Laboratory Monitoring of Direct Thrombin Inhibitors - Pathology

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

Direct Thrombin Inhibitors
! Direct thrombin inhibitors (DTIs) are
anticoagulants with a targeted specicity for
thrombin. DTIs have a predictable anticoagulant
effect with little interindividual variability,
since they do not interact with platelets
or plasma proteins and do not require
antithrombin as a cofactor. Hirudin is a natural
65 amino acid DTI produced by the salivary
glands of the medicinal leech. Recombinant
hirudin (i.e., lepirudin) and smaller
synthetic analogs of hirudin (i.e., hirulog,
bivalirudin) constituent the divalent DTIs
that interact with both the catalytic and substrate
recognition site of thrombin. Monovalent
DTIs include several small molecules
(i.e., agratoban, melagatran, ximelagatran)
that interact only with thrombin’s catalytic
site. The requirement for laboratory monitoring
of DTIs varies with the individual
agent. Although relatively new, the many
medical advantages of the DTIs portend
their widespread use in the near future for
thrombophylaxis, stroke prevention, and the
treatment of venous thromboembolic disease
and heparin-induced thrombocytopenia.
There are two general classes of direct
thrombin inhibitors: divalent inhibitors that
bind both the substrate recognition site (exosite
1) and the catalytic site of thrombin and
monovalent inhibitors that bind only the catalytic
site. Members of the bivalent class currently
available include lepirudin and desirudin
(recombinant forms of the leech extract
hirudin) and bivalirudin. Members of the
monovalent class include the currently available
argatroban and the recently developed
ximelgatran, which is not yet approved by the
FDA.
Class 1
Desirudin, Lepirudin, and Bivalirudin
Recombinant desirudin and lepirudin,
are 65 amino acids, roughly 7 kDa, polypeptides
that differ from hirudin by sulphation of
a C-terminal tyrosine and from one another
by an isoleucine to a leucine change. The
amino terminal portion of the polypeptide
forms a globular domain that binds to the
catalytic site of thrombin, while the carboxy
terminal twelve residues form an extended
strand that interacts with the fibrinogen binding
exosite 1. These peptides bind irreversibly
to thrombin and inhibit cleavage of fibrinogen
to fibrin. Binding to substrate requires
access to exosite 1, hence these peptides do
not inhibit thrombin that is already bound to
fibrinogen.
Bivalirudin is a 20 amino acid derivative
of hirudin. The amino terminus consists
of the active site inhibitory sequence, D-Phe-
Pro-Arg, which is connected by a flexible
Fig. 1. Hirudin bound to thrombin. The crystal structure
of thrombin bound to recombinant hirudin (pdb
code: 4HTC) is shown. Thrombin is depicted as a surface
model colored salmon with the active site triad
colored green. The inhibitor hirudin is shown as a ribbon
model. Residues that also form the C-terminal
portion of bivalirudin are colored cyan and the remaining
residues are colored blue.
Structure & Metabolism 2

Direct Thrombin Inhibitors
Fig. 2. Argatroban bound to thrombin. The crystal
structure of thrombin bound to argatroban (pdb
code: 1DWC) is shown. Thrombin is depicted as a surface
model colored salmon with the active site triad
colored green. The inhibitor argatroban is shown in a
stick model colored by atom type. The peptide fragment
from hirudin present in the crystal structure is
omitted from this figure for clarity.
tetra-glycine linker to twelve amino acids
from the carboxy terminus of hirudin that
bind to exosite 1. The Pro-Arg peptide bond
can be slowly cleaved by the catalytic site of
thrombin, hence bivalirudin functions as a
reversible inhibitor with a short half-life (20
to 30 minutes).
Class 2
Argatroban, Ximelagatran, and Melagatran
The monovalent inhibitor argatroban
binds with high affinity and reversibly to
thrombin. It is a small synthetic molecule that
was rationally derived by modification of N-
tosyl-L-arginine methyl ester. A crystal structure
of the complex between thrombin and
argatroban shows that the inhibitor binds in a
hydrophobic pocket in the catalytic site of
thrombin. The pro-drug ximelagatran and its
active metabolite melagatran are small synthetic
peptidomimetics that were designed to
mimic the D-Phe-Pro-Arg tripeptide sequence
of bivaluridin. Like argatroban, melagatran
binds reversibly to the catalytic cleft of
thrombin and inhibits catalysis.
Hirudin and its recombinant forms
must be administered either intravenously or
by subcutaneous injection. The plasma halflife
is either 60 minutes or 120 minutes, intravenous
or subcutaneous administration respectively,
and predominantly cleared by renal
excretion. Therefore, these drugs should
be used with caution in patients with renal
insufficiency. The usual dosage is a 0.4 mg/kg
bolus followed by 0.15 mg/kg/hr continuous
infusion. This dosing must be adjusted appropriately
in patients with renal insufficiency,
in whom the plasma half-life may be
extended to over 300 hours.
Bivalirudin is administered by intravenous
infusion with a plasma half-life of ~30
minutes. It is largely cleared by plasma peptidases
with only 20% cleared by the kidneys.
Hence, bivalirudin may be a safer alternative
in renal insufficiency.
Argatroban is also administered by
intravenous infusion and has a plasma halflife
of ~45 minutes. It is largely metabolized
by the liver so that clearance is reduced in
liver disease but not affected by renal insufficiency.
Ximelgatran is a pro-drug that is absorbed
orally and metabolized by the liver to
the active form melgatran. It has a longer
half-life than the parental direct thrombin inhibitors
and has a predictable doseanticoagulant
response. Clearance is not affected
by liver or mild to moderate renal disease.
Therefore monitoring is generally not
necessary except for in severe renal disease,
making ximelgatran an attractive alternative
for oral anticoagulation therapy.
Pharmacokinetics 3

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