MINIREVIEW
Control of the coagulation system by serpins
Getting by with a little help from glycosaminoglycans
Robert N. Pike
1,2
, Ashley M. Buckle
1,2
, Bernard F. le Bonniec
3
and Frank C. Church
4
1 Department of Biochemistry & Molecular Biology and Co-operative Research Centres for Vaccine Technology and Oral Health Sciences,
Monash University, Clayton, Victoria, Australia
2 The Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia
3 INSERM U428, Faculte
´
de Pharmacie, Universite
´
Paris V, Paris, France
4 Departments of Pathology and Laboratory Medicine, Pharmacology, and Medicine, Carolina Cardiovascular Biology Center,
The University of North Carolina at Chapel Hill, School of Medicine, NC, USA
Introduction
Efficient functioning of the coagulation system is vital
to human health [1]. However, control of this system,
in particular its regulation to prevent inappropriate,
excessive or mislocalized clotting of blood, is also vital
to prevent cardiovascular diseases such as deep vein
thrombosis.
Because many of the principal procoagulant compo-
nents of the system are serine proteases, regulation of
the system is principally by the action of serine protease

This is evident in the regulation of coagulation serine proteases, especially
the central enzyme in this system, thrombin. This review focuses on three
serpins which are known to be key players in the regulation of thrombin:
antithrombin and heparin cofactor II, which inhibit thrombin in its pro-
coagulant role, and protein C inhibitor, which primarily inhibits the throm-
bin ⁄ thrombomodulin complex, where thrombin plays an anticoagulant
role. Several structures have been published in the past few years which
have given great insight into the mechanism of action of these serpins and
have significantly added to a wealth of biochemical and biophysical studies
carried out previously. A major feature of these serpins is that they are
under the control of glycosaminoglycans, which play a key role in acceler-
ating and localizing their action. While further work is clearly required
to understand the mechanism of action of the glycosaminoglycans, the bio-
logical mechanisms whereby cognate glycosaminoglycans for each serpin
come into contact with the inhibitors also requires much further work in
this important field.
Abbreviations
AT, antithrombin; GAG, glycosaminoglycan; HC-II, heparin cofactor II; PCI, protein C inhibitor; serpin, serine protease inhibitor.
4842 FEBS Journal 272 (2005) 4842–4851 ª 2005 FEBS
(GAGs) [3]. Glycosaminoglycans such as heparin, hep-
aran sulfate and dermatan sulfate have been found to
significantly accelerate the interaction between serpins
and coagulation proteases, usually increasing the reac-
tion rates from values that are not relevant under phy-
siological conditions to rates that are relevant. This
control over the action of serpins that have the partic-
ular role of regulating procoagulant enzymes is prob-
ably vital in that it allows the enzymes to act, as they
must, to clot blood. It follows that the serpins mostly
act to localize the clotting process, which is likely to

covalent bond with the serpin. The mechanism is a sui-
cide substrate mechanism, irreversibly inactivating the
serpin. The serpin–enzyme complex is later removed
from the circulation by the action of receptors which
specifically recognize the inhibited conformation of the
serpin (reviewed in [4]).
The structure and mechanism of serpins is highly
amenable to control via binding of molecules such as
GAGs, but the same level of conformational mobility
which aids in the function of serpins also renders them
susceptible to mutations which cause the A b-sheet in
particular to become susceptible to insertion of the ser-
pin’s own RCL. This results in either a so-called latent
state, or in polymers of serpins, where the insertion of
another molecule’s RCL takes place [8]. Both of these
result in the irreversible inactivation (generally) of the
serpins, and, in the case of the anticoagulant serpins, a
lowering of the effective concentration of the serpins
and therefore diseases such as thrombosis [8].
Antithrombin
Antithrombin is arguably the major anticoagulant ser-
pin. It is a 58 kDa glycoprotein, which circulates in
blood at a concentration of  125 lgÆmL
)1
(2.3 lm)
[9]. AT inhibits a large number of serine proteases of
the coagulation system including thrombin (factor
IIa) and factors IXa, Xa, XIa and XIIa. The princi-
pal targets of the serpin are usually regarded as being
thrombin and factor Xa, although it is likely that

