stroke [65]. Reasons for this apparent effect of dipyridamole on stroke in the ESPS-2 study include the possibility that the newer formulation of the drug with improved oral bioavailability as well as the twofold higher daily dose (400 mg vs 225 mg in previous studies) resulted in a clinically detectable antiplatelet effect of the drug [5]. The findings of the ESPS-2 study have been confirmed recently by the ESPRIT trial [66].
Blockers of GPIIb/IIIa
Intravenous blockers: abciximab, eptifibatide, and tirofiban
Antagonists of GPIIb/IIIa block the final common pathway of platelet aggregation. Three classes of inhibitors have been developed: murine-human chimeric antibodies, such as abciximab; synthetic peptides, such as eptifibatide; and synthetic nonpeptide (peptidomimetic) agents, such as tirofiban [67].
The pharmacokinetics and pharmacodynamics of commercially available GPIIb/IIIa antagonists have been
reviewed recently, together with a detailed account of randomized clinical trial data that led to their regulatory approval [5]. Although abciximab currently has no place outside of the catheterization laboratory, the disappointing results of GUSTO IV-ACS [68] are also causing reassessment of the role of eptifibatide and tirofiban in patients managed conservatively. A meta-analysis of all major randomized clinical trials of GPIIb/IIIa antagonists in 31,402 patients with acute coronary syndromes who were not routinely scheduled to undergo early coronary revascularization suggests a 9% reduction in the odds of death or myocardial infarction at 30 days [69]. However, the true size of the additional benefit resulting from short term, high-grade blockade of GPIIb/IIIa combined with standard antithrombotic therapy is somewhat uncertain, since the 95% confidence interval ranged from 2% to 16% further reduction in serious vascular events [69]. Moreover, the 1% absolute difference in death or myocardial infarction was balanced by an absolute excess of 1% in major bleeding complications associated with GPIIb/IIIa antagonists versus control [69]. The PARAGON-B Investigators have reported that dose-titrated lamifiban had no significant effects on clinical outcome in patients with non-ST-elevation acute coronary syndromes and yet caused excess bleeding [70], thus reinforcing the uncertainty noted above.
Thus, the risk/benefit profile of currently available GPIIb/IIIa antagonists is substantially uncertain for patients
with acute coronary syndromes who are not routinely scheduled for early revascularization. In contrast, for patients undergoing percutaneous coronary intervention, intensification of antiplatelet therapy by adding an intravenous GPIIb/IIIa blocker is an appropriate strategy to reduce the risk of procedure-related thrombotic complications [5,49].
Oral GPIIb/IIIa blockers
The success of short-term, high-grade blockade of platelet GPIIb/IIIa with intravenous agents [5] has led to the development of several oral GPIIb/IIIa antagonists in the hope of extending this benefit to the long-term management of patients with acute coronary syndromes. To date, five large-scale clinical trials have been completed [5] and a meta-analysis of four of these has been published [71]. The consistent finding of these large-scale trials involving over 40,000 patients is that oral GPIIb/IIIa antagonists (xemilofiban, orbofiban, sibrafiban, and lotrafiban) are not more effective than aspirin or, when combined with aspirin, are not superior to placebo and may in fact increase mortality [5,71]. Several mechanisms have been put forward to explain these results. One is that the poor oral bioavailability of these compounds and the target of approximately 50% inhibition of platelet aggregation resulted in poor antiplatelet activity in many patients. This would explain a lack of clinical response but not an increase in mortality. Indeed, overall there was an increase in the frequency of bleeding and a reduced requirement of urgent revascularization, suggesting some degree of clinical efficacy [5,71].
An alternative explanation is that GPIIb/IIIa antagonists can activate platelets, at least in some individuals [72,73]. GPIIb/IIIa is not a passive receptor; rather, like all integrins, it responds to ligand binding by activating the cell. Thus, fibrinogen binding leads to signals that further activate platelets and are essential for platelet aggregation. Several studies suggest that ligands designed to bind to the receptor and prevent platelet aggregation may trigger some of these activating signals [72,73]. Moreover, the partial agonist activity may not be confined to oral drugs, as abciximab has been reported to activate platelets and promote procoagulant activity by promoting the shedding of CD40L [5].
TP antagonists
The TXA2/PGH2 (TP) receptor is a G protein-coupled receptor, which on ligand stimulation results in activation of phospholipase C and subsequent increase in inositol 1,4,5-triphosphate, diacylglycerol, and intracellular Ca2+ concentrations [21]. Potent (Kd in the low nM range) and long-lasting (half-life >20 hours) TP antagonists have been developed, including GR 32191, BMS-180291 (ifetroban), and BM 13.177 (sulotroban). Despite the antithrombotic activity demonstrated in various animal species and the interesting "cardioprotective" activity demonstrated in dogs and ferrets, these compounds have yielded disappointing results in phase II/III clinical trials [5]. Before drawing definitive conclusions on the apparent failure of this approach, however, it should be mentioned that these studies suffer from severe limitations, including:
1. unrealistic hypotheses of risk reduction being tested (e.g., a 50% reduction in the late clinical failure rate after successful coronary angioplasty),
2. heterogeneous end-points being pooled together, including "clinically important restenosis," for which no evidence of TXA2-dependence was obtained during earlier aspirin trials, and
3. an anti-ischemic effect being tested in individuals with unstable coronary syndromes treated with standard therapy, including aspirin and heparin [5].
Clinical development of GR 32191 and sulotroban has been discontinued because of these disappointing-though largely predictable-results. It would be interesting to see at least one such compound developed through phase III clinical trials with adequate end-points and realistic sample sizes. The potential advantages of potent TP antagonists compared with low-dose aspirin are related to the discovery of aspirin-insensitive agonists of the platelet TP receptor, such as TXA2 derived from the COX-2 pathway [31] and the F2-isoprostane, 8-iso-PGF2α, which is a product of free radical-catalyzed peroxidation of arachidonic acid [32]. The latter can synergize with subthreshold concentrations of other platelet agonists to evoke a full aggregatory response, thus amplifying platelet activation in those clinical settings associated with enhanced lipid peroxidation [74]. The TP antagonist, S-18886 (terutroban), has recently completed phase II clinical development and is currently being compared to low-dose aspirin in a large randomized trial (PERFORM) in patients with a recent cerebrovascular event.
ASSESSMENT OF PLATELET FUNCTION
In vitro and ex vivo assays
The first assay developed to explore hemostasis ex vivo was the bleeding time, which is a "global" index of primary hemostasis, where platelets, vessel wall reactivity, and plasma factors are involved. Moreover, several in vitro assays are currently available to investigate platelet function, measured as platelet aggregation or activation. The most commonly used tests to assess platelet aggregation include:
1. traditional optical aggregometry according to Born's method in platelet-rich plasma;
2. electrical impedance in whole blood;
3. the Platelet Function Analyzer (PFA)-100 in whole blood;
4. the Ultegra Rapid Platelet Function Assay (RPFA) in whole blood.
Some other assays of platelet aggregation in whole blood have been used, so far for experimental purposes only or in small-scale studies, such as the decay of single platelet counts. In addition, flow cytofluorimetry of different antigens expressed on platelets following activation is also used to assess platelet responsiveness in vitro or platelet activation ex vivo. Soluble indexes released by activated platelets can also be measured in vitro and ex vivo, such as TXB2 generation during whole blood clotting or plasma markers such as soluble CD40 ligand, P-selectin, and β-thromboglobulin.
We will review these assays, their limitations and potential clinical application for diagnosis, predictivity of cardiovascular events, and usefulness in monitoring antiplatelet agents.
