NEJM —The Coagulopathy of Chronic Liver Disease

Armando Tripodi, Ph.D., and Pier Mannuccio Mannucci, M.D.

N Engl J Med 2011; 365:147-156July 14, 2011DOI: 10.1056/NEJMra1011170

Article

Chronic liver disease,
particularly in the end stage, is characterized by clinical bleeding and
decreased levels of most procoagulant factors, with the notable
exceptions of factor VIII and von Willebrand factor, which are elevated.1
Decreased levels of the procoagulants are, however, accompanied by
decreases in levels of such naturally occurring anticoagulants as
antithrombin and protein C.1 In physiologic conditions, the coagulation system is balanced by these two opposing drivers (Figure 1Figure
1Coagulation with Thrombin Generation and Inhibition.), but the
mechanistic significance of the parallel decrease of both procoagulants
and anticoagulants in patients with chronic liver disease escaped
attention for many years. As a consequence, chronic liver disease is
still considered the epitome of acquired bleeding disorders and is
featured as such in most hematology textbooks. The basic laboratory
tests of coagulation (i.e., measurement of the prothrombin time and
activated partial-thromboplastin time) have been used to assess the risk
of bleeding.

However, their results are poorly correlated with
the onset and duration of bleeding after liver biopsy or other
potentially hemorrhagic procedures.2-7
These test results are also poorly correlated with the occurrence of
gastrointestinal bleeding, the prototype of hemorrhagic events in
patients with end-stage liver disease.8,9
Additional evidence that argues against the clinical relevance of the
coagulation defects as detected by conventional laboratory tests in
determining the bleeding tendency in these patients can be drawn from
the natural history of liver transplantation. In the past, this major
surgical procedure required massive transfusions of plasma and other
blood products to correct the marked abnormalities on tests of
hemostasis (assessments of coagulation, platelets, and fibrinolysis)
observed both preoperatively and perioperatively. The need for
transfusions, however, has declined considerably over time — not because
of any substantial change in medication, but rather because of improved
surgical procedures.10
Finally and most important, randomized clinical trials involving
patients with chronic liver disease have shown that powerful
procoagulant agents, such as recombinant activated factor VII, fail to
control bleeding from the upper intestinal tract11,12 or bleeding during liver transplantation,13,14 even though the postinfusion prothrombin time is considerably shortened.11
In this review, we consider the evidence regarding the balance in the
hemostatic system (involving coagulation, platelets, and fibrinolysis).

The
aforementioned observations question the validity of the
prothrombin-time test and related tests for assessing the risk of
hemorrhage and guiding the transfusion of fresh-frozen plasma or use of
procoagulant agents in patients with chronic liver disease. An old dogma
is being dispelled in favor of the newly emerging concept that blood
coagulation in such patients is rebalanced, owing to the parallel
reduction of procoagulant and anticoagulant factors (Table 1Table
1Patterns of Prohemostatic and Antihemostatic Drivers in the Different
Phases of Hemostasis in Patients with Chronic Liver Disease.). Indeed,
studies show that plasma from patients with cirrhosis generates as much
thrombin (the final enzyme of coagulation) as plasma from healthy
subjects, provided that thrombin is measured by methods that reflect the
action of both procoagulants and anticoagulants.23,24
Thrombin generation in vivo and in vitro is down-regulated by
thrombomodulin, a transmembrane protein situated on vascular endothelial
cells that acts as the main physiologic activator of protein C (Figure 2Figure
2Protein C Activation by Thrombin on the Membrane of Endothelial Cells,
and the Balance of Antihemostatic and Prohemostatic Drivers in the
Different Phases of Hemostasis.).25
Plasma and reagents that are used to measure the prothrombin time do
not contain thrombomodulin. Accordingly, this test measures the amount
of thrombin generated in plasma as a function of the procoagulant
drivers, but not the thrombin inhibited by the anticoagulant drivers,
especially protein C, which is not fully activated in the absence of
thrombomodulin. This might explain why the prothrombin-time test and
related tests do not truly represent the balance of coagulation in vivo
and are inadequate for assessing the risk of hemorrhage in those
acquired conditions, such as the coagulopathies of liver disease and
neonatal coagulopathies, in which there is a restored balance due to the
concomitant decrease of procoagulants and anticoagulants.26

