Biomedical Engineering Reference
In-Depth Information
has the same carbohydrate as the α-isoform, except at asparagine 135 where no carbohydrate moiety
is present.
75
Biosynthetic defi ciency resulting in the three-glycan β product gives a subtype in which
the heparin-binding affi nity is increased,
76
which has signifi cant implications for the likely popula-
tion mixture that may result during some ATH syntheses. Thus, there is a wide range of variants
within the AT family that have major differences in the key components of the molecule. These
alterations in the overall molecular structure have important functional effects, as will be delineated
in the following section (18.2.2).
18.2.2 F
UNCTIONAL
B
IOCHEMISTRY
OF
A
NTITHROMBIN
The AT anticoagulant has typical serpin structural features that control the action of the inhibitor
toward its protease targets. A chronology of the developments leading to our present understanding
of AT's mechanisms is illustrative in that it connects AT reactivity with its biological function
in vivo
.
Consideration of the underpinnings of AT biochemical actions affecting the patient gives credence to
the type of directions needed to construct covalent ATH complexes with advanced utility.
An early work on blood coagulation indicated that a circulating natural anticoagulant must be
present since exogenous thrombin lost activity when mixed with plasma.
59,77
Initially, this “AT”
molecule's activity in plasma was described as the “progressive AT activity.” Early research by
Robert Maclean in 1916 led to the discovery of an anticoagulant isolated from liver (i.e., UFH)
78
that effectively inhibited thrombin in the presence of an uncharacterized plasma protein. Since the
plasma protein allowing heparin to exhibit its accelerating activity for thrombin inhibition had not
been isolated, the molecule was designated as “heparin cofactor.”
79
In an attempt to systematize
classifi cation of the AT and coagulant systems, nomenclature was suggested in the 1950s
80
whereby
loss of thrombin activity due to i brin-clot binding was called AT I, heparin cofactor activity was
called AT II, and AT III was referred to progressive AT activity. In the 1960s and 1970s, purifi ca-
tion and analysis of plasma proteins yielded data showing that a single protein possessed all the
activities of the heparin cofactor and progressive AT.
59
Thus, ATs II and III were designated as AT
III. Discussions evolving from the standardization subcommittee of the International Society of
Thrombosis and Hemostasis fi nally led to the simple name of AT for this anticoagulant protein.
The mechanism of AT has been elucidated from a large wealth of investigations and it is now
one of the best-understood protease inhibitors. AT reacts to neutralize activated coagulation factors
by a stress-release mode in which initial cleavage of the inhibitor allows rapid relaxation from a
high-energy state to a vastly altered form of AT in which the protease remains covalently linked and
protected from hydrolytic release. As with many of the serpins, AT contains a reactive center loop
near the C-terminus. As it approaches the reactive center, thrombin is attracted to a binding region
close to the reactive center. Mutations discovered in AT such as Ala382
Leu407
55
have established this thrombin-binding region to involve at least amino acid residues 382
and 407. In terms of the noncovalent AT-contact points on thrombin, a substitution of Gly226 to
Val226 on the enzyme caused loss of the capacity for thrombin inhibition.
82
Molecular models have
given indication that Gly226 extends into the specifi c recognition pocket on AT since the Val side
chain was too large for the space required by the AT P
1
reactive center loop, the Arg393 R-group.
Ultimately, specifi c binding of thrombin to AT allows for concomitant covalent bond formation.
The peptide in the AT serpin that is targeted by thrombin or other proteases has been assigned the
notation P
1
-P
1
', in which the P
1
Arg393 residue confers on AT its selectivity toward the coagulation
factors.
52
Amino acids that are on the N-terminal side of P
1
add to the enzyme selectivity, such as
Gly392 at position P
2
that selects for FXa and maintains covalent linkage with AT within the resul-
tant FXa-AT complex.
27
Work is in progress to determine the signifi cance of other residues, such
as P
1
and Ser394 that are toward the C-terminus from P
1
.
Reaction steps that ensue the fi rst association of thrombin and AT are intricate. Initially, throm-
bin reacts with its AT substrate by acting on the inhibitor's Arg393 C-terminal amide bond using
the protease-active serine. Thus, an ester between the thrombin serine hydroxyl oxygen and AT's
→
Thr382
81
and Pro407
→