The bleeding time
The bleeding time is one of the oldest assays to assess hemostasis, established nearly a century ago [75]. At present, the most commonly used technique is the Ivy bleeding time, where one or two standardized incisions are made on the volar surface of the forearm with a spring-loaded device, using venostatic pressure applied on the upper arm by a sphygmomanometer [76]. The bleeding time is measured at the cessation of blood flow through the skin cuts. It reflects the function of several components of primary hemostasis including platelet number and function, von Willebrand factor levels, fibrinogen concentration, and vascular reactivity; therefore, it is not specific for any hemostatic component in particular [76,77]. Furthermore, bleeding time is significantly influenced by other variables, such as the hematocrit value and white blood cell counts [76,77]. In spite of attempts to standardize the technique (spring-loaded device, pressure of sphygmomanometer cuff, interval of wound blotting), this technique is highly dependent on the operator's skills. In fact, the orientation and size of the incision, the site of the incision, skin characteristics (e.g., temperature), and patient cooperation still heavily influence the results [76,78-80]. Thus, considering its technical limitations and its dependence on hemostatic and nonhemostatic variables, the bleeding time has considerable interoperator and interpatient variability.
Thus, it is not surprising that several studies attempting to correlate bleeding time values with clinical hemorrhage in different acquired or congenital bleeding defects have failed to demonstrate any predictive value for the bleeding time [76-78]. The bleeding time is unable to predict peri- and postoperative bleedings and is, therefore, not indicated as a routine preoperative test [81,82]. On the other hand, shorter values of the bleeding time have no value as predictors of thrombotic disorders [83]. Bleeding time has also failed to predict bleedings in patients on antiplatelet drugs, such as GPIIb/IIIa blockers [84], and revealed high variability and inadequacy in the ability to detect aspirin intake [76].
In conclusion, the bleeding time is still being used for diagnostic purposes for some congenital hemostatic disorders, such as von Willebrand disease and inherited platelet disorders. However, it appears unsuitable for the management and dosing of antiplatelet therapy and has no predictive value for thrombotic or hemorrhagic complications.
In vitro platelet aggregation in platelet-rich plasma
Optical platelet aggregometry according to Born [85] is unanimously considered the historical "gold standard" of in vitro platelet function tests and still remains an important assay to diagnose platelet disorders. This technique requires preparation of platelet-rich plasma (PRP) from whole blood, and platelet aggregation is measured photometrically by the increase in light transmission after addition of an agonist to PRP, which is stirred within a cuvette at 37°C. The agonists routinely used include ADP, collagen, arachidonic acid, thrombin-receptor-activating peptide (TRAP), and epinephrine. Platelet aggregation is measured as maximal amplitude (%) or rate (slope) of light transmission. A biphasic pattern of the aggregometric trace is observed for intermediate doses of ADP, where the initial increase in aggregation is due to primary aggregation in response to activation of the GPIIb/IIIa receptor, whereas the second wave of aggregation is the result of platelet degranulation with recruitment of additional platelet aggregates. Other agonists, such as arachidonic acid, TRAP, and collagen, usually show a single-wave aggregation (Figure 8).
 |
Figure 8. Typical aggregometric trace of platelet-rich plasma according to Born's light-transmittance method. The routinely used parameters to quantify platelet aggregation are indicated in orange. |
This technique is limited by several pre-analytical and analytical factors, which strongly influence its results, independently of the type of agonist used. These factors include: the interval between blood collection and analysis, platelet count and size in PRP, the concentration and source of the agonist, the temperature, the speed of stirring in the PRP, and the length and speed of whole blood centrifugation used to prepare PRP, which may lead to the loss of larger platelets in the final PRP, thereby resulting in the selection of a platelet population which is poorly representative of the actual circulating platelet pool [77,78,86,87]. Furthermore, international standardization of different commercial preparations of platelet agonists is not available yet. In fact, a comparison of agonists of different sources showed that even different salts of ADP give different results, and different sources of collagen produce a large variation in the aggregation response [78,88]. In addition to intrinsic technical variability, platelet aggregation responses among normal persons can vary with mental stress, age, gender, race, diet, and hematocrit level, and a person may have different responses on repeated determinations [77,78]. Altogether these technical and physiological aspects are associated with high intra- and intersubject variability of platelet aggregation measurements. Some clinical studies showed an interlaboratory variability of up to 30% [87,88]. Furthermore, aggregation resulting from ADP, collagen, or TRAP does not reflect any specific signalling pathway. Finally, aggregation in PRP in a stirred cuvette hardly simulates pathophysiological conditions of platelet response in circulating blood within arteries or veins.
On this basis, it is not surprising that optical aggregation has failed to provide a predictive index of cardiovascular events in large-scale, prospective studies [89,90]. Moreover, the relevance of changes in this index of capacity to the actual occurrence of platelet activation and inhibition in vivo is largely unknown. Platelet aggregation measurements have not been particularly useful in describing the human pharmacology of aspirin. In fact, the development of low-dose aspirin as an antiplatelet agent was largely based on measurements of serum TXB2, i.e., a mechanism-based biochemical end-point [42].
Platelet aggregation in whole blood
Whole blood aggregometry measures the impedance between electrodes immersed in citrated whole blood. Platelet aggregates cause an increase in electrical impedance [78]. Although this test has the advantage of using whole blood directly, nevertheless the results of this technique are heavily influenced by the handling and cleaning of the electrodes. Other factors influencing the results are the time between blood sampling and analysis, hematocrit, platelet count, temperature, and the final concentration of activating agents. This assay lacks predicitivity in terms of cardiovascular events [89]. Moreover, the results of optical aggregometry do not correlate with those obtained with this technique [87].
The Platelet Function Analyzer (PFA-100®) aspirates citrated whole blood through a capillary and a microscopic aperture cut into a membrane coated with collagen plus epinephrine (CEPI) or collagen plus ADP (CADP). These activators and high shear rates (5000-6000 s-1) over the membrane cause platelets to adhere, activate, and aggregate, forming a stable platelet plug at the aperture. The time required to occlude the aperture is called "closure time" (CT) and measurements are terminated after a maximum of 300 seconds [91]. This test has the advantage of rapidly assessing platelet response in a small volume of whole blood (<1 _ml29_="" without="" requiring="" sample="" processing.="" _however2c_="" several="" technical="" limitations="" exist="" that="" decrease="" reproducibility="" and="" increase="" interassay="" variability.="" in="" _fact2c_="" the="" ct="" is="" significantly="" influenced="" by="" type="" of="" anticoagulant="" _28_buffered="" vs="" unbuffered="" sodium="" _citrate29_="" final="" concentration="" citrate="" _28_129="" mm="" 100-106="" _mm29_="">78,91]. These factors are particularly critical for the CT cutoff values with CEPI cartridges, which are often used to quantitate the response to antiplatelet drugs. In fact, it is recommended that each laboratory establish its own reference range. Moreover, platelet count, hematocrit, and level of von Willebrand factor inversely affect CT values with any cartridge [91]. Duplicate measurements using both channels of the device and individual day-to-day and diurnal variability may result in variations largely exceeding 10% [78]. PFA-100 results do not correlate well with optical aggregometry [92]. Furthermore, PFA-100 is a global test system of platelet adhesion and aggregation and is not specific for any particular intraplatelet signalling pathway.
From a clinical standpoint, the preoperative screening with PFA-100 is not capable of predicting postsurgical bleedings [93]. It has a high sensitivity for von Willebrand disease but gives normal readings for other congenital defects of primary hemostasis, including mild type I von Willebrand disease [94]. Thus, it is not currently recommended as a screening tool for platelet disorders [94]. The PFA-100 is scarcely sensitive to clopidogrel [91] and data on aspirin are quite heterogeneous [91,94,95] (see below). Therefore, the PFA-100 has not been recommended for monitoring antiplatelet therapy [94].
The Ultegra Rapid Platelet Function Analyzer (RPFA®) is a turbidimetric optical detection system that measures the agglutination of fibrinogen-coated beads caused by activated platelets as an increase in light transmittance [77]. This device was originally developed to provide a simple and rapid assay to monitor anti-GPIIb/IIIa therapy and used TRAP as the activator of platelets in the cartridges [96]. The test has been adapted to measure the effect of aspirin with a modified cartridge (VerifyNow aspirin) which contains metallic cations and propyl gallate that cause platelet aggregation. It has been shown to be sensitive to aspirin incubation in vitro [97]. The instrument measures changes in light transmission automatically and thus the rate of aggregation. Results have been shown to correlate poorly with optical aggregometry and the PFA-100 [92]. It has been used as a point-of-care test to titrate GPIIb/IIIa blockers, while the published data related to aspirin intake are still limited.