As
for end-stage liver disease, another problem is that the prothrombin
time expressed as the international normalized ratio (INR) is widely
used as a prognostic index to calculate the patient's Model for
End-Stage Liver Disease (MELD) score, which is used to prioritize
candidates for liver transplantation. However, the INR was devised and
validated to standardize across laboratories the prothrombin times in
patients receiving anticoagulation therapy with vitamin K antagonists
such as warfarin and its congeners. The INR cannot be used for patients
with chronic liver disease unless an alternative system of
standardization specifically developed for them is adopted.27
This alternative system involves using a different calibration based on
plasma from patients with chronic liver disease rather than plasma from
patients receiving vitamin K antagonists.

Together, the above
observations indicate that the bleeding tendency frequently observed in
patients with end-stage liver disease should be explained by mechanisms
other than hypocoagulability, such as those triggered by underlying
conditions that favor hemorrhage (i.e., hemodynamic alterations
subsequent to portal hypertension, endothelial dysfunction, bacterial
infections, and renal failure20,28-31) (Table 2Table
2Underlying Conditions That Explain the Bleeding Tendency in Patients
with Decompensated Chronic Liver Disease.). It should also be understood
that although rebalanced, the coagulation system in patients with
chronic liver disease is not as stable as that in healthy persons, who
have an excess of both procoagulants and anticoagulants. Therefore, the
relative deficiency of both coagulation-system drivers makes the balance
fragile in patients with liver disease and may tip it toward hemorrhage
or thrombosis, depending on the prevailing circumstantial risk factors (Figure 2C).

Under
normal conditions, platelets have a dual function. They adhere to
damaged vessel walls through an interaction with the multimeric adhesive
protein von Willebrand factor, thus promoting aggregation and
ultimately the formation of the primary hemostatic plug. Platelets also
support thrombin generation by assembling activated coagulation factors
on their surfaces. Thrombocytopenia, a typical feature of chronic liver
disease,17 may therefore be another cause of bleeding (Table 1).
However, very high levels of von Willebrand factor, a common finding in
patients with chronic liver disease, may restore platelet adhesion to
the subendothelium at sites of vascular injury (Table 1), as shown by in vitro experiments carried out under flow conditions mimicking those that occur in vivo.15
Levels of ADAMTS 13, a naturally occurring plasma metalloprotease that
limits in vivo the functions of von Willebrand factor on platelets, are
reduced in patients with cirrhosis16; this may further contribute to the restoration of platelet function (Table 1). Finally, a platelet count as low as 60×10⁹
per liter in platelet-rich plasma from patients with cirrhosis is
usually sufficient to preserve thrombin generation at a level equivalent
to the lower limit of the normal range in healthy subjects.24

Fibrinolysis
is a highly regulated mechanism that, on deposition of fibrin within
the vascular system, converts the proenzyme plasminogen into the active
enzyme plasmin, which in turn degrades fibrin (Figure 3Figure
3Fibrinolysis Activation and Inhibition.). Under normal conditions,
plasminogen-to-plasmin conversion is regulated by such activators as
tissue plasminogen activator (t-PA), urokinase plasminogen activator,
and activated factor XII. These activators (profibrinolytic drivers) are
opposed by such antiactivators as t-PA inhibitors (mainly, plasminogen
activator inhibitor [PAI]), plasmin inhibitor, and thrombin-activatable
fibrinolysis inhibitor (TAFI), which cumulatively act as
antifibrinolytic drivers. Any perturbation of this balance may result in
hyperfibrinolysis, which increases the risk of hemorrhage, or
hypofibrinolysis, which increases the risk of thrombosis.

