Wednesday, July 16, 2014

Variable Platelet Response to Aspirin and Clopidogrel in Atherothrombotic Disease

Humans require rapidly responding, tightly regulated
hemostasis because of their closed high-pressure circulatory system.
Minor variation in response may predispose to
pathological bleeding or thrombosis. In the appropriate setting,
intervention with antiplatelet therapy stabilizes
the atherothrombotic phenotype, though with concomitant hemorrhagic
Populations with favorable risk–benefit ratios for
acetylsalicylic acid (aspirin) and clopidogrel therapy have nevertheless
been defined in major clinical trials. Treatment
benefit is established for secondary prevention of cardiovascular and
events, management of acute coronary syndromes, and
as an adjunct to percutaneous and surgical revascularization. There is
evidence, however, that not all individuals respond
comparably to antiplatelet drugs and hence the concept of aspirin and
clopidogrel “resistance” has arisen. The term is
misleading though because there are many determinants of failure to
to treatment.

Clinical Imperative for Consistent Platelet Inhibition

Consistent levels of platelet
inhibition are required to deliver effective therapy. Adverse
consequences of variable response
are particularly apparent when antiplatelet
drugs are used as an adjunct to coronary revascularization. During
coronary intervention (PCI), atherosclerotic
plaque is invariably disrupted, thrombosis occurs, and endothelial
healing is
delayed. Intensive periprocedural platelet
inhibition minimizes morbidity and mortality, whereas persistence of a
environment necessitates chronic antiplatelet
therapy. Failure to provide adequate platelet inhibition in all
can result in stent thrombosis, myocardial
infarction, and death.1,2 Platelet inhibition with aspirin at the time of coronary artery bypass graft surgery also provides benefit. Yet aggressive
therapy with aspirin and clopidogrel combined may increase perioperative bleeding in some cases.3 These contrasting clinical problems underlie the need for a tailored approach to therapy and illustrate the requirement for
consistent levels of platelet inhibition and a means to confirm individual response.

Platelet Adhesion, Activation, and Aggregation

Platelets adhere to sites of vascular
injury; however, endothelial disruption is not a prerequisite.
Atherosclerotic lesions
are associated with impaired endothelial
function and hence are susceptible to platelet and leukocyte adhesion.
Indeed, patients
with atherosclerosis have enhanced baseline
platelet activation, which is reflected by corresponding increases in
thromboxane (TX) metabolite excretion.4
Initially, platelets tether to the vessel wall via membrane integrins
and selectins. Subsequent rolling and firm adhesion
has been demonstrated by intravital microscopy
in experimental models of microvascular injury. Shear stress augments
receptor engagement and platelet activation
(so-called “outside-in” signaling). This in turn triggers release or
of soluble platelet activators such as TX,
adenosine diphosphate (ADP), and thrombin. A layer of activated
platelets forms
and attracts other platelets and leukocytes.
This is followed by either stable thrombus formation or rapid
Activated platelets release inflammatory and mitogenic proteins that promote leukocyte chemoattraction, vascular inflammation,
and further modify the endothelial phenotype.5 Indeed, there is growing evidence that platelet adhesion is involved in the earliest development of atherosclerotic lesions.
On activation, the most densely expressed platelet integrin αIIbβ3
(glycoprotein [GP] IIb/IIIa), undergoes conformational change, binds
soluble fibrinogen and von Willebrand factor, and facilitates
platelet aggregate formation. Notably, GP
IIb/IIIa gradually loses its binding capacity when platelets are
stimulated by ADP
alone. However, more potent agonists such as
thrombin induce persistent fibrinogen binding. The cycle of initiation,
and perpetuation of platelet activation creates
the platelet mass that forms a nidus for coagulation. Fibrin generation
release of secondary platelet agonists propagate
this process. Secondary agonists continuously activate integrins and
may be required to prevent disassembly of the
early platelet aggregate.6 Soluble ADP, TXA2, soluble CD40 ligand, and the product of growth arrest specific gene 6 are prominent in these paracrine signaling pathways.

Platelet Signaling and Thromboxane

Thromboxane and Its Platelet Receptor

Arachidonic acid is released from membrane phospholipids in response to most platelet agonists. Hydrolytic cleavage follows
activation of the enzyme phospholipase A2. On release, arachidonic acid is rapidly metabolized by prostaglandin (PG) H2 synthase, also known as cyclooxygenase (COX). Platelet COX converts arachidonic acid via PGG2 to PGH2. In turn, PGH2 is converted to an unstable, biologically active intermediate TXA2, by the downstream enzyme TX synthase. TXA2
activates the platelet via the cell membrane G-protein–coupled TX (TP)
receptor. Notably, activation of the TP receptor causes
irreversible platelet aggregation in part
through ADP release and subsequent platelet activation. Collagen,
thrombin, and
ADP all induce TXA2 synthesis and
release by platelets. Inhibitors of COX prevent platelet aggregation in
response to arachidonic acid. They
also inhibit second-wave aggregation in
response to weak platelet agonists such as epinephrine, low
concentration collagen,
and ADP, but not to potent agonists like

