Tài liệu Báo cáo khoa học: Control of the coagulation system by serpins Getting by with a little help from glycosaminoglycans pptx

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

inhibitors. One major class of serine protease inhibitors
regulating procoagulant enzymes is the serpin super-
family [2]. The principal inhibitor of procoagulant
enzymes such as thrombin and factor Xa is the serpin
antithrombin (AT). There are, however, other serpins
that act to control coagulation enzymes, such as heparin
cofactor II (HC-II), protease nexin I and C1-inhibitor.
Some serpins, such as protein C inhibitor (PCI), act to
control the action of anticoagulant enzymes, such as
activated protein C.
A feature of many of the serpins that control
enzymes in the coagulation system is that they them-
selves are under the control of glycosaminoglycans
Keywords
antithrombin; coagulation;
glycosaminoglycans; heparin cofactor II;
proteases; protein C inhibitor; serpins;
thrombin
Correspondence
R. N. Pike, Department of Biochemistry &
Molecular Biology, Monash University,
Clayton, Victoria 3800, Australia
Fax: +61 3 99054699
Tel: +61 3 99053923
E-mail:
(Received 16 December 2004, accepted 14
July 2005)
doi:10.1111/j.1742-4658.2005.04880.x
Members of the serine protease inhibitor (serpin) superfamily play import-
ant roles in the inhibition of serine proteases involved in complex systems.

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

be the crucial element in the regulation of clotting, and
also to the eventual shutting off of the clotting process,
although this latter element is most probably a com-
plex multifactorial process also involving the platelets
and endothelium.
This review will examine the basic elements of the
structure and function of the serpins involved in con-
trolling the central coagulation enzyme, thrombin, and
their control by GAGs. Control over other enzymes,
such as factor Xa, will also be mentioned where rele-
vant. We will focus on antithrombin, heparin cofactor
II and protein C inhibitor.
General mechanism of serpin action
Serpins are a highly evolved family of proteins, which
have a mechanism of action that appears to be com-
mon to most members of the family [4]. The mechan-
ism, hotly debated for many years, involves the attack
of the protease on the P1-P1¢ bond in the reactive cen-
tre loop (RCL) of the serpin [5]. The catalysis of the
peptide bond cleavage appears to be arrested at the
acyl intermediate by the unique action of the serpin,
whereby the RCL of the serpin inserts into the major
A b -sheet causing the protease to be rapidly translo-
cated from the top to the bottom of the inhibitor [6].
In the process, the structure of the protease appears to
be deformed by being ‘crushed’ against the bottom of
the inhibitor [7]. In particular, this deformation of the
protease affects the geometry of the catalytic triad,
preventing the completion of catalysis beyond the acyl
intermediate and therefore trapping the protease in a

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

inhibition of the other enzymes by this serpin is
also important.
The serpin has a structure (Fig. 1) which is highly
similar to that of other serpins, with a few important
features. In its native state the RCL is partially inser-
ted into the top of the A b-sheet of the molecule [10].
Upon the addition of heparin, the RCL is expelled
from the A b-sheet by a closing of the sheet caused by
a conformational transition in the molecule following
the binding of a specific heparin pentasaccharide
sequence to a highly positively charged cluster located
at the D-helix of the serpin [11,12]. The pentasaccha-
ride sequence of heparin on its own is able to induce
the conformational change in AT [13,14] and this
change in the structure of the serpin is apparently able
to substantially accelerate the interaction with serine
proteases such as factor Xa, but not enzymes such as
thrombin [15]. It is thought that the overall increase in
the rate of interaction with factor Xa brought about
by the heparin pentasaccharide-mediated conforma-
tional change occurs through a combination of
the changes in the structure of the RCL, allowing the
interaction of residues on the RCL with subsites in the
active site of fXa [16,17], and the exposure of a new
exosite on the serpin for interaction with the protease
[18,19]. Given the plasma concentration of AT and the
rates of interaction in the presence and absence of hep-
arin pentasaccharide (Table 1), one can calculate that
the half-life of enzyme activity in the absence of hep-
arin pentasaccharide would be 133 s (full lifetime,