Plasma
hyperfibrinolysis has been reported in patients with chronic liver
disease, but its mechanistic role in bleeding is still debated.20
Uncertainty rests mainly on the lack of appropriate laboratory tests
for its evaluation, because most observations are based on the
measurement of the individual components of the system rather than on
the overall activity stemming from the action of both profibrinolytic
and antifibrinolytic drivers. Cirrhosis has been variably associated
with laboratory changes favoring hyperfibrinolysis, such as increased
levels of t-PA and reduced levels of plasmin inhibitor and TAFI, but
also with changes favoring hypofibrinolysis, such as reduced levels of
plasminogen and increased levels of PAI (Table 1).
Hence, although contrasting results have been reported, the balance of
fibrinolysis is probably restored in patients with liver disease by the
parallel changes in profibrinolytic and antifibrinolytic drivers.21,22

Overall,
the aforementioned observations suggest that patients with chronic
liver disease are not naturally “autoanticoagulated,” as previously
believed. This concept is reinforced by clinical evidence indicating
that they are not protected from32,33 and may even be at increased risk for34 thrombosis, particularly but not exclusively in the portal venous system,35,36 and especially in the presence of inherited prothrombotic mutations.37

Laboratory signs of a procoagulant imbalance, which was not evident in the previous studies,23,24 have been reported in association with chronic liver disease.18,19 As noted above, thrombin generation in vivo and in vitro is down-regulated by thrombomodulin (Figure 2),25
which effectively quenches thrombin generation when added to plasma
from healthy subjects but is much less effective when added to plasma
from patients with chronic liver disease.18
This indicates that in such patients, the plasma is partially resistant
to anticoagulation mediated by thrombomodulin. This resistance is
evident only when the results of thrombin-generation tests are expressed
as the ratio of thrombin activity in the presence of thrombomodulin to
thrombin activity in its absence. The resistance is probably the result
of two alterations typically found in patients with chronic liver
disease18,19: markedly increased plasma levels of factor VIII (one of the most potent drivers of thrombin generation38)
and the concomitant decrease in levels of protein C (one of the most
potent anticoagulant drivers in quenching thrombin generation25).
Although protein C is reduced owing to the impaired synthetic capacity
of the liver, the increased levels of factor VIII are likely to be
explained by decreased clearance of this moiety from plasma,39
mediated by two mechanisms, one involving von Willebrand factor, and
the other the low-density lipoprotein receptor–related protein.39 Von Willebrand factor binds factor VIII in vivo and protects it from cleavage by plasma proteases and from premature clearance.40 High plasma levels of von Willebrand factor in patients with cirrhosis15
may be mechanistically involved in maintaining high plasma levels of
factor VIII through the stabilization of its procoagulant activity. The
low-density lipoprotein receptor–related protein, a multifunctional
ligand that mediates the cellular uptake and subsequent degradation of
factor VIII,41 is inadequately expressed in patients with cirrhosis39 and, in conjunction with high levels of von Willebrand factor, may help sustain the high plasma levels of factor VIII.

The
procoagulant imbalance associated with chronic liver disease can be
detected by measuring thrombin generation in plasma in the presence and
absence of thrombomodulin.18 An alternative method uses a snake-venom extract (Protac, Pentapharm)19
that acts as a surrogate activator of protein C in a manner similar to
that of thrombomodulin. Whereas the results of the first test are
expressed as the ratio of the thrombin concentration generated in the
presence of thrombomodulin to the concentration generated in its
absence,18
the results of the second test are expressed as the percentage of
extract-induced coagulation inhibition, measured as the amount of
thrombin generated in the presence versus the absence of the venom
extract.19
By definition, the higher the ratio or the lower the percentage of
extract-induced coagulation inhibition, the greater the degree of
procoagulant imbalance. As detected by these assays in the context of
chronic liver disease, the procoagulant imbalance is negatively
correlated with levels of plasma protein C and positively correlated
with levels of factor VIII.18,19 Furthermore, the degree of imbalance increases with the severity of cirrhosis as assessed by the Child–Pugh score.18,19
Whether the procoagulant imbalance detected in the laboratory as
thrombomodulin resistance is a risk factor for thrombosis in patients
with chronic liver disease remains to be established by prospective
studies. It must be recognized that although thrombin-generation tests
mimic the conditions operating in vivo much more closely than do
conventional tests, they remain artificial because they use
platelet-free plasma and the amount of thrombomodulin added in vitro is
chosen arbitrarily, not on the basis of the density of the protein on
endothelial cells.

