Laboratory Monitoring of Warfarin Therapy - Pathology

Laboratory Monitoring
of Warfarin Therapy
David Williams, M.D., Ph.D.
Roger S. Riley, M.D., Ph.D.
Ann Tidwell, M.T. (ASCP) SH

Warfarin Monitoring
Crystalline warfarin sodium (Coumadin,
Panwarfin, Sofarin, Coufarin, Athrombin-K) is
the most widely used oral anticoagulant in
the world. Warfarin interferes with the hepatic
synthesis of the vitamin-K dependent
coagulation factors by interfering with the
vitamin K cycle. Laboratory monitoring of
warfarin therapy is mandatory, since the
agent has a relatively narrow therapeutic
range. The therapeutic efficacy is of warfarin
is reduced with an inadequate dose, while there
is a serious risk of bleeding with an excess
amount of the drug. In addition, the pharmacokinetics
of warfarin are significantly influenced
by the diet, other medications, and
many other factors. During the past two decades,
several developments in laboratory science
have helped the clinician to ensure a
proper dosage intensity that decreases the
risk of bleeding while maintaining therapeutic
efficacy. These developments include improved
laboratory reagents and instrumentation,
as well as a standardized method for reporting
the prothrombin time (i.e., International
Normalized Ratio, INR). However, a comprehensive
system of quality assurance is still
necessary for optimal patient care.
It is imparative that physicians monitoring
warfarinized patients must understand the biological
effect and pharmacokinetics of warfarin,
its interaction with other medications, and the
factors affecting the prothrombin time/INR assay,
including the recently discovered impact of
the CYP2C9 and VKORC1 genetic polymorphisms.
The physician must also work with
other members of the patient care team to ensure
that patient identification procedures are
followed and the proper type and amount of
specimen is submitted for laboratory testing.
Warfarin is a synthetic derivative of the
naturally occurring anti-coagulant dicumarol.
The anti-coagulant properties of dicumarol
were first observed in cattle that suffered a
hemorrhagic disorder after being fed spoiled
sweet clover hay during the 1920s. Karl Link
identified the causative agent by 1939 and
later developed warfarin as a rat poison before
it was used in humans in the 1950s. However
the anticoagulant mechanism of warfarin
was not elucidated until twenty years
later.
Warfarin is a specific inhibitor of the vitamin
K epoxide reductase necessary for the
regeneration of vitamin K from vitamin K
2,3-epoxide in vivo (Fig. 1). Vitamin K acts as
a cofactor for !-glutamyl carboxylase, which
carboxylates specific glutamic acid residues
in vitamin K dependent proteins. The vitamin
K dependent proteins include coagulation
factors V, VII, IX, and X as well as protein C
and protein S. This post-translational modification
forms !-carboxyglutamic acid residues
that chelate metal ions and allow the proteins
to bind specific cofactors on phospholipid
surfaces necessary for normal coagulation.
The carboxylation reaction generates vitamin
K 2,3-epoxide, which must be converted back
to vitamin K by an epoxide reductase to
maintain stores. Inhibition of vitamin K epoxide
reductase reduces the availability of vitamin
K, which leads to a decrease in the active
forms of vitamin K dependent coagulation
factors and ultimately anti-coagulation.
Warfarin is currently the only FDA approved
orally available anti-coagulant medication.
It is rapidly absorbed from the gastrointestinal
tract, reaching maximum concentration
90 minutes after administration, with
a half-life of 36 to 42 hours. However, the
pharmacokinetics of warfarin can by highly
variable. It circulates bound to albumin and is
metabolized in the liver. Genetic variation in
a cytochrome P450 enzyme alters the dose-
Warfarin Structure, Metabolism, Pharmacokinetics 2

Warfarin Monitoring
O
OH
O
O
CH 3
Fig. 1. The biochemistry of warfarin.
(A) The molecular structure of warfarin.
(B) A simplied schematic of the
vitamin K cycle, showing the effect of
warfarin on the synthesis of vitamin-K
dependent coagulation factors. In the
cycle, vitamin K1 or K2 are reduced to
their hydroquinone forms (KH2). A further
stepwise oxidation of KH2 to vitamin
K epoxide (KO) occurs, during
which descarboxyprothrombin
(descarboxy-II) is converted to
prothrombin by carboyxlation of glutamate
residues (Glu) to g-
carboxyglutamate (Gla). Enzymatic
reduction of the epoxide requires reduced
nicotinamide adenine dinucleotide
(NADH) as a cofactor and is sensitive
to warfarin (Stop sign). The R
sidechain on the vitamin K molecule is
a 20-carbon phytyl in vitamin K1 and a
5- to 65-carbon prenyl side chain in
vitamin K2.
response relationship as well as a number of
environmental, dietary, and disease related
factors. A long list of commonly prescribed
medications can either enhance or interfere
with warfarin anti-coagulation by various
mechanisms. For example, amiodarone inhibits
clearance via hepatic metabolism and
enhances anti-coagulation while carabamezepine,
barbiturates, and rifampicin
increase metabolic clearance and inhibit
anti-coagulation. Interestingly, there are specific
inherited mutations of factor IX that
render it particularly sensitive to decreased
carboxylation such that the anti-coagulation
effect of warfarin is enhanced without prolonging
the prothrombin time. Furthermore,
dietary changes can have a profound effect
on anti-coagulation. Vitamin K is largely
found in green vegetables such that increased
intake of leafy green vegetables can
provide vitamin K bypassing the need to regenerate
stores with epoxide reductase.
The variable pharmacokinetics of warfarin
requires careful monitoring and dose
adjustments to achieve a therapeutic range.
Even after stabilizing within a therapeutic
range, monitoring is still necessary since so
Warfarin Monitoring 3