 22 min), and this would be  1.33 s in the presence
of heparin pentasaccharide (full lifetime, 0.22 min).
This action of the synthetic heparin pentasaccharide is
R. N. Pike et al. Control of the coagulation system by serpins
FEBS Journal 272 (2005) 4842–4851 ª 2005 FEBS 4843
apparently effective enough, and has allowed its intro-
duction as a new antithrombotic drug [20].
Heparin pentasaccharide on its own does not sub-
stantially increase the rate of inhibition of some coagu-
lation enzymes, such as thrombin, indicating that the
conformational change in AT alone does not cause
much acceleration in the rate of interaction [15]. For
full acceleration of the rate of inhibition of enzymes
such as thrombin, full-length heparin (> 26 saccharide
units in length) is required. The longer chains of hep-
arin appear to accelerate the interaction between AT
and thrombin by ‘templating’ the serpin and enzyme,
binding to both molecules (via an exosite on the prote-
ase) and facilitating their diffusion towards each other
in solution [21]. This accelerates the interaction of AT
with thrombin 1000-fold and with fXa 10 000-fold
[22]. With regard to the latter interaction, it is clear
that calcium ions are required to overcome the negat-
ive effects of the Gla-domain of factor Xa on the tem-
plating interaction mediated by heparin. For thrombin,
this means that AT controls the enzyme in 0.27 s in
the presence of heparin, compared to 4.4 min in the
absence of heparin. Clearly this is important, as the
impairment of heparin binding on mutants of AT has
disease-causing consequences [23]. Recently, the struc-

rather than actually shutting down clotting. It would
appear that AT might localize to clots due to the expo-
sure of heparan sulfate chains on the endothelium
following vascular disruption or the localized release
of heparin from the granules of mast cells which are
found lining the vasculature [28,29]. Thus the AT may
act as a sentinel to prevent escape of active procoagu-
lant enzymes from their site of action, allowing clot-
ting to proceed where it is required, but not allowing it
to spread.
Antithrombin is clearly critical to survival. Homo-
zygous null mutants of AT die in utero [30] and hetero-
zygous mutants which have about 50% of the normal
concentrations of AT are predisposed towards disease
[31]. The experiments using the genetically manipulated
mice have confirmed a host of studies which reveal that
mutations of AT which impair its normal function
predispose patients to thrombotic disorders [8], partic-
ularly when found in combination with other predispo-
sing factors [32].
Heparin cofactor II
Heparin cofactor II mRNA has been detected only in
human liver, and the normal concentration of HC-II
in blood plasma is  1.2 lm and the mature protein is
65.6 kDa [33]. The HC-II reactive site peptide bond is
Leu444-Ser445 [34,35]. Intriguingly, HC-II is a very
specific inhibitor of thrombin, but no other serine pro-
tease in blood coagulation; however, it does exhibit
some inhibitory action to the chymotrypsin-like pro-
teases, cathepsin G and chymotrypsin [36,37].

ing exosite-1 [44,46,47,51]. An alternative allosteric
mechanism has been suggested based on the recently
described crystal structures of both native HC-II
(Fig. 1) and HC-II complexed with catalytically inac-
tive S195A thrombin [40]. In a surprising revelation,
the native HC-II structure showed that the hinge of
the reactive centre loop is partially inserted into the A
b-sheet, similar to the situation seen in native AT, and
the short segment of the amino terminus that was visi-
ble suggested that this region might be interacting with
an alternative basic site on the serpin near the reactive
loop [40]. Thus, GAG activation of HC-II was
Table 1. Second order rates of association (k
ass
) values for the
reaction of serpins with proteases in the presence and absence of
a range of GAGs (values are representative of those reported in a
range of publications cited in this article).
Serpin Enzyme Glycosaminoglycan k
ass
(M
)1
Æs
)1
)
Antithrombin Thrombin – 1 · 10
4
Heparin 4 · 10
7
Heparan 2 · 10