The in vitro procoagulant imbalance associated with chronic liver disease18,19
may have clinical implications. First, it calls into question the
unrestricted use of plasma infusion to correct the results of
conventional coagulation tests in patients undergoing invasive
procedures. This is still a common practice, despite a lack of evidence
from controlled, randomized trials and the recent guidelines of the
American Association for the Study of Liver Diseases, which warn against
the indiscriminate use of plasma therapy before liver biopsy.42 Second, the procoagulant imbalance18,19
may help explain mechanistically why these patients are not protected
from clinical events such as peripheral-vein thrombosis, portal-vein
thrombosis, atherothrombosis, and the progression of liver fibrosis. In
the next sections, these potential clinical implications are discussed.

Retrospective
studies showed that patients with chronic liver disease are not
protected from venous thromboembolism (deep-vein thrombosis and
pulmonary embolism).32,33 Recently, a nationwide, population-based case–control study34
involving 99,444 patients with venous thromboembolism and 496,872
controls showed that patients with liver disease had an increased
relative risk of venous thromboembolism, with the risk being greater for
deep-vein thrombosis than for pulmonary embolism and for cirrhosis than
for noncirrhosis liver disease. However, other studies have shown a low
prevalence of venous thromboembolism among patients with chronic liver
disease.43,44
The retrospective design of all these studies makes it difficult to
assess the true risk of venous thromboembolism among such patients. It
is clear, however, that patients with chronic liver disease are not
autoanticoagulated and may eventually have clinical manifestations of
thromboembolism, even though the abnormal results of conventional
coagulation tests would suggest the opposite.

Thrombosis in
patients with chronic liver disease might become an emerging issue owing
to their increasing life expectancy and changing lifestyle, which
expose them much more than in the past to such circumstantial risk
factors as tumors, surgery, obesity, prolonged hospitalization, and
inadequate physical activity. Thus, the logical consequence is that
patients with chronic liver disease who have peripheral-vein thrombosis
should be treated with anticoagulants just as any other patient would;
it is important to note that the long-term safety of this approach has
not been studied. Furthermore, the in vitro procoagulant imbalance
associated with chronic liver disease, confirmed by many independent
studies,45-47
suggests that these patients are eligible for antithrombotic
prophylaxis when exposed to such risky situations as major surgery and
prolonged immobilization. This notion contradicts current clinical
practice, whereby patients with cirrhosis often receive no or suboptimal
prophylaxis because of the perceived risk of bleeding.48 Clinical studies are needed to determine the appropriate care of these patients.

Even
though it is not firmly established that patients with chronic liver
disease have an increased risk of arterial thrombosis (i.e., coronary
artery disease and stroke), they are not free from these and other
clinical manifestations of atherothrombosis.49
Furthermore, the occurrence of hepatic-artery occlusion after liver
transplantation worsens the prognosis for these patients. Therefore,
early detection of this complication is important.50
Whether aspirin or other antiplatelet agents are indicated in the
primary prophylaxis of this complication warrants evaluation in clinical
trials.

The prevalence of portal-vein thrombosis
in patients with cirrhosis increases with the severity of the disease:
approximately 1% among patients with compensated cirrhosis35 but 8 to 25% among those who are candidates for liver transplantation.36 Because not only reduced flow velocity51 but also procoagulant imbalance and vessel-wall abnormalities (Virchow's triad)52
are mechanistic factors in this complication, antithrombotic therapy
(low-molecular-weight heparin or vitamin K antagonists) is commonly
used.53,54 This approach is relatively safe,36 but varices may need to be treated (with vasoactive drugs or endoscopic ligation)28
before patients start taking anticoagulants. Portal-vein thrombosis
worsens the post-transplantation prognosis, so primary prevention with
low-molecular-weight heparin or vitamin K antagonists should be
considered in patients awaiting liver transplantation. Randomized
clinical trials to test the efficacy of these drugs are under way.55
However, because of the mechanistic role played by low levels of
protein C in the balance of coagulation in patients with chronic liver
disease,18,19
vitamin K antagonists are perhaps not the ideal drugs. Protein C is a
vitamin K–dependent protein, and treatment with vitamin K antagonists
might therefore further reduce levels of this naturally occurring
anticoagulant in patients with end-stage liver disease, increasing the
risk of thrombosis.