Other assays in whole blood
The cone-and-plate-analyzer measures whole blood platelet adhesion to a plastic plate, on exposure to defined shear conditions [77,78]. It appears sensitive to von Willebrand factor and fibrinogen as "adhesive" proteins, and to GPIb, GPIIb/IIIa, and platelet activation events. Data related to this new device are still limited.
The Plateletworks®is based on the reduction of the number of free platelets due to the formation of aggregates following platelet stimulation. The test is performed on whole blood, by comparing platelet counts in an EDTA-anticoagulated control sample to a sample treated with agonists (ADP or collagen). Although Plateletworks is quite simple, data on platelet disorders and following antiplatelet drug therapy are still limited. Furthermore, free platelet decrease after ADP has been shown to be largely GPIIb/IIIa-sensitive but aspirin-insensitive in vitro [98].
Membrane markers of platelet activation
Flow cytometry is a sophisticated procedure to quantify platelet surface markers that are exposed by platelets following activation. P-selectin (also known as GMP-140, PADGEM, CD62p), activated GPIIb/IIIa, and CD40-ligand are some of the platelet surface antigens that are activation-dependent and can be measured following exposure to agonists in vitro or directly ex vivo [77,78]. This technique has the advantage of requiring few mL of whole blood and of being largely independent of the platelet count. However, it is expensive, time-consuming, requires well-trained operators, and is very sensitive to platelet activation occurring during and after blood sampling. Therefore, pre-analytical handling is crucial and prone to artefacts. Flow cytometry has been useful to diagnose platelet disorders [77]. However it is not used routinely and its relevance to antiplatelet drug management remains to be established.
Soluble indexes of platelet activation: serum TXB2
The analytical tools for investigating platelet-derived TXA2 metabolism in vivo and ex vivo are largely based on the characterization of the catabolic pathways of TXA2 in humans and on the unique physiological features of platelets with survival spanning 7 to 10 days. COX-1-dependent TXA2 formation is triggered by several platelet agonists, and thrombin is one of the most potent inducers of TXA2 release. TXA2 is chemically unstable and has a very short half-life of approximately 30 seconds; it undergoes a rapid, nonenzymatic hydrolysis to TXB2, a biologically inactive but chemically stable derivative. TXB2 can be measured by immunoassays. In the "whole blood clotting" test, originally described by Patrono et al [99], whole blood is withdrawn without anticoagulants, placed in a glass test tube (glass has the property of activating the coagulation cascade, allowing the formation of thrombin, a potent platelet agonist), incubated for 1 hour at 37°C and subjected to high-speed centrifugation [99]. TXB2 produced in vitro reflects the maximal biosynthetic capacity of circulating platelets activated by thrombin generated during whole blood clotting [99]. The assay is quite simple and quantitative, given the very high concentration of serum TXB2 (200-400 ng/mL) [99]. Pre-analytical and analytical variables affecting the final results are quite limited, due to the simplicity of the procedure. Furthermore, the use of RIA or ELISA to measure TXB2 confers high reproducibility on this assay. It has been used extensively both in vitro and ex vivo to measure platelet-derived prostanoids (e.g., TXB2 and PGE2) and their pharmacological inhibition [45,46,99]. Finally, it explores the function of one specific metabolic pathway in platelets, i.e., AA metabolism via the COX-1 pathway.
In vivo assays
Plasma indexes of platelet activation
Several platelet-derived, circulating substances have been studied to assess platelet function in vivo. Soluble P-selectin, β-thromboglobulin, platelet factor 4, and GP V have been used as platelet release markers. However, artefactual platelet activation can occur during blood withdrawal and plasma separation (high-speed centrifugation), the specificity of these measurements is very low and their correlation with clinical outcomes has never been demonstrated convincingly [100].
Soluble CD40-ligand appears to be a promising marker of platelet activation. Its circulating plasma levels have recently been shown to be platelet-dependent, highly correlated with in vivo markers of platelet activation (i.e., urinary metabolites of thromboxane; see following section) and significantly reduced by low-dose aspirin in patients with type 2 diabetes mellitus [101]. Further studies are needed in other clinical settings.
Urinary indexes of platelet activation
Measurement of the actual rate of TXA2 biosynthesis in vivo is based on characterization of the metabolic disposition of systemically infused TXB2 in humans [102,103]. TXB2 undergoes two major enzymatic degradation pathways: β-oxidation, which results in the formation of 2,3-dinor-TXB2, and transformation through 11-OH-dehydrogenase into 11-dehydro-TXB2. Both metabolites are chemically stable, have an extended plasma half-life, and are excreted in the urine of mammalian species, including humans [102,103]. As compared to 2,3-dinor-TXB2, 11-dehydro-TXB2 has a longer half-life in plasma and is a more abundant metabolite of TXB2 in human urine. Measurement of thromboxane metabolite (TXM) excretion provides a reliable index of the actual rate of TXA2 biosynthesis in vivo [103]. This technique is based on urine extraction and quantitation of TXM by immunoassays or gas-chromatography/mass spectrometry. Pre-analytical artefacts are almost nonexistent, given the noninvasive nature of this procedure, and this technique is highly reproducible. Specificity depends on the characteristics of the individual antisera employed as well as on the chromatographic steps involved in the analytical procedure.
Human studies assessing the effects of low-dose aspirin administration on urinary TXM excretion suggest that in healthy subjects TXA2 is largely derived from platelets [42]. Therefore, measurement of urinary TXM provides a noninvasive approach for studying TXA2-dependent platelet activation in various clinical conditions.
The maximal biosynthetic capacity of platelets to generate TXA2 when challenged ex vivo, as reflected by measurements of serum TXB2, exceeds the endogenous rate of production of TXA2 in vivo by several thousandfold in man [102]. It is important to emphasize that when measuring the inhibition of platelet COX-1 activity ex vivo and the reduction in TXM excretion in vivo the correlation between the two sets of measurements is strikingly nonlinear (see Figure 6).Urinary TXM measurements have been used extensively to monitor platelet activation in vivo in a variety of clinical settings where platelet TXA2 production is transiently or persistently enhanced in response to pathophysiological stimuli. Persistently enhanced TXM excretion has been reported in association with major cardiovascular risk factors, such as cigarette smoking [104], type IIa hypercholesterolemia [105], type 2 diabetes mellitus [106], severe hyperhomocysteinemia [107], and visceral obesity [108]. Moreover, myeloproliferative disorders such as polycythemia vera [109] and essential thrombocythemia [110] are also characterized by markedly enhanced TXM excretion. Episodic increases in TXM excretion have been reported in patients with acute coronary syndromes [111,112] and in the acute phase of ischemic stroke [113]. These are likely to reflect episodes of platelet activation in response to acute vascular injury and are largely, though not completely, suppressed by low-dose aspirin [112].
ASPIRIN "RESISTANCE"
At the end of 1994, Helgason et al published a paper in Stroke entitled "Development of aspirin resistance in persons with previous ischemic stroke" [114]. In this study, the authors administered increasing doses of aspirin (from 325 to 1300 mg daily) to patients with previous ischemic stroke and determined the extent of inhibition of platelet aggregation after 2 weeks and thereafter at approximately 6-month intervals. Over a period of 33 months, 306 patients had platelet aggregation measured at baseline. Of these, 228 (75%) had complete and 78 (25%) had partial inhibition of platelet aggregation at initial testing. At the time of writing, 119 of those who had complete inhibition and 52 of those who had partial inhibition had undergone repeat testing at least once. Approximately one-third of the patients with complete inhibition at initial testing had lost part of the antiplatelet effect of aspirin and converted from complete to partial inhibition without change in aspirin dosage. Of the 52 with partial inhibition at initial testing, 35 achieved complete inhibition either by aspirin dose escalation (in 325 mg increments) or fluctuations of response at the same dosage, but 8 of those 35 had reverted to partial inhibition when tested again. Overall, 8% of patients ultimately exhibited aspirin "resistance" to 1300 mg daily-8 of 52 with partial inhibition and 6 of 119 with complete inhibition at initial testing.