 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-

ture of AT templated to a genetically modified form of
thrombin by a synthetic heparin has been solved
[24,25]. These structures have supported much of what
Native Antithrombin - Heparin Native Antithrombin
Cleaved Protein C InhibitorNative Heparin Cofactor 2
Fig. 1. The structures of serpins controlling
thrombin. The structures of AT with and
without the heparin pentasaccharide bound,
heparin cofactor II and cleaved protein C
inhibitor are shown as indicated. In all of the
structures, the A b-sheet is shown in red,
the B b-sheet in green and the C b-sheet in
yellow. The reactive centre loop is shown in
magenta and the D-helix in dark blue. The
H-helix is shown in dark pink in the protein
C inhibitor structure. Positively charged resi-
dues on the D- and H-helices and the hep-
arin pentasaccharide are shown in ball and
stick format.
Control of the coagulation system by serpins R. N. Pike et al.
4844 FEBS Journal 272 (2005) 4842–4851 ª 2005 FEBS
has been published before in terms of the mechanism
by which templating occurs and have provided addi-
tional insights into the conformation of the reactive
centre loop of AT when it is in complex with a target
protease.
It is interesting to note that clot bound thrombin
and factor Xa are protected from inactivation by AT
[26,27]. This is consistent with the role of AT in local-
izing the clot and preventing it from spreading too far,

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].

Heparin cofactor II rapidly inhibits thrombin fol-
lowing binding to GAGs (Table 1). However, the
GAG specificity of HC-II is much less discriminating
than that of AT. While both heparin ⁄ heparan sulfate
and dermatan sulfate GAGs are physiological activa-
tors of HC-II, many different polyanions, including
polyphosphates, polysulfates and polycarboxylates, are
able to accelerate HC-II inhibition of thrombin [38,39].
The GAG binding site of HC-II has been identified as
the D-helix region [40–52]. The effects of mutagenesis
of thrombin anion-binding exosites-1 and -2 on GAG
acceleration of the HC-II–thrombin reaction suggest
that the template mechanism makes only a minor con-
tribution to heparin acceleration and no contribution
to dermatan sulfate acceleration [42,45,49,50,53].
Instead, the major mechanism of GAG enhancement
appears to be allosteric and uses conformational acti-
vation of the serpin. Heparin cofactor II possesses a
unique amino-terminal extension that contains two
tandem repeats rich in acidic amino acids with two sul-
fated tyrosines (contained in the region encompassed
by residues 54–75). The acidic region repeats of HC-II
are significantly homologous to the carboxyl-terminal
sequence of hirudin (the thrombin inhibitor from the
medicinal leech), which binds to thrombin anion-bind-
ing exosite-1 [54,55]. Glycosaminoglycan binding to
HC-II is thought to allosterically activate the serpin
by displacing the acidic amino terminus from an intra-
molecular interaction with the basic GAG binding site
and freeing it for binding to the thrombin anion-bind-

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

7
High affinity heparin 4 · 10
7
Heparin pentasaccharide 2 · 10
4
Factor Xa – 2 · 10
3
Heparin 4 · 10
7
Heparan 2 · 10
7
High affinity heparin 4 · 10
7
Heparin pentasaccharide 5 · 10
5
HCII Thrombin – 7 · 10
2
Dermatan sulfate 1 · 10
7
Heparin 1 · 10
7
PCI Thrombin Heparin 5 · 10
5
fXa Heparin 3 · 10
3
aPC – 1 · 10
4
Heparin 2 · 10
4
Heparin ⁄ calcium 3 · 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

confirmed that the antithrombotic effect of exogenously
added dermatan sulfate is due to its interaction with
HC-II [66]. Collectively, these findings imply that
HC-II has a major role in thrombin regulation at extra-
vascular tissue sites following vessel injury.
Protein C inhibitor (also named PAI-3)
Protein C inhibitor antigen is found in human blood
plasma (3.6–6.8 lgÆmL
)1
or  90 nm) [67], numerous
other human tissues, in urine and several other body
fluids (e.g. tears, saliva, cerebral spinal fluid, amniotic
fluid), and in seminal fluid at  200 lgÆmL
)1
, which is
almost 40 times the amount in plasma [68–76]. The
mature protein is 57 kDa [77,78]. The PCI reactive site
peptide bond is Arg354-Ser355, and PCI displays a
protease inhibition profile for numerous ‘arginine-
specific’ serine proteases, including trypsin, thrombin
(in the absence and presence of thrombomodulin), acti-
vated protein C, acrosin, kallikrein, urokinase, tissue
plasminogen activator and factor XIa [69–71,74,77–
91]. The GAG binding site in protein C inhibitor
appears to be localized not to the D-helix as in AT
and HC-II, but to the H-helix region, with possible
contributions from the N-terminal A
+
-helix region
[92–96]. Both regions have sequences of basic residues