The newer direct thrombin inhibitors and inhibitors of activated factor X56
(e.g., dabigatran, rivaroxaban, and apixaban) may be attractive
alternatives to vitamin K antagonists because they do not reduce protein
C levels. Moreover, they do not require regular laboratory monitoring
to adjust the dosage, whereas vitamin K antagonists require monitoring
with the use of the INR, the validity of which has been questioned in
patients with chronic liver disease.57
Other potential advantages of these new drugs over low-molecular-weight
heparin are their oral route of administration and their mechanism of
action, which is independent of antithrombin (low in these patients).
However, specially designed clinical trials are needed because patients
with chronic liver disease are usually excluded from the randomized
clinical trials of these drugs.58-60

Another
consequence of procoagulant imbalance in chronic liver disease pertains
to liver fibrosis and its progression. Two hypotheses are currently
considered for the pathogenesis of this condition. Both involve
coagulation, and they might be synergistic. One hypothesis centers on
the role of microemboli. Obliterative lesions in the portal and hepatic
veins frequently occur in patients with cirrhosis, owing to the
formation of microthrombi that lead to tissue ischemia, cell death, and
fibrosis through parenchymal extinction.61

Another
hypothesis suggests that coagulation activation within the liver's
vascular system may play a role in the development and progression of
the fibrotic process. Thrombin, besides being a potent procoagulant, has
many cellular effects that are mediated by a family of widely expressed
G-protein–coupled receptors called protease-activated receptors (PARs).62
Thrombin signaling through PARs expressed on hepatic stellate cells,
which are responsible for tissue repair, might therefore play a crucial
role in the mechanisms and progression of fibrosis.63
The degree of thrombin-receptor expression is associated with the
severity of liver disease, and it has also been observed that humans64 and mice65
with hypercoagulability due to a gain-of-function mutation in the
factor V gene (factor V Leiden) have an accelerated progression of liver
fibrosis. PAR1 antagonists can provide protection against experimental
liver fibrosis in rodents,63 and anticoagulant drugs slow fibrosis progression in mice.65
Furthermore, low-molecular-weight heparin prevents hepatic fibrogenesis
caused by the injection of carbon tetrachloride in rodents.66 These observations are consistent with the hypothesis that thrombin generation and fibrosis are directly associated.65
Accordingly, a controlled, randomized clinical trial is being carried
out to investigate whether vitamin K antagonists can influence the
progression of fibrosis in patients with hepatitis C (ClinicalTrials.gov
number, NCT00180674).

Undoubtedly, patients
with end-stage liver disease have prominent bleeding symptoms,
particularly in the gastrointestinal tract. Yet evaluation of this
bleeding tendency solely on the basis of abnormal levels of the
conventional coagulation biomarkers should be reconsidered. When
patients are assessed by means of global tests such as the
thrombin-generation test, the results do not show hypocoagulability.18,19,45-47
Thus, the main culprits for the bleeding tendency observed in patients
with end-stage liver disease should be sought among underlying
conditions that favor hemorrhage, such as portal hypertension,
endothelial dysfunction, bacterial infection, and renal failure20,28-31 (Table 2).