When comparing the fingerprints of "aspirin resistance," as characterized in this paper, it becomes immediately apparent they are very different from those of "drug resistance" as defined above [1]. Thus, the phenomenon described by Helgason et al [114] was not related to a change in drug target, was fluctuating over time, and was, at least in part, reversible with increasing dosage of the drug. Furthermore, the clinical significance of this phenomenon remained undefined.
Since then, the number of publications dealing with "aspirin resistance" has increased exponentially (Figure 9), approaching 80 publications in the year 2005. Over the past 10 years, additional uncertainty and misunderstandings have derived from the use of the designation "aspirin resistance" to describe a variety of different phenomena. In fact, "resistance" has been defined, on the basis of heterogeneous end-points, as the inability of aspirin to: (1) protect individuals from thrombotic complications (clinical end-point); (2) cause a prolongation of the bleeding time or produce an anticipated effect on one or more in vitro tests of platelet aggregation (functional end-point); (3) inhibit TXA2 biosynthesis (biochemical end-point) [5].
 |
Figure 9. Number of publications per year on aspirin "resistance" since 1993. |
Clinical aspirin resistance
The fact that some patients may experience recurrent vascular events despite aspirin therapy should be properly labelled "treatment failure," rather than "resistance." Indeed, before the appearance of the term "aspirin resistance" [114], this phenomenon was accurately described as "aspirin treatment failure." Thus, in 1990, Chyatte and Chen, and later Bornstein et al [115,116], performed prospective observational studies on patients who developed ischemic neurological symptoms while on aspirin and indicated this phenomenon as "aspirin treatment failure." In clinical practice, treatment failure is a common phenomenon, well known by physicians, occurring with any cardiovascular drug (e.g., lipid-lowering or antihypertensive drugs) used to prevent atherothrombosis. Therefore, the clinical diagnosis of an atherothrombotic event in a patient while on a therapeutic dose of aspirin should be more appropriately classified as treatment failure, rather than "clinical resistance" to therapy.
The meta-analysis by the Antithrombotic Trialists' Collaboration has shown that aspirin can prevent up to about 25% to 30% of all ischemic vascular events in high-risk patients [48]. Given the multifactorial nature of atherothrombosis, it is not surprising that only a fraction (one-quarter to one-third) of all vascular complications can be prevented by any single preventive strategy.
The clinical occurrence of an atherothrombotic event while on aspirin is highly nonspecific in terms of potential mechanisms, which range from noncompliance to intrinsic, mechanism-based limitations of the prescribed drug. As a consequence, changing an evidence-based therapeutic strategy is not justified on the basis of the occurrence of a recurrent vascular event, because the efficacy and safety of such a change has never been tested.
Aspirin resistance defined on the basis of functional end-points
Several platelet function assays have been used in different studies to measure platelet response to aspirin in healthy volunteers or vascular patients: bleeding time, Born's light-transmittance aggregometry (LTA), PFA-100 in whole blood, whole blood impedentiometry, RPFA (see also above). Overall, the majority of these studies were characterized by the following major limitations:
1. biochemical or witnessed verification of patient's adherence to the prescribed therapy was absent;
2. there was a single measurement of any given test;
3. intra- and intersubject variability and stability of the assay over time was usually not reported;
4. the criteria to define the normal versus the "aspirin resistant" range and the assay conditions differed among studies;
5. doses of aspirin were heterogeneous, ranging from 75 to 1300 mg;
6. none of these studies was properly controlled.
Lack of biochemical assessment of compliance is a major issue for the majority of studies assessing platelet function in response to aspirin, and this aspect is crucial in studies investigating aspirin "unresponsiveness."
Interestingly, a recent study in 190 patients with a history of myocardial infarction compared AA-induced platelet aggregation in patients while receiving their usual aspirin therapy, after 7 days of withdrawal, and 24 hours after a single witnessed intake of aspirin, 325 mg [117]. While 9% of patients who declared having taken their usual therapy failed to show inhibition of platelet aggregation, this percentage dropped to <_125_ _28_1="" patient="" out="" of="" _19029_="" after="" a="" witnessed="" dose="">117]. Furthermore, this single patient admitted nonsteroidal anti-inflammatory drug (NSAID) intake, 12 hours before testing. Similar results were reported in the study by Lev et al, where, after a witnessed dose of 325 mg of aspirin, the mean of AA-induced LTA became <_2025_ _28_the="" established="" limit="" to="" define="" _22_resistance22_29_="" in="" formerly="" resistant="" patients="">118]. Other studies have reported up to 40% noncompliance with chronic aspirin use [119,120]. It is therefore clear that questionnaires cannot be a reliable parameter to assess the compliance to any given treatment, including aspirin, and that studies not relying on salicylate measurements or serum TXB2 have a major, intrinsic bias, seriously hampering the interpretation of results. Furthermore, the few studies directly comparing different functional assays failed to find any significant agreement between tests, generating the disappointing conclusion that aspirin nonresponsiveness might be highly test-specific.
These biases, together with the fact that none of these techniques directly reflects the mechanism of action of aspirin, i.e., COX-1 inhibition (see above), contribute to a highly variable detection rate of "aspirin-nonresponsive" patients (Figure 10).
 |
Figure 10. Percentage of aspirin "resistance" according to the different assays used in a total of 33 studies listed in Tables 1 to 4. Horizontal bars indicate means.
|
PFA-based studies
Studies investigating platelet function during aspirin intake by the PFA method, and in particular by the CEPI cartridge, are characterized by a wide range of "functional unresponsiveness," ranging from 9% to 51% (Tables 1 and 2). Several studies have demonstrated that aspirin-unresponsiveness based on the CEPI test is significantly associated with higher levels of von Willebrand factor [92,121,122], lower dose (i.e., 81 mg) of aspirin [123-125], female gender [123,125,127-128], ageing [123,125,127], and high platelet count or lower hematocrit [125,128-129]. One study where aspirin intake (500 mg) was witnessed, showed absence of aspirin resistance by CEPI [126]. The effect of female gender on CEPI might be related to the lower hematocrit value of women. None of the above-mentioned associations (higher efficacy at higher doses, gender-related differences, lower efficacy with ageing, high platelet count or low hematocrit) has ever been demonstrated in controlled clinical trials. A high assay variability (12%-15%) has been reported on repeated measurements of aspirin-treated patients [125,129]. More worrying is the fact that there was no correspondence of the CEPI values obtained from samples of the same subjects after either in vitro incubation with aspirin (30 mg/mL) or after 10 days of 150 mg/d aspirin intake [130].
 |
TABLE 1. Studies using one single type of platelet function assay (PFA, RPFA, light-transmittance aggregometry [LTA], bleeding time), without clinical outcomes |
 |
TABLE 2. Studies using multiple functional tests without clinical outcomes |
Moreover, studies comparing different assays in aspirin-treated subjects consistently failed to show any correlation between aspirin unresponsiveness defined by CEPI, LTA, and RPFA (Tables 2 and 3). For instance, in the study of Gonzalez-Conejero et al, while 33% of healthy volunteers taking 100 mg/d were "resistant" to aspirin based on CEPI measurements, the same subjects were full responders based on collagen- or AA-induced LTA [126]. A lack of agreement between LTA and PFA has also been reported in a larger study in 325 patients with stable ischemic heart disease [129].
 |
TABLE 3. Studies using TXA2-related indexes, either urinary TX metabolites (TXM) or in vitro TXB2 formation |
Finally, the clinical significance of CEPI-based aspirin "resistance" remains unproven. Crowe et al showed no correlation with coronary disease severity [129], while Borna et al showed no correlation of CEPI values with plasma ADP, an index of tissue damage, in patients with chest pain developing ST-elevation myocardial infarction [131] (Table 4).
 |
TABLE 4. Studies of functional data and clinical outcomes or biochemical markers of cardiovascular disorders |
LTA-based studies
Light-transmittance aggregometry in response to collagen, ADP, and/or AA has been often used to define aspirin resistance. Overall, with different combinations of the 3 agonists, "resistance" ranged from as low as <_125_2c_ including="" no="" evidence="" of="" _22_resistance22_="">117-118,126,132], to as high as 40% in postcoronary artery bypass patients [133]. Notably, platelet agonist concentrations are not the same in different studies, and the upper limit of percent aggregation to define resistance is variable for collagen and ADP, while it is quite constant (≥20%) for AA (1 or 1.6 mM final concentration). Overall, studies using LTA have a more rigorous assessment of treatment compliance and, coincidentally, studies where aspirin was administered in-hospital or by a witnessed intake showed the lowest (between 0% and 7%) incidence of "resistance" [117-118,126,132,134].