Acetylsalicylic Acid (Aspirin)

Aspirin and Cyclooxygenase-1

There are at least 2 isoforms of the COX enzyme, COX-1 and COX-2. Both are membrane-bound homodimeric molecules, although
mouse studies suggest that PGH2 synthase–1 and PGH2 synthase–2 may heterodimerise.7
COX-1 is constitutively expressed and regulates house-keeping cellular
functions such as vascular hemostasis, gastric mucosal
integrity, and renal blood flow. COX-2 is
largely absent from normal tissues; however, it is induced by cytokines
and growth
factors to regulate inflammation and cell
growth. COX-1 and COX-2 coexist in the vasculature and macrophages, and
is induced in atherosclerotic plaque. Both
isoforms are present in mature megakaryocytes, but mature platelets
express COX-1. In conditions of high platelet
turnover, a proportion of platelets may also express COX-2.8
Aspirin covalently modifies both
COX-1 and COX-2, although its affinity for COX-1 is 50 to 100 times that
for COX-2. Aspirin
acetylates a serine hydroxyl group at
position 529 in a narrow region of COX-1’s hydrophobic pocket and
thereby sterically
inhibits the passage of arachidonic acid to
the so-called active site of the enzyme. Platelets are anucleate
cytoplasts and
largely lack transcriptional activity.
Therefore, aspirin induces an irreversible defect in TX synthesis, which
persists for
the lifespan of the platelet (8 to 10 days).
Only 10% of the platelet pool is replenished daily, so despite the short
of aspirin (15 to 20 minutes), plain low-dose
aspirin can fully inhibit platelet COX-1 on repeat daily dosing.9
Inhibition of TX biosynthesis is understood to be the principal mode by
which aspirin prevents vascular thrombosis. This
apparently dose-independent effect on
platelet function contrasts with the clearly dose-dependent
aspirin-induced gastrointestinal

Platelet Capacity for Thromboxane Synthesis

Being anucleate, the platelet has
finite capacity to generate TX; however, in vivo biosynthesis varies
considerably. Capacity
for platelet TX synthesis in response to
physical and chemical stimuli is approximately 1000-fold greater than
plasma levels.11
Interestingly, TX biosynthesis in patients with stable coronary artery
disease (CAD) is similar to that of healthy individuals.
However, patients with greater
atherosclerotic burden, such as those with severe peripheral vascular
disease, have markedly
increased in vivo TX biosynthesis.12 Enhanced platelet activation and de novo TX biosynthesis by vascular cells and monocytes may contribute to what is largely
a COX-1–mediated process.13
Phasic increases in TX synthesis occur in subjects with unstable angina
and acute stroke, and occur during PCI, which presumably
reflects transiently increased platelet

Inhibition of Thromboxane Generation and Platelet Aggregation by Aspirin

Aspirin inhibits in vitro platelet aggregation triggered by exogenous arachidonic acid (metabolized to TXA2) and low-dose ADP, but not platelet response to stronger agonists such as thrombin. Capacity of platelets to generate TXA2 can be estimated by the measurement of its stable metabolite TXB2 in blood clotted at 37°C for 45 minutes. Aspirin inhibits serum TXB2 formation in a dose-dependent manner; however, 95% inhibition is the minimum required to achieve full platelet inhibition.
Indeed, the relationship between serum TXB2 level and suppression of platelet aggregation is nonlinear, and maximum inhibition of aggregation and prolongation of the
bleeding time may require 99% serum TXB2 inhibition (Figure). It is important, therefore, to note that minimal residual capacity to generate TX may be enough to sustain TX-dependent
platelet activation.11 Thus, although low concentrations of the TX analog U46619 or epinephrine alone may fail to activate aspirin-treated platelets,
inhibition is overcome when the 2 agonists are combined.16 Consistent with these findings, 99% inhibition of serum TXB2 was required to suppress platelet aggregation fully in a population with stable CAD.17

Relationship between TXB2 levels in serum (ELISA) and maximal arachidonic acid–induced platelet aggregation measured by light transmission aggregometry
in patients with stable coronary artery disease who were taking aspirin (75 mg). Serum TXB2 levels are represented on the X-axis in logarithmic scale. Reproduced from Maree et al17 with permission from the American College of Cardiology. Copyright 2005.