On
the other hand, the restored balance of hemostasis afforded by the
concomitant reduction of procoagulant and anticoagulant factors,
together with increased levels of factor VIII (Table 1),
might explain why patients with chronic liver disease are not protected
from arterial and venous thrombosis. This apparent clinical paradox may
be explained by the findings that these patients have a procoagulant
imbalance in vitro owing to resistance to thrombomodulin18,19,45-47
and that their thrombocytopenia is compensated for by increased plasma
levels of the adhesive protein von Willebrand factor. Another dogma is
being challenged by the finding that platelet activation plays a crucial
role in the immune-mediated progression of liver disease in an animal
model of viral hepatitis.67

In
conclusion, the reassessment of hemostasis in patients with chronic
liver disease challenges the dogma that the major coagulopathy in these
patients leads consistently to bleeding. Other changes that accompany
chronic liver disease may restore the balance of anticoagulant and
procoagulant effects (Figure 2C).
In certain circumstances, the risk of thrombotic events may be greater
than the risk of hemorrhage. We speculate that drugs that are often
regarded as contraindicated in patients with chronic liver disease may
instead prove beneficial and should be tested in appropriate clinical
trials.

HAS-BLED bleeding risk score

Clinical feature /   Points

H Hypertension -( systolic blood pressure >160 mmHg) (Points: 1 )

A Abnormal renal function ( defined as the presence of chronic dialysis or renal transplantation                   or serum creatinine 200µmol/L (>~2.3 mg/dL)) (Points: 1 )     Abnormal liver function ( defined as chronic hepatic disease (eg. cirrhosis) or biochemical               evidence of significant hepatic derangement (eg. bilirubin >2x upper limit of normal, in association               with AST/ALT/ALP >3x upper limit normal) (Points: 1 )

S Stroke (Previous history of stroke) (Points: 1 )

B Bleeding (Major bleeding history (anemia or predisposition to bleeding))  (Points: 1 )

L Labile INRs (refers to unstable/high INRs or poor time in therapeutic range(eg<60%))(Points: 1)

E Elderly (age >/= 65) (Points: 1 )

D Drug Therapy (concomitant therapy such as antiplatelet agents, NSAID's)   (Points: 1 )     Alcohol intake (consuming 8 or more alcoholic drinks per week)   (Points: 1)

Background

HAS-BLED: acronym of the major factors associated with bleeding risk in patients with atrial fibrillation receiving oral anticoagulation.  Clinical Characteristics Composing the HAS-BLED Bleeding Risk Score Letter  Clinical  Characteristic Points Awarded H Hypertension 1 A Abnormal renal and liver function (1 point each) 1 or 2 S Stroke 1 B Bleeding 1 L Labile INRs 1 E Elderly 1 D Drugs or alcohol (1 point each) 1 or 2 Maximum possible score is 9 The risk of major bleeding within one year in atrial fibrillation patients enrolled in the Euro Heart Survey. HAS-BLED, acronym: Hypertension [uncontrolled, >160 mmHg systolic), Abnormal renal/liver function, Stroke, Bleeding history or predisposition [anemia], Labile INR [i.e. therapeutic time in range <60%], Elderly (>65) and Drugs/alcohol concomitantly [antiplatelet agents, non-steroidal anti-inflammatory drugs] [Maximum score 9]. HAS-BLED score n Bleeds, n Bleeds/100 patients* 0 798 9 1.13 1 1286 13 1.02 2 744 14 1.88 3 187 7 3.74 4 46 4 8.70 5 8 1 12.50 Any score 3071 48 1.56

Bleeding Complications With Dual Antiplatelet Therapy Among Patients With Stable Vascular Disease or Risk Factors for Vascular Disease

Background—
Uncertainty exists about the frequency, correlates, and clinical
significance of bleeding with dual antiplatelet therapy
(DAPT), particularly over an extended period in a
stable population. We sought to determine the frequency and time course
of bleeding with DAPT in patients with
established vascular disease or risk factors only; identify correlates
of bleeding;
and determine whether bleeding is associated
with mortality.