LTA-based studies showed a significantly lower incidence of "resistance" as compared to PFA assay (Figure 10), and no concordance could be demonstrated between the two assays in several studies, independently of the agonist used for LTA measurements (Table 2). Although it has been claimed that AA-induced aggregation might reflect more specifically the COX-1-related pathway, the criteria used to define LTA unresponsiveness are based on ≥2 agonists, one being AA, the other being ADP and/or collagen. Under these circumstances, there is no clear correlation among different agonists. In a recent study by Harrison et al comparing different methods, "resistance" based on ADP-induced LTA was 14%, while it was 21% based on AA-induced LTA, with an overlap of 4% [92]. Studies on repeated determinations of LTA while on aspirin showed a large variability over time, with 67% of formerly defined "resistant" subjects being responsive on subsequent determinations [114]. Thus, the variability of this functional assay remains too wide (see also above) to be confident on the significance of a single measure, as reported in the vast majority of studies.
LTA-based "resistance" does not show distinctive features compared to the responsive group (e.g., gender, hematocrit, aspirin dose), as reported with the CEPI assay. In a recent study, Maree et al showed that the GCGCC haplotype of the COX-1 promoter was associated with an LTA >20% in response to AA [135]. This observation has been recently confirmed by Lepantalo et al in patients with stable coronary artery disease [136]. In the same study, aspirin unresponsiveness detected by PFA or AA-induced LTA showed no concordance at all. However, the same haplotype was not correlated with enhanced TXB2 biosynthesis in vitro. Zimmermann et al used a "confirmation" test for the "resistant" population, such as in vitro incubation with a known concentration of aspirin for samples from patients showing lower-than-expected LTA response ex vivo [133]. This technique always lowers the number of "resistant" subjects based on ex vivo measurements.
Some studies tried to associate LTA response with clinical outcome (Table 4). Aspirin "resistance" assessed by collagen and ADP was unrelated to the incidence of recurrent stroke in 291 patients with previous ischemic stroke or TIA, followed for 4 years [134]. The same study assessed aspirin "resistance" after a witnessed dose of aspirin, and measurements were repeated 3 times/year, with 4% of "resistant" patients on repeated measurements. At variance with this study, Gum et al reported a hazard ratio of 3.12 for a combination of death, myocardial infarction, and cerebrovascular accident associated with aspirin "resistance," as defined by AA- and ADP-induced LTA [148]. However, this study assessed compliance by patient interview only, without confirmation assays or witnessed administration.
RPFA-based studies
Fewer studies assessed the functional response to aspirin by the RPFA method, alone or in combination with other functional assays, due to the quite recent availability of this instrument (Tables 1 and 2). Overall, RPFA-based aspirin "resistance" was 18.5±3% (Figure 10), significantly lower than PFA but not significantly different from LTA (P=.3). Four out of 5 studies with RPFA assessed compliance by interview only. However, the concordance between this assay and PFA or LTA was poor [92]. Conflicting data have been reported on the dependence of RPFA-based unresponsiveness on gender, aspirin dosage, age, platelet count, or hematocrit levels. The effect of female gender and hematocrit might reflect the fact that this is a whole blood assay, and red blood cells might influence the principle of the test, independently of the phenomenon under study.
Two studies evaluated functional and clinical end-points (Table 4). In one study, aspirin "resistance" by the RPFA was associated with higher creatine kinase-myocardial band and troponin I elevation post-percutaneous coronary intervention [137]. The same association between high creatine kinase-myocardial band and aspirin resistance has been reported in a different study, where platelet function was assessed after a witnessed administration of 325 mg of aspirin, but 3 criteria were used to define aspirin "resistance": LTA by ADP and AA as well as RPFA response [118].
Aspirin resistance defined on mechanism-based biochemical end-points
In order to assess aspirin resistance, some studies measured in vitro TXB2, formed during whole blood clotting or following collagen-induced platelet aggregation, in association with other functional tests (Table 3). Two studies could not find any evidence of aspirin nonresponsiveness on the basis of TXB2 formed ex vivo in patients while on aspirin [138-139]. Interestingly, Andersen et al, in a large study of 202 post-MI patients, used TXB2 to check compliance, using the CEPI assay to define "resistance." While all patients showed profound inhibition of TXB2, CEPI identified 35% of aspirin "resistant" patients, whose soluble P-selectin was higher as well. Therefore, it appears that there is no concordance between serum TXB2 and CEPI results. Furthermore, this study showed no differences in major cardiovascular events over 4-year follow-up between aspirin "resistant" and responsive patients [138]. Fontana et al have recently shown similar results in 96 healthy subjects treated with 100 mg/d aspirin for 1 week [140]. Only 1 out of 96 subjects had abnormal values of AA-induced LTA and poor suppression of serum TXB2 after a low-dose aspirin regimen, while 30% of the same subjects were "resistant" based on PFA data.
Relatively few studies measured urinary TXM excretion. Gonzalez-Conejero et al found no evidence of "resistance" in 24 healthy subjects taking 100 mg/d of aspirin on the basis of TXM inhibition, while 33% of the same group were "resistant" using the CEPI assay [126]. Therefore, TXM and PFA assays showed no correlation. The clinical relevance of aspirin-resistant TXA2 biosynthesis has been explored by Eikelboom et al [141] and Bruno et al [142]. Eikelboom performed a nested case-control study of baseline TXM excretion in relation to the occurrence of major vascular events in aspirin-treated high-risk patients enrolled in the Heart Outcome Prevention Evaluation (HOPE) trial. After adjusting for baseline differences, the odds for the composite outcome of myocardial infarction, stroke, and cardiovascular death increased with each increasing quartile of 11-dehydro-TXB2 excretion, with patients in the upper quartile having a 1.8-times-higher risk than those in the lower quartile. However, in this study aspirin compliance was not biochemically assessed and variability of the assay over time was not verified. These data have not been confirmed by Bruno et al, who studied a subset of patients enrolled in the African American Antiplatelet Stroke Prevention Study [142]. TXM measurements were repeated over 2 years, with no association between TXM levels and subsequent vascular events over a 4 to 23 month follow-up. Repeated TXM measurements showed very little variability. However, this study had a considerably smaller sample size, 61 post-stroke patients on aspirin and 6 events, as compared to Eikelboom's study (n=488), and this might in part account for the apparent discrepancy.
At least three potential mechanisms may underlie the occurrence of aspirin-insensitive TXA2 biosynthesis, as observed in the HOPE sub-study. The transient expression of COX-2 in newly formed platelets [10] in clinical settings of enhanced platelet turnover is a potentially important mechanism that deserves further investigation. Extraplatelet sources of TXA2 (e.g., monocyte/macrophage COX-2 or COX-1) may contribute to low-dose-aspirin-insensitive TXA2 biosynthesis in acute coronary syndromes. Moreover, a pharmacodynamic interaction with over-the-counter analgesics (e.g., ibuprofen and naproxen) may impair the antiplatelet effect of low-dose aspirin (see below).
CLOPIDOGREL "RESISTANCE"
The terms clopidogrel "resistance," "nonresponsiveness," and "hyporesponsiveness" have been used interchangeably to indicate a less-than-expected inhibition of ADP-induced platelet aggregation/activation following standard clopidogrel therapy [151]. However, the issue of a less-than-expected platelet response to clopidogrel is somehow less heterogeneous, as compared to aspirin, possibly because the assays used to explore this phenomenon are all ADP-based and, thus, more closely related to the mechanism of action of clopidogrel, i.e., the inactivation of the platelet ADP receptor P2Y12.