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

C pathway, because the thrombin-thrombomodulin
complex initiates this pathway by the activation of
zymogen protein C to an anticoagulant serine prote-
ase. Interestingly, the increased rate of thrombin
inhibition when bound to thrombomodulin that is
measured with PCI does not involve the chondroitin
sulfate moiety of thrombomodulin, but rather is
apparently promoted by the epidermal growth factor-
like domains of thrombomodulin.
Like HC-II, PCI has a broad GAG ⁄ polyanion
specificity for acceleration of protease inhibition reac-
tions [61,98]. A variety of GAGs and polyanions
[including heparin, low molecular weight heparin,
heparan sulfate, fucoidan, and other polyanions
(phosvitin)] accelerate both thrombin and activated
protein C inhibition by PCI; this is consistent with a
relatively nonspecific heparin-binding site in protein
C inhibitor. In contrast to AT, there is no evidence
for any sequence-specific binding of heparin ⁄ heparan
sulfate to PCI. In a cell-derived series of studies,
heparan sulfate-containing proteoglycans were
involved in binding to PCI using cultured human
epithelial kidney tumor cells (TCL-598); furthermore,
Control of the coagulation system by serpins R. N. Pike et al.
4846 FEBS Journal 272 (2005) 4842–4851 ª 2005 FEBS
dermatan sulfate-containing proteoglycans were impli-
cated in binding PCI in the extracellular matrix
[70,100]. However, identification of the physiological
proteoglycan responsible for acceleration of PCI’s
activity in vivo has not been clearly identified.

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

Grants HL-06350 and HL-32656 from the National
Institutes of Health (to FCC), the Institut National
de la Sante
´
et de la Recherche Me
´
dicale of France
and the Foundation pour la Recherche Me
´
dicale of
France (to BFLB).
References
1 Spronk HM, Govers-Riemslag JW & ten Cate H
(2003) The blood coagulation system as a molecular
machine. BioEssays 25, 1220–1228.
2 Silverman GA, Bird PI, Carrell RW, Church FC,
Coughlin PB, Gettins PGW et al. (2001) The serpins
are an expanding superfamily of structurally similar
but functionally diverse proteins: Evolution, mechan-
ism of inhibition, novel functions, and a revised
nomenclature. J Biol Chem 276, 33293–33296.
3 Huntington JA (2003) Mechanisms of glycosaminogly-
can activation of the serpins in hemostasis. J Thromb
Haemost 1, 1535–1549.
4 Gettins PG (2002) Serpin structure, mechanism, and
function. Chem Rev 102, 4751–4804.
5 Ye S, Cech AL, Belmares R, Bergstrom RC, Tong Y,
Corey DR, Kanost MR & Goldsmith EJ (2001) The
structure of a Michaelis serpin-protease complex. Nat
Struct Biol 8, 979–983.

6 Kaslik G, Kardos J, Szabo E, Szilagyi L, Zavodszky P,
Westler WM, Markley JL & Graf L (1997) Effects of
serpin binding on the target proteinase: global stabili-
zation, localized increased structural flexibility, and
conserved hydrogen bonding at the active site.
Biochemistry 36, 5455–5464.
7 Huntington JA, Read RJ & Carrell RW (2000) Struc-
ture of a serpin-protease complex shows inhibition by
deformation, Nature 407.
8 Stein PE & Carrell RW (1995) What do dysfunctional
serpins tell us about molecular mobility and disease?
Struct Biol 2, 96–113.
9 Conard J, Brosstad F, Lie Larsen M, Samama M &
Abildgaard U (1983) Molar antithrombin concentra-
tion in normal human plasma. Haemostasis 13, 363–
368.
10 Skinner R, Abrahams JP, Whisstock JC, Lesk AM,
Carrell RW & Wardell MR (1997) The 2.6 A structure
of antithrombin indicates a conformational change at
the heparin binding site. J Mol Biol 266, 601–609.
11 Huntington JA, Olson ST, Fan B & Gettins PG (1996)
Mechanism of heparin activation of antithrombin.
Evidence for reactive center loop preinsertion with
expulsion upon heparin binding. Biochemistry 35,
8495–8503.
12 Jin L, Abrahams JP, Skinner R, Petitou M & Pike RN
(1997) The anticoagulant activation of antithrombin by
heparin. Proc Natl Acad Sci USA 94, 14683–14688.
13 Choay J, Petitou M, Lormeau JC, Sinay P, Casu B &
Gatti G (1983) Structure-activity relationship in