Aspirin Beyond Thromboxane Inhibition

Aspirin benefit in patients with
atherothrombosis may exceed that which is explained by platelet TX
inhibition alone. It has
been proposed that aspirin inhibits platelets
independent of COX acetylation, has anticoagulant properties,
suppresses vascular
inflammation, and enhances fibrinolysis.18 Indeed, aspirin does not acetylate proteins selectively and thus may modify platelet and erythrocyte membrane proteins and
thereby alter their conformation and decrease membrane fluidity.19
Very low doses of aspirin are effective, and prevention of clinical
events appears to be dose-independent. This finding supports
the theory that platelet COX suppression is
the primary mechanism by which benefit is derived. However, some aspirin
may occur downstream from platelet
inhibition. Proteins secreted by activated platelets adhere to the
vessel wall and promote
atherosclerosis and thrombosis.20 Low-dose aspirin downregulates soluble CD40 ligand, a platelet inflammatory mediator. Soluble CD40 ligand expression closely
correlates with urinary 11-dehydro-TXB2, a marker of in vivo platelet activation, and hence is mediated in part by platelet COX.21 Moreover, aspirin indirectly suppresses the peroxidase function of COX and thereby inhibits hydroperoxide generation and
vascular nitric oxide inactivation.22

Aspirin to Prevent Cardiovascular Disease

The role of aspirin in secondary
prevention of cardiovascular disease is well established. A recent
meta-analysis concluded
that aspirin therapy reduces the combined end
point of serious vascular events by one quarter, nonfatal myocardial
by one third, nonfatal stroke by one quarter,
and vascular mortality by one sixth in high-risk patients with vascular
disease.23 Furthermore, benefit accrued is proportional to absolute cardiovascular risk of the population studied, and reflects the
degree to which antithrombotic potential exceeds associated hemorrhagic risk.24 In the context of myocardial infarction, the number of vascular events avoided with aspirin therapy is approximately 100
times the number of major hemorrhagic complications.23
Absence of benefit when aspirin is used for primary prevention of
cardiovascular events presumably reflects the narrower
risk-to-benefit ratio in this setting. A
primary preventative role in higher risk subpopulations remains to be

Variable Platelet Response to Aspirin

Treatment Failure

Aspirin does not prevent the majority of cardiovascular events.26
This is not surprising because aspirin blocks only one of several
pathways of platelet activation and aggregation. In some
cases however, failure to respond to aspirin
may be caused by an inadequate primary pharmacological effect. This has
been referred to as “aspirin resistance.”
Depending on the population studied, the assay used, and the definition
prevalence of aspirin resistance is estimated
to be between 5% and 65%. Disparity in the reported frequency of
aspirin resistance
reflects the diverse nature of the
populations studied, the wide variety of tests used, and the arbitrary
cut-off values imposed
(Table 1).

TABLE 1. Prospective Studies of Variable Platelet Response to Aspirin and Clinical Events

Measures of Aspirin Response

Platelet Function Assays

Pharmacokinetic studies of aspirin
are not particularly informative. Aspirin is unstable and rapidly
hydrolyzed to salicylate,
which is an inactive and more stable product.
This conversion occurs initially in the gut, so plasma salicylate is a
measure of drug bioavailability. Aspirin
exerts much of its effect in the presystemic circulation before its
in the liver. Thus it may have had an
antiplatelet effect despite failure to detect aspirin in the systemic
Platelet response to aspirin can be determined with the use of a variety of assays (Table 1).
Most assays measure response to agonists in vitro. Weak agonists or low
concentration of strong agonists depend on platelet
TX generation to produce aggregation.
Similarly, platelet aggregation to exogenous arachidonic acid is
dependent on TX generation.
Incomplete inhibition of arachidonic
acid–induced platelet aggregation, or failure to prevent the
TX-dependent second wave
of platelet aggregation in response to weak
agonists, indicates incomplete platelet COX inhibition.5

Thromboxane Assays

The primary pharmacological effect
of aspirin, which is understood to prevent thrombosis, is almost
complete inactivation
of platelet COX-1 and consequent inhibition
of TX biosynthesis. Assays that detect platelet COX-1 function best
aspirin response.9,35 Ex vivo determination of TXB2 in serum reflects maximal capacity of activated platelets to synthesize TX via the COX-1 pathway and is a sensitive measure
of aspirin response. Levels of the urinary TX metabolite 11-dehydro TXB2 reflect in vivo TX biosynthesis. Though less specific for TX generated by platelet COX-1, this assay has been correlated
with clinical outcome.30 The relationship between serum TXB2 and 11-dehydro TXB2 in urine is nonlinear, and profound continuous inhibition of the former is necessary to suppress the urinary metabolite.11 This nonlinear response may reflect the contribution of extraplatelet (vascular and renal) TX sources or TX generated by
COX-2.4 Plasma levels of TXB2 are very low, and plasma assays generally lack the sensitivity and specificity to estimate the effect of aspirin. Of greater
concern is the fact that plasma TXB2 levels are readily confounded by inadvertent ex vivo platelet activation, which occurs readily during sample collection and