The majority of the published studies used either LTA or flow cytometry of platelet activation antigens, and, for both assays, ADP has been used as the agonist. These ADP-based assays consistently showed a significant positive correlation in several studies where more than one test had been performed (Table 5). However, at least three important elements should be considered: (1) the intrinsic limitations in terms of intra- and interindividual variability associated with both LTA and flow cytometry (see above), (2) the fact that platelets express two receptors for ADP, both contributing to the aggregation response (see above), and that clopidogrel inactivates only one of them, and (3) neither of the above-mentioned assays is standardized. These fundamental aspects are also well reproduced in the heterogeneity of ADP-induced LTA measurements both in patients with inherited defects of the P2Y12 receptor and after in vitro incubation of normal samples with saturating concentrations of the antagonist [152].
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TABLE 5. Studies on clopidogrel "resistance" based on functional assays only, without clinical outcomes |
In general, the assessment of compliance in clopidogrel studies is superior to that in aspirin studies, due to the in-hospital administration of the drug in the majority of reports. One aspect of the response to clopidogrel is quite consistent among different studies, independent of the type of assay used and of the population under study (healthy volunteers vs cardiac patients): clopidogrel is characterized by a very wide range of responses that follow a normal, bell-shaped curve of distribution [153-156]. This phenomenon has been described at all doses of the drug, with a trend toward a narrower distribution at the highest dose (600 mg) [153]. This fact can be largely accounted for by interindividual variations in the formation of the active metabolite of clopidogrel (see above). At variance with aspirin, clopidogrel requires hepatic transformation by the CYP3A4 enzyme system in order to generate its active metabolite (see above). Indeed, residual platelet aggregation after clopidogrel administration has been shown to be inversely correlated with the activity of CYP3A4 [157].
The incidence of "resistance" or hyporesponsiveness to clopidogrel varies from 5% to 46% (see Tables 5 and 6).
 |
TABLE 6. Studies on clopidogrel "resistance" with clinical outcomes |
Many factors may contribute to this wide range of inadequate responses. First, there is no standard definition or algorithm to describe this phenomenon. Independently of the type of assay, different ways of quantifying "resistance" have been used, based on the standard deviations of mean values of inhibition, on percentiles of the normal distribution of post-clopidogrel aggregation, and on the percentage of inhibition of pre- minus post-clopidogrel values reported as absolute difference or as percentage of pre-clopidogrel measurement. In addition, both dose- and time-dependent variability has been reported in the response to clopidogrel assessed by LTA and by flow cytometric analysis of platelet activation antigens. With few exceptions [159], several studies have shown that time to maximal platelet inhibition after 600 mg is at least 4 hours and is similar to that seen after a 300 mg dose; however, the 600 mg dose increases the level of inhibition of platelet aggregation as compared with the 300 mg dose and decreases the number of low responders [153-159] (Table 5). The time point used to measure the response and therefore the "resistant" population varies in different studies (between 4 and 24 h after a loading dose, or at 5 to 7 days after 75 mg/d). Furthermore, the concentrations (from 1 up to 20 mM) and sources of ADP are not standardized, and the fraction of non- and low-responders appears directly dependent on the concentration of ADP used in vitro [153,161]. Furthermore, most studies are based on a single determination, which is largely inadequate given the well-established high variability of light-transmittance aggregometry (see above). Reproducibility over time has been studied by Serebruany et al, who reported up to 50% change in status from hypo- to normal responder on repeated measurement [154]. The majority of the studies have not shown any distinctive features of the non- or low-responder group as compared to responders, in terms of underlying disorder, medical history, demographics, or levels of hemostatic factors such as fibrinogen or von Willebrand factor [118,153-155,160] (Table 5).
More recently, a flow cytometric assay has been described, based on the quantification of the vasodilator-stimulated phosphoprotein (VASP) phosphorylation to evaluate the antiplatelet effect of thyenopyridines [161]. This assay is potentially more specific than traditional LTA for this class of drugs. The level of VASP phosphorylation is closely related to the degree of inhibition of ADP-induced GPIIb/IIIa activation via the P2Y12 receptor, and, therefore, represents a mechanism-based biochemical end-point [161]. The VASP-P measurements are highly correlated with ADP-induced LTA measurements [153,161]. However, large studies are needed to ascertain the superiority of this costly assay, as compared to traditional ADP-based tests, especially considering that it is based on flow cytometry, which has many intrinsic limitations when applied to platelet studies (see above).
The clinical significance of resistance/unresponsiveness to clopidogrel remains largely unknown. Two studies of small sample size suggested that clopidogrel nonresponders may be at higher risk for thrombotic events [162-163]. Gurbel et al have recently reported a significantly higher platelet reactivity in clopidogrel-treated patients who had developed an acute stent thrombosis (n=20) as compared to patients without such an event (n=100) [164]. However, platelet function was assessed on average 218 days after the event. Larger and prospective studies are needed, together with more specific mechanism-based platelet function assays, using standardized conditions in order to understand the clinical significance of a hyporesponsiveness to clopidogrel as assessed ex vivo.
DETERMINANTS OF THE INTERINDIVIDUAL VARIABILITY IN RESPONSE TO ANTIPLETELET DRUG
Introduction: therapy as a science
Practicing physicians have long recognized that individual patients show wide variability in response to the same drug. Antiplatelet drugs make no exception. Strategies are needed to deal with interindividual variability in the clinical setting, and progress is required to identify the specific sources of variability for each antiplatelet drug. As described in this section, pharmacokinetic and pharmacodynamic factors may largely account for the main sources of variability in response to antiplatelet drugs.
Pharmacokinetic variability
For any given drug, there may be wide variations in its pharmacokinetics among individuals. The processes of pharmacokinetics involve drug absorption, distribution, metabolism (biotransformation), and elimination [166]. Each of these steps contributes to the quite variable plasma concentrations of the drug and/or its active metabolite(s) in different individuals receiving the same therapeutic regimen. In principle, one would expect that a threshold drug concentration is required in order to achieve a critical level of inhibition of a platelet enzyme or receptor. A standard therapeutic dose of the antiplatelet drug may or may not achieve such a threshold concentration in the individual patient because of the pharmacokinetic factors outlined in Figure 1, even in the presence of ideal compliance with the prescribed regimen.
The following examples illustrate the potential contribution of some of these factors to variability in response to antiplatelet drugs. Dipyridamole is a pyrimidopyrimidine derivative with vasodilator and antiplatelet properties [5] (see above). The absorption of dipyridamole from conventional formulations is quite variable and may result in low systemic bioavailability of the drug. A modified-release formulation of dipyridamole with improved bioavailability has been developed in association with low-dose aspirin [167]. Dipyridamole is eliminated primarily by biliary excretion as a glucuronide conjugate and is subject to enterohepatic recirculation. A terminal half-life of 10 hours has been reported. This is consistent with the twice-a-day regimen used in recent clinical studies. Although the clinical efficacy of dipyridamole (75 mg tid), alone or in combination with aspirin, has been questioned on the basis of earlier randomized trials [168], the whole issue has been reopened by the reformulation of the drug to improve bioavailability and the results of the ESPS-2 study of the new preparation in 6602 patients with prior stroke or transient ischemic attack (TIA) [65]. Whether the favorable results obtained in ESPS-2 reflect the higher dose (400 vs 225 mg daily) and improved systemic bioavailability of modified-release dipyridamole compared with conventional formulations, or the substantially larger sample size and statistical power of the study as compared with previous trials, remains to be established.
A second example of pharmacokinetic variability is provided by the poor oral bioavailability of GPIIb/IIIa antagonists (see above) that, combined with the target of approximately 50% inhibition of platelet aggregation, resulted in poor antiplatelet activity in many patients [5].