heparin: a synthetic pentasaccharide with high affinity
for antithrombin III and eliciting high anti-factor Xa
activity. Biochem Biophys Res Commun 116, 492–499.
14 Desai UR, Petitou M, Bjork I & Olson ST (1998)
Mechanism of heparin activation of antithrombin.
Role of individual residues of the pentasaccharide
activating sequence in the recognition of native and
activated states of antithrombin. J Biol Chem 273,
7478–7487.
15 Olson ST, Bjork I, Sheffer R, Craig PA, Shore JD &
Choay J (1992) Role of the antithrombin-binding penta-
saccharide in heparin acceleration of antithrombin-
proteinase reactions. Resolution of the antithrombin
R. N. Pike et al. Control of the coagulation system by serpins
FEBS Journal 272 (2005) 4842–4851 ª 2005 FEBS 4847
conformational change contribution to heparin rate
enhancement. J Biol Chem 267, 12528–12538.
16 Quinsey NS, Whisstock JC, Le Bonniec B, Louvain V,
Bottomley SP & Pike RN (2002) Molecular determinants
of the mechanism underlying acceleration of the inter-
action between antithrombin and factor Xa by heparin
pentasaccharide. J Biol Chem 277, 15971–15978.
17 Rezaie AR, Yang L & Manithody C (2004) Mutagen-
esis studies toward understanding the mechanism of
differential reactivity of factor Xa with the native and
heparin-activated antithrombin. Biochemistry 43, 2898–
2905.
18 Chuang YJ, Swanson R, Raja SM & Olson ST (2001)
Heparin enhances the specificity of antithrombin for
thrombin and factor Xa independent of the reactive

center loop sequence. Evidence for an exosite determi-
nant of factor Xa specificity in heparin-activated
antithrombin. J Biol Chem 276, 14961–14971.
19 Izaguirre G, Zhang W, Swanson R, Bedsted T & Olson
ST (2003) Localization of an antithrombin exosite that
promotes rapid inhibition of factors Xa and IXa
dependent on heparin activation of the serpin. J Biol
Chem 278, 51433–51440.
20 The Rembrandt Investigators. (2000) Treatment of
proximal deep vein thrombosis with a novel synthetic
compound (SR90107A ⁄ ORG31540) with pure anti-fac-
tor Xa activity: a phase II evaluation. Circulation 102,
2726–2731.
21 Olson ST & Shore JD (1982) Demonstration of a two-
step reaction mechanism for inhibition of alpha-throm-
bin by antithrombin III and identification of the step
affected by heparin. J Biol Chem 257, 14891–14895.
22 Rezaie AR (1998) Calcium enhances heparin catalysis
of the antithrombin-Factor Xa reaction by a template
mechanism: Evidence that calcium alleviates Gla
domain antagonism of heparin binding to Factor Xa.
J Biol Chem 273, 16824–16827.
23 Lane DA, Olds R & Thein S-L (1992) Antithrombin and
its deficiency states. Blood Coag Fibrinol 3, 315–341.
24 Li W, Johnson DJ, Esmon CT & Huntington JA
(2004) Structure of the antithrombin-thrombin-heparin
ternary complex reveals the antithrombotic mechanism
of heparin, Nat Struct Mol Biol 11, 857–862.
25 Dementiev A, Petitou M, Herbert JM & Gettins PG
(2004) The ternary complex of antithrombin-anhydro-