Warfarin Monitoring
many environmental and dietary factors can
change the dose-response. Measuring the
prothrombin time (PT) remains the primary
mechanism by which warfarin anticoagulation
is monitored. Prolongation of
the PT, however, depends on the responsiveness
of the thromboplastin used to initiate
coagulation. Hence, the WHO has currently
established and international standard by
which manufacturers or individual labs can
determine an international sensitivity index
(ISI) for each lot of thromboplastin. A therapeutic
range of PT is then defined by the international
normalized ratio (INR), which is
the ratio between the patients PT to the mean
normal PT for the lab raised to the power of
the ISI:
Fig. 2. A nomogram for obtaining
an INR from a measured
prothrombin ratio (i.e., ratio of
patient PT/mean normal PT). The
ISI is provided by the manufacturer
of each lot of prothrombin
reagent (thromboplastin). In the
example shown, an INR of 3.0 is
obtained from a plasma sample
with a PTR of 2.0 if the ISI is 1.6.
INR = (Patient PT/Mean Normal PT) ISI
where the “mean normal PT” is the geometric
mean prothrombin time for the laboratory.
The ISI is a correction factor, specific for each
lot of laboratory prothrombin reagent, that
correlates the activity of the reagent to a
standard maintained by the World Health Organization.
A smaller ISI reflects a more sensitive
thromboplastin reagent such that a
more prolonged PT will be observed for the
same therapeutic effect.
In most medical institutions, the INR is
automatatically calculated by the laboratory
information system. The INR can also be calculated
from the above formula or derived
from an INR nomogram (Fig. 2).
The dosing of warfarin is driven by
monitoring the prothrombin time to achieve
a PT in the appropriate therapeutic range.
Typically 5 to 10 mg of warfarin is administered
daily with daily monitoring of the INR.
The dose is changed gradually until therapeutic
and then the INR is followed on a regular
but less frequent basis. Adjustments in dosage
should be based on the weekly dose since
Warfarin Monitoring 4

Warfarin Monitoring
warfarin has a long half-life. For most
indications the INR should be maintained
between 2.0 to 3.0. Exceptions include
anticoagulation in patients with
mechanical prosthetic heart valves and
patients with the antiphospholipid antibody
syndrome, in which the INR is usually
kept between 2.5 and 3.5. However, it
must be remembered that the INR was
developed for deep venous thrombosis
prophylaxis and may not be the most appropriate
monitoring mechanism for
other situations. Furthermore, inhibitors
such as lupus anticoagulants can prolong
the PT obscuring the appropriate therapeutic
range such that it may be necessary
to use alternative assays such as the
direct quantitation of the plasma warfarin
concentration.
Fig. 3. Recommended therapeutic
ranges for oral anticoagulant therapy.
Most recent recommendations
of the American College of chest
Physicians and the National Heart,
Lung, and Blood institute. From
Ansell et al. 2004.
As with all anticoagulant medications,
the primary risk for warfarin therapy is
hemorrhage. The risk is greater the
higher the INR, highlighting the importance
of regular monitoring. The risk of
a major bleed is 1 to 2 percent per year
with only a 0.1 to 0.5 percent per year
risk of intracranial hemorrhage. If a pa-
Warfarin Risks & Side Effects 5

Warfarin Monitoring
Table I
Clinical Management of the Supratherapeutic INR*
*Data from Ansell, 2004 and other sources. The purpose of this table is to convey general guidelines for the treatment of a
supratherapeutic INR. Clinical judgment is required for the individual patient.
Warfarin Risks and Side Effects 6

Warfarin Monitoring
tient is actively bleeding and the INR needs to
be corrected acutely, fresh frozen plasma can
be administered with vitamin K and in extreme
12cases prothrombin complex concentrates
can be given. When a patient is found
to have a markedly elevated PT without active
bleeding, a more conservative approach is
warranted such as simply skipping dosages
until the INR returns to the appropriate range
(Table I).
Warfarin-induced skin necrosis is a rare
but significant side effect. It follows administration
by one to ten days, is heralded by
parasthesia and an erythematous flush, which
is followed by petechiae and hemorrhage
which leads to skin necrosis. Skin necrosis is
most commonly associated with proctein C
deficiency and occasionally protein S deficiency.
Protein C has a very short half-life
and a rapid decline in activity from over 50%
to less than 5% apparently initiates the development
of skin necrosis in these patients.
Therefore, screening for protein C and S deficiencies
in patients with deep venous thrombosis
may help to identify those with an increased
risk of skin necrosis.
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