Platelet Aggregation Assays

Inhibition of platelet aggregation
is frequently used to measure antiplatelet response. Multiple agonists
of varying concentrations
have been used to assess aspirin response.
Different agonists, however, reflect COX-1–dependent platelet activation
to varying
degrees. Arachidonic acid is the substrate
for COX-1–dependent TX generation in platelets, so aggregation response
reflects platelet COX-1 activity. The
inhibitory effect of aspirin on arachidonic acid–induced platelet
aggregation, however,
is nonlinear and may reflect release of
secondary agonists that act in synergy with TX. This finding may also
explain the
modest correlation observed between serum TXB2 levels and arachidonic acid–induced platelet aggregation in patients with stable CAD (Figure). However, arachidonic acid–induced platelet activation ex vivo correlates with baseline circulating platelet activity, which
suggests that it does parallel in vivo platelet activation.36

Flow Cytometry

Surface expression of P-selectin
and activated GP IIb/IIIa receptor by flow cytometry may also be used to
determine platelet
inhibition by aspirin or clopidogrel.
However, these assays need to be performed using facilities that are not
widely available.

Semiautomated Point-of-Care Assays

Advent of newer antiplatelet drugs
and emergence of the concept of aspirin and clopidogrel resistance
coincide with the development
of semiautomated point-of-care platelet
function assays. Potential advantages of these systems include ease of
use and the
ability to rapidly assay platelet function in
whole blood. Devices employ different assays to determine platelet
These include response to combined platelet
agonists, agglutination to fibrinogen-coated beads, and adhesion and
under arterial flow conditions.37 Small studies have explored the utility of these assays to determine drug response (Tables 1 and 2).
However, correlation with clinical outcome in large prospective trials
is required, so these devices currently remain research
tools. Ultimately, a sensitive and specific,
yet rapid and inexpensive screening test that detects predisposition to
or bleeding, be it sensitive to aspirin,
thienopyridine, or GP IIb/IIIa antagonists, may prove clinically useful.

TABLE 2. Studies of Variable Platelet Response to Clopidogrel and Clinical Outcome

Determinants of Platelet Response to Aspirin

Clinical Relevance of Incomplete Platelet Inhibition by Aspirin

Significance of incomplete platelet
COX inhibition by aspirin has been evaluated in small clinical studies
with a variety
of aspirin sensitive assays. One study
followed high-risk cardiovascular patients on chronic aspirin therapy
and found that
subjects with high levels of urinary
11-dehydro TXB2 had a nearly 2-fold increased risk of myocardial infarction, cerebrovascular accident, or cardiovascular death. Although
urinary 11-dehydro TXB2 is aspirin-sensitive, aspirin-insensitive nonplatelet TX sources may contribute. Tissue COX-2 activity in atherosclerotic
plaque confounds the assay, which therefore does not solely reflect platelet response to aspirin.30 Another study detected an association between suboptimal inhibition of platelet aggregation by aspirin and increased risk
of death, myocardial infarction, or cerebrovascular accident.31 To date, however, evidence that links aspirin resistance and clinical outcome is weak, and insufficient numbers of patients
have been studied to draw definitive clinical conclusions (Table 1). Nevertheless, it is not unreasonable to expect a drug to have its intended pharmacological effect.

Mechanisms That Underlie Incomplete Platelet Response to Aspirin

Incomplete platelet response to
aspirin, so called aspirin resistance, likely reflects a composite of
processes. These can
broadly be divided into pharmacokinetic or
pharmacodynamic mechanisms. Pharmacokinetic determinants of an
incomplete aspirin
response include noncompliance, inadequate
dosing with various aspirin formulas, and interactions with other COX
Pharmacodynamic factors result from failure
to inhibit platelet COX despite adequate plasma levels. Enhanced
platelet turnover,
transcellular metabolism of PG precursors,
and genetic variants of COX-1 may obviate platelet COX inhibition.
which are non–enzymatic oxidation products of
arachidonic acid, may activate the platelet TP receptor, which thereby
circumvents COX inhibition.