thrombin-heparin reveals the basis of inhibitor specifi-
city. Nat Struct Mol Biol 11, 863–867.
26 Weitz JI, Hudoba M, Massel D, Maraganore J &
Hirsh J (1990) Clot-bound thrombin is protected from
inhibition by heparin-antithrombin III but is suscepti-
ble to inactivation by antithrombin III-independent
inhibitors. J Clin Invest 86, 385–391.
27 Rezaie AR (2001) Prothrombin protects factor Xa in
the prothrombinase complex from inhibition by the
heparin-antithrombin complex. Blood 97, 2308–2313.
28 Mertens G, Cassiman J-J, Van den Berghe H, Vermy-
len J & David G (1992) Cell surface heparan sulfate
proteoglycans from human vascular endothelial cells:
core protein characterization and antithrombin III
binding properties. J Biol Chem 267, 20435–20443.
29 Scully MF, Ellis V, Shah N & Kakkar V (1989) Effect
of a heparan sulphate with high affinity for antithrom-
bin III upon inactivation of thrombin and coagulation
factor Xa. Biochem J 262, 651–658.
30 Ishiguro K, Kojima T, Kadomatsu K, Nakayama Y,
Takagi A, Suzuki M et al. (2000) Complete antithrom-
bin deficiency in mice results in embryonic lethality.
J Clin Invest 106, 873–878.
31 Yanada M, Kojima T, Ishiguro K, Nakayama Y,
Yamamoto K, Matsushita T et al. (2002) Impact of
antithrombin deficiency in thrombogenesis: lipopolysac-
charide and stress-induced thrombus formation in het-
erozygous antithrombin-deficient mice. Blood 99, 2455–
2458.
32 Beauchamp NJ, Pike RN, Daly M, Butler L, Markis

M, Daffron TR et al. (1998) Antithrombins Wibble
and Wobble (T85M ⁄ K): Archetypal conformational
diseases with in vivo latent-transition, thrombosis and
heparin activation. Blood 92, 2696–2706.
33 Tollefsen DM & Pestka CA (1985) Heparin cofactor II
activity in patients with disseminated intravascular coa-
gulation and hepatic failure. Blood 66, 769–774.
34 Blinder MA, Marasa JC, Reynolds CH, Deaven LL &
Tollefsen DM (1988) Heparin cofactor II: cDNA
sequence, chromosome localization, restriction frag-
ment length polymorphism, and expression in Escheri-
chia coli . Biochem 27, 752–759.
35 Griffith MJ, Noyes CM, Tyndall JA & Church FC
(1985) Structural evidence for leucine at the reactive
site of heparin cofactor II. Biochemistry 24, 6777–6782.
36 Church FC, Noyes CM & Griffith MJ (1985) Inhibi-
tion of chymotrypsin by heparin cofactor II. Proc Natl
Acad Sci USA 82, 6431–6434.
37 Parker KA & Tollefsen DM (1985) The protease specifi-
city of heparin cofactor II. Inhibition of thrombin gener-
ated during coagulation. J Biol Chem 260, 3501–3505.
38 Pratt CW, Whinna HC, Meade JM, Treanor RE &
Church FC (1989) Physicochemical aspects of heparin
cofactor II. Ann NY Acad Sci 556, 104–114.
39 Tollefsen DM (1997) Heparin cofactor II. Adv Exp
Med Biol 425, 35–44.
40 Baglin TP, Carrell RW, Church FC, Esmon CT &
Huntington JA (2002) Crystal structures of native and
thrombin-complexed heparin cofactor II reveal a multi-
step allosteric mechanism. Proc Natl Acad Sci USA 99,

11079–11084.
41 Blinder MA & Tollefsen DM (1990) Site-directed
mutagenesis of arginine 103 and lysine 185 in the
proposed glycosaminoglycan-binding site of heparin
cofactor II. J Biol Chem 265, 286–291.
Control of the coagulation system by serpins R. N. Pike et al.
4848 FEBS Journal 272 (2005) 4842–4851 ª 2005 FEBS
42 Fortenberry YM, Whinna HC, Gentry HR, Myles T,
Leung LL & Church FC (2004) Molecular mapping of
the thrombin-heparin cofactor II complex. J Biol Chem
279, 43237–43244.
43 Jeter ML, Ly LV, Fortenberry YM, Whinna HC,
White RR, Rusconi CP, Sullenger BA & Church FC
(2004) RNA aptamer to thrombin binds anion-binding
exosite-2 and alters protease inhibition by heparin-
binding serpins. FEBS Lett 568, 10–14.
44 Liaw PCY, Austin RC, Fredenburgh JC, Stafford AR
& Weitz JI (1999) Comparison of heparin- and derma-
tan sulfate-mediated catalysis of thrombin inactivation
by heparin cofactor II. J Biol Chem 274, 27597–27604.
45 Myles T, Church FC, Whinna HC, Monard D & Stone
SR (1998) Role of thrombin anion-binding exosite-I in
the formation of thrombin-serpin complexes. J Biol
Chem 273, 31203–31208.
46 Ragg H, Ulshofer T & Gerewitz J (1990) On the acti-
vation of human leuserpin-2, a thrombin inhibitor, by
glycosaminoglycans. J Biol Chem 265, 5211–5218.
47 Ragg H, Ulshofer T & Gerewitz J (1990) Glycosamino-
glycan-mediated leuserpin-2 ⁄ thrombin interaction:
Structure · function relationships. J Biol Chem 265,