Methods and Results—
We analyzed 15 603 patients enrolled in the Clopidogrel for High
Atherothrombotic Risk and Ischemic Stabilization, Management,
and Avoidance (CHARISMA) trial, a double-blind,
placebo-controlled, randomized trial comparing long-term clopidogrel 75
mg/d
versus placebo; all patients received aspirin
(75 to 162 mg) daily. Patients had either established stable vascular
disease
or multiple risk factors for vascular disease
without established disease. Median follow-up was 28 months. Bleeding
was assessed
with the use of the Global Utilization of
Streptokinase and t-PA for Occluded Coronary Arteries (GUSTO) criteria.
Severe bleeding
occurred in 1.7% of the clopidogrel group versus
1.3% on placebo (P=0.087); moderate bleeding occurred in 2.1% versus 1.3%, respectively (P<0.001).
The risk of bleeding was greatest the first year. Patients without
moderate or severe bleeding during the first year
were no more likely than placebo-treated
patients to have bleeding thereafter. The frequency of bleeding was
similar in patients
with established disease and risk factors only.
In multivariable analysis, the relationship between moderate bleeding
and
all-cause mortality was strong (hazard ratio,
2.55; 95% confidence interval, 1.71 to 3.80; P<0.0001), along with myocardial infarction (hazard ratio, 2.92; 95% confidence interval, 2.04 to 4.18; P<0.0001) and stroke (hazard ratio, 4.20; 95% confidence interval, 3.05 to 5.77; P<0.0001).

Conclusions— In CHARISMA, there was an increased risk of bleeding with long-term clopidogrel. The incremental risk of bleeding was greatest
in the first year and similar thereafter. Moderate bleeding was strongly associated with mortality.

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,
pharmacological
intervention with antiplatelet therapy stabilizes
the atherothrombotic phenotype, though with concomitant hemorrhagic
risk.
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
cerebrovascular
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
respond
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
percutaneous
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
prothrombotic
environment necessitates chronic antiplatelet
therapy. Failure to provide adequate platelet inhibition in all
individuals
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
urinary
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
adhesion
receptor engagement and platelet activation
(so-called “outside-in” signaling). This in turn triggers release or
generation
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
resolution.

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 α_IIbβ3
(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,
propagation,
and perpetuation of platelet activation creates
the platelet mass that forms a nidus for coagulation. Fibrin generation
and
release of secondary platelet agonists propagate
this process. Secondary agonists continuously activate integrins and
importantly
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
thrombin.

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
expression
is induced in atherosclerotic plaque. Both
isoforms are present in mature megakaryocytes, but mature platelets
predominantly
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
half-life
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
toxicity.¹⁰

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
endogenous
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
activation.^14,15

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
benefit
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
infarction
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
established.²⁵

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
sometimes
been referred to as “aspirin resistance.”
Depending on the population studied, the assay used, and the definition
applied,
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
poor
measure of drug bioavailability. Aspirin
exerts much of its effect in the presystemic circulation before its
inactivation
in the liver. Thus it may have had an
antiplatelet effect despite failure to detect aspirin in the systemic
circulation.⁵

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
represent
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
processing.

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
closely
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
function.
These include response to combined platelet
agonists, agglutination to fibrinogen-coated beads, and adhesion and
aggregation
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
thrombosis
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
inhibitors.
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.
Isoprostanes,
which are non–enzymatic oxidation products of
arachidonic acid, may activate the platelet TP receptor, which thereby
directly
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
recovering
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
incidence
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
CAD.

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
inactivated
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
absorbed.
Enteric-coated preparations, however, deliver
aspirin into the almost neutral pH environment of the small intestine
where
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
have
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
target
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
regulate.

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
inhibition
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
potential
to influence antiplatelet response. COX-1
haplotype modulates platelet response to aspirin determined by in vitro
platelet
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
concentrations.⁶⁰

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
component
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
transduction.⁶³

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.
Pharmacokinetic
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
bioavailability
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
within
6 hours. However, full clinical benefit may
not be achieved for 24 hours. Maximum antiplatelet response is attained
approximately
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
cardiovascular
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
varying
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
vasodilator-stimulated
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
irreversible
effect expected during repeat daily dosing
with clopidogrel may be undermined by even moderately poor compliance.
Noncompliance
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
inhibition
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.

Pharmacogenetics

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.

Conclusion

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.