6
R. N. Pike et al. Control of the coagulation system by serpins
FEBS Journal 272 (2005) 4842–4851 ª 2005 FEBS 4845
proposed to resemble AT, where GAG binding to the
D-helix causes the expulsion of the buried reactive cen-
tre loop hinge from the A b-sheet, which in turn alters
the amino-terminal tail interaction to promote binding
to the thrombin anion-binding exosite-1. Regardless of
which allosteric mechanism turns out to be more cor-
rect, it is obvious that release of the amino-terminal
portion of HC-II to bind to thrombin anion-binding
exosite-1 is a primary part of the allosteric activation
mechanism.
For many years, the physiological activator of HC-II
has been assumed to be extravascular dermatan sulfate
[56–64], which would complement the intravascular
effect of heparan sulfate binding to AT. Maimone and
Tollefsen [60] described the structure of a high affinity
dermatan sulfate hexasaccharide that bound to HC-II.
Furthermore, dermatan sulfate proteoglycans on the
surface of cultured fibroblasts and vascular smooth
muscle cells and purified biglycan and decorin derma-
tan sulfate proteoglycans accelerate the rate of throm-
bin inhibition by HC-II [58,62]. Dermatan sulfate
proteoglycans in the extracellular matrices and on cer-
tain cell surfaces may localize HC-II to sites appropri-
ate for inhibiting thrombin. The murine knock-out
studies of HC-II revealed a role for this serpin in regu-
lating thrombin formation, especially in the arterial cir-
culation [65]. Recent studies using HC-II deficient mice

consistent with a general heparin-binding consensus
sequence motif. Mutagenesis of four basic residues in
the H-helix, Lys266, Arg269, Lys270 and Lys273, in
recombinant PCI has shown that all of these residues
are important for heparin binding. With the recent
report of the crystal structure of cleaved-PCI (Fig. 1)
[97], there are clearly other basic residues near the pri-
mary H-helix GAG binding site that probably contri-
bute to GAG ⁄ polyanion binding (including Arg26,
Arg27, Arg213, Arg234, Arg229, Lys255 and Arg362).
In contrast to both AT and HC-II, there is no
evidence for an allosteric activation mechanism and
instead the mechanism appears to involve only a
ternary complex with heparin bridging the serpin
and protease [61,98]. As found for other serine pro-
teases with c-carboxyglutamic acid domains, heparin
bridging of PCI and activated protein C is only
modest unless calcium ions are present to bind the
acidic domain and prevent its interaction with the
heparin-binding site of the protease [89,91]. Throm-
bin is also inhibited by PCI and the inhibition is
accelerated by heparin, but the heparin-enhanced rate
does not appear to be physiologically relevant when
compared to thrombin inhibition rates by both
AT-heparin ⁄ heparan sulfate and HC-II-heparin ⁄
dermatan sulfate. A more physiologically significant
rate of thrombin inhibition by PCI results when
thrombin binds to thrombomodulin, the endothelial
cell receptor ⁄ proteoglycan [84,86,90,99]. This is con-
sistent with PCI regulating the anticoagulant protein

Overall conclusions and future
directions
It is readily apparent that the action of the three ser-
pins discussed here is highly controlled by interactions
with GAGs. There are differences in the way which
each of the three serpins bind the GAGs, but common
to each is that GAGs increase the rate of interaction
with target proteases. The interaction of AT with hep-
arin is clearly the most understood in structural terms,
although a number of elements of the conformational
change brought about in AT by heparin remain a little
unclear. Further structural studies are obviously
required to fully understand the interaction of HC-II
and PCI with cognate GAGs.
The action of GAGs, in particular that of heparin
on AT, has been very successfully exploited in clinical
practice and this has been brought to even greater
sophistication by the introduction of synthetic ana-
logues of heparin. There is still a great need to fully
understand the situation in the physiological setting,
however. It is not completely clear when each serpin
comes into contact with the GAGs that modulate its
activity and how this leads to the vital regulation
which evidently occurs. This is a major area of basic
research for the immediate and medium term future.
Acknowledgements
This work was supported by the National Health &
Medical Research Council of Australia, the Austra-
lian Research Council, the National Heart Founda-
tion of Australia (to RNP and AMB), Research

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