22386–22391.
48 Rogers SJ, Pratt CW, Whinna HC & Church FC
(1992) Role of thrombin exosites in inhibition by
heparin cofactor II. J Biol Chem 267, 3613–3617.
49 Sheehan JP, Tollefsen DM & Sadler JE (1994) Heparin
cofactor II is regulated allosterically and not primarily
by template effects. Studies with mutant thrombins and
glycosaminoglycans. J Biol Chem 269, 32747–32751.
50 Sheehan JP, Wu Q, Tollefsen DM & Sadler JE (1993)
Mutagenesis of thrombin selectively modulates inhibi-
tion by serpins heparin cofactor II and antithrombin
III. Interaction with the anion-binding exosite deter-
mines heparin cofactor II specificity. J Biol Chem 268,
3639–3645.
51 VanDeerlin VMD & Tollefsen DM (1991) The N-term-
inal acidic domain of heparin cofactor II mediates the
inhibition of a-thrombin in the presence of glycosami-
noglycans. J Biol Chem 266, 20223–20231.
52 Whinna HC, Blinder MA, Szewczyk M, Tollefsen
DM & Church FC (1991) Role of lysine 173 in
heparin binding to heparin cofactor II. J Biol Chem
266, 8129–8135.
53 Verhamme IM, Bock PE & Jackson CM (2004) The
preferred pathway of glycosaminoglycan-accelerated
inactivation of thrombin by heparin cofactor II. J Biol
Chem 279, 9785–9795.
54 Inhorn RC & Tollefsen DM (1986) Isolation and char-
acterization of a partial cDNA clone for heparin cofac-
tor II. Biochem Biophys Res Commun 137, 431–436.
55 Ragg H (1986) A new member of the plasma protease

inhibitor gene family. Nuc Acids Res 14, 1073–1087.
56 Tollefsen DM, Pestka CA & Monafo WJ (1983) Acti-
vation of heparin cofactor II by dermatan sulfate.
J Biol Chem 258, 6713–6716.
57 Tollefsen DM, Peacock ME & Monafo WJ (1986)
Molecular size of dermatan sulfate oligosaccharides
required to bind and activate heparin cofactor II.
J Biol Chem 261, 8854–8858.
58 McGuire EA & Tollefsen DM (1987) Activation of
heparin cofactor II by fibroblasts and vascular smooth
muscle cells. J Biol Chem 262, 169–175.
59 Tollefsen DM, Maimone MM, McGuire EA & Pea-
cock ME (1989) Heparin cofactor II activation by der-
matan sulfate. Ann N Y Acad Sci 556, 116–122.
60 Maimone MM & Tollefsen DM (1990) Structure of a
dermatan sulfate hexasaccharide that binds to heparin
cofactor II with high affinity. J Biol Chem 265, 18263–
18271.
61 Pratt CW, Whinna HC & Church FC (1992) A com-
parison of three heparin-binding serine proteinase inhi-
bitors. J Biol Chem 267, 8795–8801.
62 Whinna HC, Choi HU, Rosenberg LC & Church FC
(1993) Interaction of heparin cofactor II with biglycan
and decorin. J Biol Chem 268, 3920–3924.
63 Shirk RA, Church FC & Wagner WD (1996) Arterial
smooth muscle cell heparan sulfate proteoglycans accel-
erate thrombin inhibition by heparin cofactor II. Arter-
ioscler Thromb Vasc Biol 16, 1138–1146.
64 Shirk RA, Parthasarathy N, San Antonio JD, Church
FC & Wagner WD (2000) Altered dermatan sulfate