Drug Compliance

When failure to respond to aspirin
is assessed, noncompliance with therapy must be assumed from the outset.
Regardless of
disease process, prognosis, or symptoms, many
patients routinely miss medication doses. A recent study of patients
from ischemic stroke showed that >10% were
noncompliant with aspirin.44
Clinical implications of aspirin noncompliance have been studied in
patients with prior myocardial infarction. Noncompliance,
detected by serum TX assay and on interview,
occurred in 16% of the population and was associated with 4-fold higher
of death, reinfarction, or rehospitalization
at 12 months of follow-up.45 Consistent with these findings, poorly compliant patients in the Physicians Health Study derived less benefit than compliant
patients (17% versus 51% reduction in myocardial infarction relative to placebo).46 Others have associated aspirin withdrawal for any reason with hospitalization for an acute coronary syndrome and specifically
late stent thrombosis.47

Aspirin Dose

Recommended drug doses are
generally based on population rather than individual dose-response
analysis, and considerable interindividual
variability occurs. Dose-dependent
variability in platelet response to aspirin has been determined with
various biochemical
assays. Indeed, there is evidence to suggest
that response to low-dose aspirin varies with anatomic distribution of
atherothrombosis.48 Secondary prevention studies in large populations, however, fail to show additional clinical benefit of higher aspirin doses.23
Indeed, evidence that gastrointestinal injury increases as aspirin dose
exceeds the dosage required for an antiplatelet effect,
as well as the increasing prescription of
combined antiplatelet therapy, may underlie the recent downward revision
of recommended
aspirin maintenance doses in patients with

Aspirin Formulation

Initial aspirin dose finding
studies were performed with plain aspirin, which is rapidly absorbed
from the stomach and small
intestine, has a bioavailability of about 50%
and achieves peak plasma levels in 30 to 40 minutes. It is then rapidly
in the liver and gut and excreted mainly in
urine. Platelet exposure and COX inhibition occur initially in the
portal circulation,
and as a result, antiplatelet activity has
occurred before aspirin enters the systemic circulation. As a
consequence of slow
platelet turnover, doses of plain aspirin as
low as 30 mg inhibit platelet TX formation in healthy subjects.9 Indeed, a sophisticated controlled release aspirin was developed to limit aspirin activity to the portal circulation and
thus spare systemic PGI2 biosynthesis.49
It is assumed that all low-dose
aspirins are created equal; however, there is evidence to the contrary.
“Aspirin” now encompasses
a myriad of formulations; various salts,
polymer-coated, controlled or rapid-release (compressed, soluble),
buffered and enteric-coated
preparations. Indeed, low-dose enteric-coated
aspirin preparations are increasingly prescribed in an attempt to
reduce gastrointestinal
side effects. However, differences in
formulation influence bioavailability of a drug that is now administered
in critically
low doses to individuals who respond
variably. Plain preparations release aspirin (a weak acid, pKa=3) into
the acidic environment
of the stomach where it is protected from
deacetylation, remains nonionized and lipid-soluble, and thus is rapidly
Enteric-coated preparations, however, deliver
aspirin into the almost neutral pH environment of the small intestine
absorption is delayed (peak plasma levels
occur in 2 to 4 hours), and bioavailability is reduced.17,35 Studies among healthy volunteers and patients with stable CAD indicate that some subjects treated with low-dose enteric-coated
aspirin fail to achieve minimum thresholds of effective platelet inhibition (>95% serum TXB2 inhibition).11
An inverse relationship between patient weight and level of platelet
inhibition was detected in both populations. Among healthy
volunteers with a suboptimal treatment
response, superior platelet inhibition was demonstrated with plain
aspirin. In patients
with stable CAD, younger heavier subjects and
those with a history of prior myocardial infarction were most likely to
evidence of incomplete COX inhibition.17,35

Pharmacodynamic Interaction With Nonsteroidal Antiinflammatory Drugs

Some nonsteroidal antiinflammatory
drugs may interact with aspirin and interfere with its antithrombotic
effect. Inhibitors
of COX-1 such as ibuprofen and naproxen share
a common docking site with aspirin and prevent acetylation of aspirin’s
serine residue within the hydrophobic pocket
of the enzyme.50 Indeed, use of high-dose nonselective nonsteroidal antiinflammatory drugs by patients who take aspirin for secondary prevention
has been linked to adverse cardiovascular events.51
Although medical professionals are increasingly aware of this potential
interaction, direct access to over-the-counter nonsteroidal
antiinflammatory drugs is difficult to

Enhanced Platelet Turnover, COX Regeneration, and Aspirin-Insensitive Eicosanoid Biosynthesis