Ticlopidine and clopidogrel are structurally related thienopyridines which require in vivo hepatic transformation to an active metabolite(s) for their antiplatelet effects. The pharmacokinetics of clopidogrel are somewhat different from those of ticlopidine. Thus, after administration of single oral doses (up to 200 mg) or repeated doses (up to 100 mg daily), unchanged clopidogrel was not detectable in peripheral venous plasma. Concentrations of 1 to 2 ng/mL were measured in the plasma of patients who received 150 mg/d of clopidogrel for 16 days. The main systemic metabolite of clopidogrel is the carboxylic acid derivative SR 26334. Based on measurements of circulating levels of SR 26334, it has been inferred that clopidogrel is rapidly absorbed and extensively metabolized. The plasma elimination half-life of SR 26334 is approximately 8 hours. As noted above, clopidogrel, inactive in vitro, is metabolically transformed by the liver into a short-lived active platelet inhibitor. However, the interindividual variability in this metabolic activation is still being assessed, and there are no published data on whether liver impairment decreases the ability of clopidogrel to inhibit platelet function.
As the cytochrome P450 isozymes CYP3A4 and 3A5 metabolize clopidogrel faster than other human P450 isozymes and are the most abundant P450s in human liver, they are predicted to be predominantly responsible for the activation of clopidogrel in vivo [170]. When clopidogrel and atorvastatin, a CYP3A4 substrate, are present at equimolar concentrations in vitro, clopidogrel metabolism is inhibited by >90% [170]. Clopidogrel exhibited marked interindividual variability in inhibiting platelet function in three different studies of patients undergoing elective percutaneous coronary intervention (PCI) and stenting [156,160,165]. A variable proportion of these patients was considered to be clopidogrel "nonresponder" or to have clopidogrel "resistance" based on ADP-induced platelet aggregation (see above). Three separate studies [165,170-171] suggest that concurrent treatment with lipophilic statins (e.g., atorvastatin and simvastatin) may interfere with the inhibitory effects of clopidogrel on platelet function. In the study of Lau et al [171], atorvastatin, but not pravastatin, attenuated the antiplatelet effect of clopidogrel in a dose-dependent manner. Approximately 50% to 60% of the available drugs are metabolized by the CYP3A4 [172], which has a low selectivity for its numerous and structurally unrelated ligands. Indeed the complex molecular structure of CYP3A4 has been recently determined, revealing an active site of sufficient size and topography to accommodate both large ligands and multiple smaller ligands [173]. Drug interaction with CYP3A4 may result in its inhibition or in enhanced activity (Table 7) [172-173]. Therefore, it is likely that other drugs (e.g., cyclosporine and erythromycin) may modify the systemic bioavailability of the active metabolite of clopidogrel and therefore may affect its clinical efficacy. Furthermore, hormones, environmental chemicals, and food are known to affect the CYP3A4 activity [172]. Ethnicity is also related to preferential expression of some CYP3A4 polymorphisms linked to variation in its metabolic activity [172]. Moreover, variable metabolic activity of CYP3A4 linked to exogenous factors or to genetic polymorphisms may contribute to the interindividual variability in the platelet inhibitory effects of clopidogrel [174]. In fact, percent platelet aggregation after clopidogrel inversely correlated with CYP3A4 activity [157].
 |
TABLE 7. Common drug substrates, inhibitors, and inducers of CYP3A, according to drug class |
Although ex vivo measurements of ADP-induced platelet aggregation have suggested a pharmacokinetic interaction between atorvastatin and clopidogrel, post hoc analyses of placebo-controlled studies of clopidogrel have failed to detect a statistically significant clinical interaction between the two [175-176]. However, it should be emphasized that retrospective post hoc analyses have limitations that preclude definitive conclusions. Moreover, the lack of information on statin daily doses used in these trials notably restricts our ability to assess the dose-dependence of potential drug interactions.
Insufficient bioavailability has been recently described for low-dose enteric coated aspirin. In 131 patients with stable cardiovascular diseases [177] treated with this aspirin preparation, an incomplete inhibition of TXB2 generation and/or AA-induced platelet aggregation was observed in 44% of patients. However, in all cases the addition of exogenous aspirin to samples of "resistant" patients resulted in a complete inhibition of platelet aggregation, indicating a low bioavailability of this preparation of aspirin, rather than a real resistance to chronic aspirin treatment. Younger age and higher body weight were independent predictors of incomplete response to aspirin.
Pharmacodynamic variability
Considerable interindividual variation in the response to drugs remains after the concentration of the drug in plasma has been adjusted to a target value. Pharmacodynamics relates to all the processes involved in the mechanism of drug action, i.e., interactions with macromolecules and target(s), which result in a complex and often unpredictable relationship between a given concentration and the magnitude of the observed functional or clinical response. For some drugs, this pharmacodynamic variability accounts for much of the total variation in response among individuals [166].
Genetic factors are major determinants of the normal variability of any drug effect and strongly influence pharmacological activity. Genetic variability of the enzyme or receptor targeted by antiplatelet drugs may also alter the expected pharmacological response. Thus, an increased number of ADP receptors associated with the H2 haplotype of the P2Y12 gene may diminish the antiplatelet effect of thienopyridines that only provide partial P2Y12 blockade [178]. Several polymorphisms of COX-1 [135,179-180] and -2 [181] have been described. Fourteen variants within the coding region of the COX-1 gene failed to show any association with recurrence of stroke in 68 aspirin-taking patients [180]. However, the A-842G polymorphism within the COX-1 promoter, has been consistently associated with higher AA-induced platelet aggregation in patients on aspirin, in two different studies [135-136], although the same polymorphism did not affect TXB2 generation. The clinical influence of this haplotype carried by 12% of the population [135] on the efficacy of low-dose aspirin remains currently unknown. In a small study on healthy subjects, the C50T polymorphism of the COX-1 gene was associated with approximately double values of pre- and post-aspirin urinary 11-dehydro-TXB2 in carriers of the 50T allele as compared to the 50C carriers, even though the effect of low-dose aspirin as percentage of inhibition of 11-dehydro-TXB2 excretion was similar for the two types of carriers [136]. In the same study, the COX-2 G-765C polymorphism was associated with a different sensitivity of 11-dehydro-TXB2 reduction by low-dose aspirin. The PlA1/A2 polymorphisms of the GPIIIa have been associated with a variable response to aspirin [127,182], although both studies evaluated the functional response to aspirin by functional assays, such as the PFA-100 [127] and the bleeding time [182]. Recent studies did not confirm such observations using AA-induced platelet aggregation or TXB2 measurements [136,140]. Interestingly, Lepantalo et al showed an influence of different polymorphisms on aspirin response, when evaluated by different methods: the COX-1 A-842G polymorphism influenced only the response detected by AA-induced aggregation, while the GPVI/C13254T polymorphism influenced the response as measured by PFA-100 [136]. These data further highlight the need for a mechanism-based evaluation of aspirin response (see above).
On the other hand, several lines of evidence suggest that the PlA1/A2 polymorphism of platelet GPIIb/IIIa may explain some of the variability in response to GPIIb/IIIa antagonists [183-185]. In a substudy of the OPUS-TIMI 16 (orbofiban in patients with unstable coronary syndromes-thrombolysis in myocardial infarction 16) trial, there was a significant interaction between treatment (placebo and orbofiban) and the PlA polymorphism for bleeding [183]. Thus, while orbofiban increased bleeding in noncarriers in a dose-dependent fashion, it did not increase bleeding events in PlA2 carriers [183]. There was no interaction between treatment and the PlA polymorphism for the primary efficacy end-point. However, in the patients receiving orbofiban, there was a higher risk of a primary event and myocardial infarction in PlA2 carriers compared with noncarriers [183].
Whether the influence of the PlA genotype in modifying the clinical response to an oral GPIIb/IIIa antagonist applies also to short-term intravenous administration of this class of antiplatelet drugs is as yet unknown, though ex vivo and in vitro studies demonstrate reduced inhibition by abciximab in platelets with PlA2 polymorphism [184].
A pharmacodynamic interaction potentially affecting the antiplatelet effect of aspirin is related to the two-step mechanism of COX-1 inactivation by the drug [5]. Thus, concomitant administration of NSAIDs, such as ibuprofen [186] and naproxen [187], may interfere with the irreversible inactivation of platelet COX-1 by low-dose aspirin. This is due to competition between NSAIDs and aspirin for a common docking site within the COX-1 channel (Arg120), which aspirin binds to with weak affinity prior to irreversible acetylation of Ser529 [5]. This pharmacodynamic interaction does not occur with NSAIDs endowed with some degree of COX-2 selectivity, including diclofenac [186].