structure and reduced heparin cofactor II-stimulating
activity of biglycan and decorin from human athero-
sclerotic plaque. J Biol Chem 275, 18085–18092.
65 He L, Vicente CP, Westrick RJ, Eitzman DT & Tollef-
sen DM (2002) Heparin cofactor II inhibits arterial
thrombosis after endothelial injury. J Clin Invest 109,
213–219.
66 Vicente CP, He L, Pavao MS & Tollefsen DM (2004)
Antithrombotic activity of dermatan sulfate in heparin
cofactor II-deficient mice. Blood 104, 3965–3970.
67 Suzuki K, Deyashiki Y, Nishioka J & Toma K (1989)
Protein C inhibitor: structure and function. Thromb
Haemostas 61, 337–342.
68 Laurell M & Stenflo J (1989) Protein C inhibitor from
human plasma: characterization of native and cleaved
inhibitor and demonstration of inhibitor complexes
with plasma kallikrein. Thromb Haemostasis 62, 885–
891.
69 Espana F, Gruber A, Heeb M, Hanson S, Harker L &
Griffin J (1991) In vivo and in vitro complexes of acti-
vated protein C with two inhibitors in baboons. Blood
77, 1754–1760.
70 Geiger M, Priglinger U, Griffin JH & Binder BR
(1991) Urinary protein C inhibitor. Glycosaminocly-
cans synthesized by the epithelial kidney cell line
R. N. Pike et al. Control of the coagulation system by serpins
FEBS Journal 272 (2005) 4842–4851 ª 2005 FEBS 4849
TCL)598 enhance its interaction with urokinase. J Biol
Chem 266, 11851–11857.
71 Ecke S, Geiger M, Resch I, Jerabek I, Stingl L, Maier

M & Binder BR (1992) Inhibition of tissue kallikrein
by protein C inhibitor. Evidence for identity of protein
C inhibitor with the kallikrein binding protein. J Biol
Chem 267, 7048–7052.
72 Laurell M, Christensson A, Abrahamsson PA, Stenflo
J & Lilja H (1992) Protein C inhibitor in human body
fluids. Seminal plasma is rich in inhibitor antigen deriv-
ing from cells throughout the male reproductive sys-
tem. J Clin Invest 89, 1094–1101.
73 Radtke KP, Fernandez JA, Greengard JS, Tang WW,
Wilson CB, Loskutoff DJ, Scharrer I & Griffin JH
(1994) Protein C inhibitor is expressed in tubular cells
of human kidney. J Clin Invest 94, 2117–2124.
74 Hermans JM, Jones R & Stone SR (1994) Rapid inhi-
bition of the sperm protease acrosin by protein C inhi-
bitor. Biochemistry 33, 5440–5444.
75 Espana F, Sanchez-Cuenca J, Fernandez PJ, Gilabert
J, Romeu A, Estelles A, Royo M & Muller CH (1999)
Inhibition of human sperm-zona-free hamster oocyte
binding and penetration by protein C inhibitor.
Andrologia 31, 217–223.
76 Uhrin P, Dewerchin M, Hilpert M, Chrenek P, Schofer
C, Zechmeister-Machhart M et al. (2000) Disruption of
the protein C inhibitor gene results in impaired sperma-
togensis and male infertility. J Clin Invest 106, 1531–
1539.
77 Suzuki K, Nishioka J & Hashimoto S (1983) Protein C
inhibitor: purification from human plasma and charac-
terization. J Biol Chem 258, 163–168.
78 Suzuki K, Deyashiki Y, Nishioka J, Kurachi K, Akira

M, Yamamoto S & Hashimoto S (1987) Characteriza-
tion of a cDNA for human protein C inhibitor. A new
member of the plasma serine protease inhibitor super-
family. J Biol Chem 262, 611–616.
79 Suzuki K (1984) Activated protein C inhibitor. Semin
Thrombos Hemostas 10, 154–161.
80 Heeb MJ, Espana F, Geiger M, Collen D, Stump DC
& Griffin JH (1987) Immunological identity of heparin-
dependent plasma and urinary protein C inhibitor and
plasminogen activator inhibitor-3. J Biol Chem 262,
15813–15816.
81 Geiger M, Heeb MJ, Binder BR & Griffin JH (1988)
Competition of activated protein C and urokinase for
a heparin-dependent inhibitor. FASEB J 2, 2263–2267.
82 Meijers JCM, Kanters DHAJ, Vlooswijk RAA, van
Erp HE, Hessing M & Bouma BN (1988) Inactivation
of human plasma kallikrein and factor XIa by protein
C inhibitor. Biochemistry 27, 4231–4237.
83 Heeb MJ, Espana F & GJ (1989) Inhibition and com-
plexation of activated protein C by two major inhibi-
tors in plasma. Blood 73, 446–454.
84 Rezaie AR, Cooper ST, Church FC & Esmon CT
(1995) Protein C inhibitor is a potent inhibitor of the
thrombin-thrombomodulin complex. J Biol Chem 270,
25336–25339.
85 Elisen MG, van Kooij RJ, Nolte MA, Marquart JA,
Lock TM, Bouma BN & Meijers JC (1998) Protein C
inhibitor may modulate human sperm–oocyte interac-
tions. Biol Reprod 58, 670–677.
86 Elisen MG, dem Borne PA, Bouma BN & Meijers