Regeneration of COX-1 and COX-2 occurs in conditions associated with enhanced platelet turnover and may overcome the inhibitory
response to aspirin.8 Continued TX formation despite aspirin therapy was detected in patients after coronary artery bypass graft surgery.52
Addition of terbogrel, a combined TX synthase and TP receptor
inhibitor, further reduced TX generation. Platelet COX-2 was
also detected; however, selective inhibition
of COX-2 did not prevent TX generation, which points to incomplete
of the COX-1 pathway as the mechanism that
underlies persistent TX formation.
Mature platelets are anucleate and
therefore should not be able to regenerate COX. However, a recent study
introduced the
novel concept that platelets may splice
endogenous pre-mRNA in response to external signals. Thus, platelets may
have the
ability to translate mature mRNAs into
biologically active proteins and thereby regenerate COX-1 de novo in
response to cellular
activation.53 In a study of healthy volunteers, TXA2 biosynthesis in response to thrombin and fibrinogen recovered in a time-dependent manner and was abrogated by translational
inhibitors such as rapamycin.54 This finding may explain observed temporal trends toward loss of platelet inhibition despite chronic aspirin therapy.55
Mechanisms have been proposed in which platelet TX is generated despite COX-1 inhibition. Precursors of PGH2
generated by vascular tissue and metabolized by platelet TX synthase
may bypass platelet COX inhibition. Such transcellular
metabolism could occur at sites of
atherothrombosis or via platelet-leukocyte aggregates. More simply,
local release of vascular
TX or PG endoperoxides may activate the
platelet TP receptor and act in synergy with weak platelet agonists such
as epinephrine
or subthreshold levels of stronger agonists.16 A recent study detected arachidonic acid–induced platelet activation independent of COX activity that was partially mediated
by ADP.36 Finally, isoprostanes generated nonenzymatically by arachidonic acid oxidation are insensitive to aspirin, yet can partially
activate the TP receptor in a COX-independent manner.56

Enhanced Platelet Aggregability and Genetic Determinants of Aspirin Response

Variation in genes, which encode
enzymes or receptor targets of antiplatelet drugs, may modulate
pharmacological response.
In effect, genetic variation in any platelet
signaling component, whether directly targeted by a drug or not, has the
to influence antiplatelet response. COX-1
haplotype modulates platelet response to aspirin determined by in vitro
function assays.57
The precise mechanism involved, be it modulation of COX-1 enzyme
expression, biochemical function, interaction with pharmacological
agents, or an unrelated process, remains to
be established.

Thienopyridines (Ticlopidine and Clopidogrel)

Adenosine Diphosphate and Its Platelet Receptor

ADP is released actively from platelet-dense granules and passively by damaged erythrocytes and vascular cells. It activates
platelets via 2 surface-expressed G-protein–coupled receptors, P2Y1 and P2Y12.
Each acts through a distinct signaling cascade, and coordinated
activation of both is required to induce full platelet aggregation.
At low concentrations, ADP is a relatively
weak agonist whose activity is reinforced by platelet synthesis of TXA2, which causes granule secretion and secondary platelet aggregation.58 Soluble ADP, however, amplifies response to other platelet agonists, which makes it an important drug target. Signaling via
P2Y1 induces platelet shape change, reversible aggregation, and initial GP IIb/IIIa activation. Signaling through P2Y12 perpetuates GP IIb/IIIa activation, maintains its high affinity state, and appears critical for stable platelet aggregate
formation. Importantly, P2Y12 antagonism may not only prevent platelet aggregation but also promote disaggregation.6
Indeed, hereditary human ADP receptor deficiency results in a mild
hemorrhagic phenotype characterized by prolongation of
the bleeding time, impaired platelet
aggregation, and spreading and formation of unstable platelet
aggregates.59 Mouse models of P2Y1 or P2Y12 deficiency demonstrate impaired platelet aggregation to ADP, TX/endoperoxide analogs, and thrombin, particularly at low agonist
The role of the ADP receptor extends beyond platelet activation. Antagonism of P2Y12
may also attenuate CD40L and P-selectin expression, inhibit
platelet-leukocyte aggregate formation, and abrogate periprocedural
rise in C-reactive protein in patients who
undergo revascularization. Furthermore, both ADP receptors have also
been linked
to rapid activation of intravascular tissue
factor, the main initiator of physiological coagulation and a central
of pathological thrombosis.61 Thus, ADP antagonism may modulate coagulation and vascular inflammation in addition to platelet thrombosis.