Oral GPIIb/IIIa antagonists can activate platelets, at least in some individuals, acting as partial agonists, usually at concentrations below the therapeutic range [72-73,188]. GPIIb/IIIa is not a passive receptor, rather like all integrins it responds to ligand binding by activating the cell. Thus, fibrinogen binding leads to signals that further activate platelets and are essential for platelet aggregation. Several studies suggest that ligands designed to bind to the receptor and prevent platelet aggregation may paradoxically activate the receptor so that it can bind ligand or perhaps directly trigger some activating signals [72-73,188].
The same phenomenon may occur with TP antagonists. Whether agonistic activity of these compounds is differentially expressed as a function of variable concentration of the endogenous TP agonists remains to be established.
An additional factor contributing to pharmacodynamic variability is related to the extent and duration of platelet receptor blockade. In fact, a relatively constant level of high grade inhibition throughout the dosing interval appears to represent an essential requirement for translating the antiplatelet effect into reduced risk of vascular events. This is ensured by: (1) the covalent nature of drug-receptor interaction, resulting in permanent inactivation of a platelet protein that cannot be resynthesized during 24 hours, as in the case of aspirin and clopidogrel; (2) the constant plasma levels being maintained through continuous infusion of the antagonist, as in the case of abciximab, epifibatide, and tirofiban, or through an extended release formulation, as in the case of dipyridamole [5].
At variance with the requirements for antithrombotic efficacy, those associated with bleeding complications (most often from pre-existing upper gastrointestinal lesions) most likely involve transient, high-grade inhibition of the same receptors that may occur occasionally in a small percentage of the exposed individuals. This may explain the apparent paradox of two distinct classes of drugs endowed with same antiplatelet activity causing excess bleeding complications in the absence of any antithrombotic efficacy, i.e., traditional NSAIDs and oral GPIIb/IIIa antagonists.
A potential role for alternative sources of the endogenous platelet agonist that is targeted by the antiplatelet drug is exemplified by aspirin-insensitive sources of TXA2. Thus, aspirin-insensitive TXA2 biosynthesis has been described in patients with unstable angina [44,74,112] as well as in patients with post-stroke dementia [189]. Both COX-2 expression in inflammatory cells endowed with TX-synthase [190] and in newly formed platelets [10] could account for TXA2 biosynthesis in these settings.
Finally, disease-related mechanism(s) might cause acquired modifications of the structure of the drug target. Broad, nonspecific protein glycosylation is a major effect of poorly controlled type II diabetes mellitus. Watala et al have recently described a reduced incorporation of the aspirin acetyl residue into proteins of platelets from diabetic patients, increased platelet protein glycation as well as impaired response to aspirin assessed by whole blood-based assays [191]. Type II diabetes was significantly more represented in patients with lower-than-expected platelet inhibition by low-dose aspirin [136]. Similar findings have been reported for the response to clopidogrel [192]. Although several mechanisms might contribute to an altered platelet response to aspirin in diabetes, including higher COX-2 expression due to enhanced platelet turnover [193], a disease-related transient modification of the drug target is biologically plausible and deserves further investigation.
CONCLUSIONS
Variability of the response to antiplatelet drugs: implications for the practicing physician
A test of platelet function should not be performed simply because an assay is available. In fact, no test of platelet function is currently recommended to assess the antiplatelet effects of aspirin or clopidogrel in the individual patient [5,49]. At present, there is no convincing evidence for an association with clinical events conditioning cost-effective changes in antiplatelet therapy [194].
Increased awareness of the distinct factors potentially interfering with the desired antiplatelet effects of aspirin or clopidogrel, particularly avoidable drug interactions, may ultimately result in better patient management than requesting unnecessary, costly tests of platelet function.
As with any drug (antithrombotic, lipid-lowering, or antihypertensive) used to prevent atherothrombosis, treatment "failure" can occur with aspirin or clopidogrel, perhaps not surprisingly, given the multifactorial nature of atherothrombosis.
There is no scientific basis to change antiplatelet therapy in the face of a treatment "failure," as we cannot be sure whether a second vascular event occurring in the same patient will reflect the same pathophysiological event that led to the first. Moreover, we have no controlled evidence that changing therapy is a more effective strategy than maintaining an evidence-based therapy.
Variability of the response to antiplatelet drugs: implications for the clinical investigator
We have a pretty detailed molecular understanding of how aspirin and clopidogrel work in preventing arterial thrombosis [5]. Thus, new studies addressing the interindividual variability in response to these antiplatelet agents should rely upon mechanism-based biochemical end-points rather than platelet aggregation measurements [194-195]. Serum TXB2 and urinary 11-dehydro-TXB2 provide reliable information on the maximal biosynthetic capacity of circulating platelets ex vivo and on the actual rate of TXA2 biosynthesis in vivo, respectively [196]. These measurements have been used extensively to characterize the clinical pharmacology of aspirin as an antiplatelet drug [45,46]. In patients treated with low-dose aspirin, serum TXB2 levels reflect the adequacy of platelet COX-1 inhibition and its duration, while urinary TX-metabolite excretion provides a noninvasive, time-integrated index of aspirin-insensitive sources of TXA2 biosynthesis [5,190].
Similarly, because the extent of residual P2Y1-dependent platelet aggregation induced by ADP varies considerably among patients with congenital P2Y12 deficiency [197] or healthy subjects in whom P2Y12 function has been completely blocked in vitro by saturating concentrations of specific antagonists [194], ADP-induced platelet aggregation is not the most appropriate test to evaluate the interindividual variability in response to clopidogrel [194]. Thus, measurement of ADP-induced inhibition of adenylyl cyclase, which is uniquely mediated by P2Y12, would provide a mechanism-based biochemical end-point for assessing the adequacy of clopidogrel pharmacodynamics [194].
Reliable assessment of the adequacy and persistence of the expected pharmacodynamic effect of aspirin on platelet COX-1 would require a long-term, controlled study comparing aspirin to another antiplatelet drug in a sizeable group of stable patients requiring antiplatelet therapy. Clopidogrel would be an ideal comparator, because of remarkable similarities in the mechanism of action (permanent inactivation of a platelet protein), pharmacokinetics (short half-life of the active moiety), and dosing regimen (once daily). It should be noted, however, that while aspirin is currently used at doses that represent a 2.5- to 10-fold excess over the dose of 30 mg necessary and sufficient to fully inactivate platelet COX-1 activity on repeated daily dosing [45], clopidogrel is used routinely at or near the threshold dose that appears to cause a ceiling effect on ADP-induced platelet aggregation on repeated daily dosing [5].
Finally, given the size of the relative risk reduction (typically, 25%-30% in high-risk patients) associated with long-term antiplatelet prophylaxis, novel studies aiming to detect an attenuation or loss of this protective effect, as a function of specific causes of interindividual variability in response to aspirin or clopidogrel, should have both the sensitivity and specificity necessary to detect such a small "signal." None of the studies published so far meets these requirements, and estimates of relative risk of recurrent atherothrombotic events associated with aspirin [148] or clopidogrel [163] "resistance" are simply unrealistic.
Similarly, because of limitations with post hoc analyses and observational studies of drug interactions (e.g., between ibuprofen and aspirin or between atorvastatin and clopidogrel), well-designed clinical trials are needed to specifically evaluate the clinical readouts of these interactions.
Table 8 proposes an algorithm for how to investigate a less-than-expected response to aspirin. In this algorithm, the definition of true aspirin resistance is consistent with a widely accepted definition of resistance to chemotherapy (see Chapter The concept of drug resistance as exemplified by resistance to chemotherapy). The other mechanisms reflect the interindividual variability in response to aspirin based on pharmacokinetic and pharmacodynamic factors. A true aspirin resistance has not been demonstrated so far.
 |
TABLE 8. How to investigate mechanisms of aspirin response based on TXA2 formation both in vivo and in vitro |
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