JC (1998) Protein C inhibitor acts as a procoagulant
by inhibiting the thrombomodulin-induced activation
of protein C in human plasma. Blood 91, 1542–1547.
87 Elisen MGLM, Bouma BN, Church FC & Meijers
JCM (1998) Inhibition of serine proteases by reactive
site mutants of protein C inhibitor (plasminogen acti-
vator inhibitor-3). Fib Prot 12, 283–291.
88 Shen L, Villoutreix BO & Dahlback B (1999) Involve-
ment of Lys 62 (217) and Lys 63 (218) of human anti-
coagulant protein C in heparin stimulation of inhibition
by the protein C inhibitor. Thromb Haemost 82, 72–79.
89 Friedrich U, Blom AM, Dahlba
¨
ck B & Villoutreix BO
(2001) Structural and energetic characteristics of the
heparin-binding site in antithrombotic protein C. J Biol
Chem 276 , 24122–24128.
90 Mosnier LO, Elisen MG, Bouma BN & Meijers JC
(2001) Protein C inhibitor regulates the thrombin-
thrombomodulin complex in the up- and down regula-
tion of TAFI activation. Thromb Haemost 86, 1057–
1064.
91 Rezaie AR (2003) Exosite-dependent regulation of the
protein C anticoagulant pathway. Trends Cardiovasc
Med 13, 8–15.
92 Kuhn LA, Griffin JH, Fisher CL, Greengard JS, Bou-
ma BN, Espana F & Tainer JA (1990) Elucidating the
structural chemistry of glycosaminoglycan recognition
by protein C inhibitor. Proc Natl Acad Sci USA 87,
8506–8510.

93 Shirk RA, Elisen MGLM, Meijers JCM & Church FC
(1994) Role of the H helix in heparin binding to pro-
tein C inhibitor. J Biol Chem 269, 28690–28695.
94 Cooper ST, Whinna HC, Jackson TP, Boyd JM &
Church FC (1995) Intermolecular interactions between
protein C inhibitor and coagulation proteases. Bio-
chemistry 34, 12991–12997.
95 Elisen MGLM, Maseland MHH, Church FC, Bouma
BN & Meijers JCM (1996) Role of the A+ helix in
heparin binding to protein C inhibitor. Thromb Hae-
most 75, 760–766.
96 Neese LL, Wolfe CA & Church FC (1998) Contribu-
tion of basic residues of the D and H helices in heparin
binding to protein C inhibitor. Arch Biochem Biophys
355, 101–108.
97 Huntington JA, Kjellberg M & Stenflo J (2003) Crystal
structure of protein C inhibitor provides insights into
Control of the coagulation system by serpins R. N. Pike et al.
4850 FEBS Journal 272 (2005) 4842–4851 ª 2005 FEBS
hormone binding and heparin activation. Structure
(Camb) 11, 205–215.
98 Pratt CW & Church FC (1992) Heparin binding to
protein C inhibitor. J Biol Chem 267, 8789–8794.
99 Yang L, Manithody C, Walston TD, Cooper ST &
Rezaie AR (2003) Thrombomodulin enhances the reac-
tivity of thrombin with protein C inhibitor by provid-
ing both a binding site for the serpin and allosterically
modulating the activity of thrombin. J Biol Chem 278,
37465–37470.
100 Priglinger U, Geiger M, Bielek E, Vanyek E &

Binder BR (1994) Binding of urinary protein C
inhibitor to cultured human epithelial kidney tumor
cells (TCL-598). The role of glycosaminoglycans pre-
sent on the luminal cell surface. J Biol Chem 269,
14705–14710.
FEBS Journal 272 (2005) 4842–4851 ª 2005 FEBS 4851
R. N. Pike et al. Control of the coagulation system by serpins