Ticlopidine and Clopidogrel

Ticlopidine and clopidogrel block
the ADP pathway and suppress its amplifying effect on platelet response
to other agonists.
Both agents inhibit platelet aggregation
induced by ADP, TX analogs, collagen, low-dose thrombin, and shear, but
strong agonists
such as high-dose thrombin can overcome
inhibition. Both drugs prolong the bleeding time (1.5- to 2-fold longer
than baseline),
impair clot retraction, and render
thrombin-induced platelet aggregates susceptible to disaggregation.
Ticlopidine and clopidogrel are prodrugs that require oxidation by the hepatic cytochrome P450-1A enzyme system to acquire
activity (CYP2C19 for ticlopidine and CYP3A4 for clopidogrel), and in turn both drugs inhibit CYP2B6.62 Both are selective noncompetitive inhibitors of the P2Y12
receptor. The active metabolites of ticlopidine and clopidogrel induce a
permanent defect that involves a single platelet-signaling
pathway for the lifetime of the cell via
cumulative inhibition at low doses in a manner similar to the
pharmacodynamics of
aspirin. Recent evidence indicates that P2Y12
receptors exist in homo-oligomeric complexes associated with platelet
cell membrane lipid rafts and that the active metabolite
of clopidogrel partitions the receptor out of
the rafts to disrupt these oligomers, which thereby prevents signal
Ticlopidine is rapidly absorbed and
extensively metabolized, and onset of platelet inhibition (250 mg
twice-daily PO) occurs
within 24 to 48 hours with maximal effect at 3
to 5 days. Food enhances absorption, whereas antacids slow the process.
variability may reflect interindividual
variation in metabolic clearance.64 Poor tolerance of larger loading doses (>500 mg) precludes this approach to achieve earlier platelet inhibition.62 Diarrhea, nausea, and vomiting are common side effects (30% to 50%). Skin rash is also a frequent problem. Neutropenia is
reported in approximately 2% of recipients and has resulted in fatality.65 Because of these factors, ticlopidine use is now largely reserved for patients who are unable to tolerate clopidogrel.
Much of the clopidogrel dose
undergoes esterase deactivation, and therefore only a small portion is
metabolized to its active
moiety in the liver. After hepatic
metabolism, peak plasma metabolite concentrations occur at 1 hour, and
is unaffected by food.65
Ex vivo inhibition of platelet aggregation is dose- and time-dependent,
and, in the absence of loading, a maximal effect
(40% to 60% inhibition of ADP-induced
aggregation ex vivo) occurs after 3 to 5 days. Platelet function
recovers 3 to 5 days
after drug withdrawal. With a loading dose of
300 mg clopidogrel, maximum inhibition of platelet aggregation occurs
6 hours. However, full clinical benefit may
not be achieved for 24 hours. Maximum antiplatelet response is attained
2 hours after a loading dose of 600 mg, which
is generally well tolerated and appears optimal.66

Clinical Trials With Clopidogrel

Clear benefit from ADP receptor blockade has been established in the secondary prevention of cardiovascular disease, independent
of COX pathway inhibition.67
Furthermore, complementary mechanisms of action of aspirin and
clopidogrel translate into additive benefit in certain populations
(Table 3). An additive effect on bleeding time is also apparent. Particular consideration of risk versus benefit is therefore necessary
when prolonged therapy in lower risk patients is considered.

TABLE 3. Randomized Clinical Trials of Clopidogrel Use to Treat Vascular Disease

Clopidogrel Resistance or Nonresponse

Variable Response to Clopidogrel

The concept of an incomplete
clopidogrel response has arisen because multiple studies demonstrate
interindividual variability
in platelet response, and several small
studies have associated an incomplete treatment response with recurrent
events (Table 2).
Furthermore, incomplete inhibition of ADP-induced platelet aggregation
has been demonstrated in several studies of patients
after stent thrombosis has occurred. It is
unclear, however, if incomplete response to clopidogrel, aspirin, or
both agents
contributes to this complication (Table 2).
The definition of “clopidogrel
resistance” and assay specifications vary from study to study. The
predominant assay employed
is ADP-induced platelet aggregation measured
by light transmittance aggregometry. Nonstandardized methods, use of
doses of ADP, and determination of either
absolute difference, final, or maximum aggregation makes comparison of
study results
difficult. Furthermore, ADP-induced platelet
aggregation is mediated by both P2Y1 and P2Y12,
and the relative contribution of these receptors is known to vary
between individuals and thus may confound assays of clopidogrel
response. Flow cytometric evaluation of
P-selectin, activated GP IIb/IIIa expression, or phosphorylated
phosphoprotein are less widely available
alternative assays (Table 2).
In a manner similar to aspirin response, variable platelet response to
clopidogrel probably represents a composite of processes,
which include noncompliance, variable
absorption, metabolism and receptor sensitivity, and enhanced baseline
platelet reactivity.

Dosing, Compliance, and Platelet Reactivity

Studies indicate that the minimum
daily dose of clopidogrel required to achieve optimal platelet
inhibition is 60 mg, and
most patients receive 75 mg clopidogrel
daily. In contrast, daily dosing with 30 mg plain aspirin inhibits
platelet COX in
healthy volunteers. However, most patients
are maintained on a dose at least 2-fold greater. Thus, the cumulative
effect expected during repeat daily dosing
with clopidogrel may be undermined by even moderately poor compliance.
with clopidogrel therapy may be a frequent
problem and may be associated with significant morbidity and mortality.77
Others have detected a relationship between platelet aggregability at baseline and variability in clopidogrel response.78
Optimized dosing may partially attenuate this effect. However,
platelets in unstable high-risk patients are simultaneously
exposed to multiple agonists and lack
redundancy in their signaling pathways, which may enhance baseline
aggregability and
modulate drug response.79 On-treatment platelet reactivity and response to single or combined antiplatelet therapy have been evaluated with several
small studies demonstrating platelet reactivity, which correlated with cardiovascular morbidity.43
Healthy volunteers have variability in clopidogrel response and respond in a manner that is dose- and time-dependent.80 Patients post-PCI who take standard-dose clopidogrel (300 mg loading and 75 mg daily maintenance) also respond heterogeneously,
and time-dependence of response indicates inadequate clopidogrel loading.81 Indeed, response to single-bolus clopidogrel is dose-related, and more rapid platelet inhibition is achieved with a higher
loading dose (600 mg), which may be associated with improved outcomes in patients who undergo PCI.82
Clopidogrel response ex vivo assayed by platelet aggregometry forms a normal bell-curve distribution.83
However, unlike the profound antagonism detected in aspirin and GP
IIb/IIIa receptor blocker assays, standard-dose clopidogrel
(300 mg loading and 75 mg once-daily
maintenance) achieves maximally 40% to 50% inhibition of ADP-induced
platelet aggregation.
Addition of a clopidogrel bolus during
chronic clopidogrel therapy (75 mg per day) achieves additional platelet
and may indicate the need for higher
maintenance doses in some individuals.84 Indeed, the recent updated AHA guidelines for PCI provide for higher loading and maintenance doses in certain settings.


Pharmacodynamic heterogeneity
occurs with most drugs to varying degrees. Genotypic variation is known
to modulate platelet
reactivity and thus may influence clopidogrel
response. Several genetic mutations that modulate both P2Y12 function and expression have been identified.85,86 Furthermore, small studies of common sequence variation in the genes that encode the P2Y1 and P2Y12 receptor have detected an association with platelet response to ADP in vitro, predominantly at lower agonist concentrations.87 An effect on clopidogrel response, however, has not been discerned.88 Correlation between carriage of the human platelet alloantigen membrane GP IIIa variant (PLA2), and the antithrombotic effect of clopidogrel in patients with CAD has also been explored. However, data from these studies
are conflicting.89,90

Pharmacokinetic Variability

Marked interindividual variability
in clopidogrel pharmacokinetics has been confirmed after high loading
doses. Differences
in oral absorption, variable metabolism,
failure to clear the active metabolite, and differing ADP receptor
reactivity may
each contribute. Evidence supports variable
oral absorption as a prominent factor.91
Two of the more abundant CYP450
isozymes in the liver, CYP3A4 and CYP3A5, appear to metabolize
clopidogrel most rapidly and
are therefore credited with its
transformation to the active metabolite. Indeed, a correlation between
CYP3A4 activity and
platelet inhibition by clopidogrel has been
demonstrated.92 Existence of a clinically relevant interaction between clopidogrel and CYP3A4-metabolized statins is proposed, though this
association is contentious and requires further evaluation.92,93
Relative substrate concentration and binding site affinity determine
competitive inhibition. Clopidogrel is a reversible
competitive inhibitor of CYP3A4. Therefore,
potential for interaction exists particularly when lower clopidogrel
doses coincide
with higher statin doses. Furthermore, in
vitro, clopidogrel metabolism is inhibited by >90% when clopidogrel
and atorvastatin
are present at equimolar concentrations.94

Alternative Adenosine Diphosphate Inhibitors

Additional P2Y12 receptor antagonists are under development and may provide more predictable levels of ADP inhibition. Prasugrel (CS-747,
LY 640315) is an oral irreversible P2Y12
inhibitor that requires metabolism to acquire activity in a similar
manner to clopidogrel. It is a more potent drug and achieves
more rapid and consistent platelet
inhibition.95 Cangrelor (AR-C69931MX) and AZD6140 are reversible and direct P2Y12 inhibitors. AZD6140 is administered orally, and Cangrelor is administered parenterally.96,97 Rapid onset and offset of platelet inhibition with Cangrelor makes its use attractive in the acute setting and as an adjunct
to PCI. With the absence of a clinically correlated and desirable level of P2Y12 receptor inhibition and lessons learned regarding risk–benefit margins in thienopyridine trials, consistent rather than potent
platelet inhibition over shorter durations may be a prudent initial goal.


Aspirin and clopidogrel provide
significant benefit in patients with cardiovascular disease; however,
evidence of variable
platelet response has led to the concept of
aspirin and clopidogrel “resistance.” Rather than true “resistance” to
these antiplatelet
agents, there is a variable response to aspirin
and clopidogrel that reflects a variety of mechanisms.

No comments:

Post a Comment