Journal of Thrombosis and Haemostasis, 9 (Suppl. 1): 26–34
INVITED REVIEW
Serpin structure, function and dysfunction
J. A. HUNTINGTON
Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK
To cite this article: Huntington JA. Serpin structure, function and dysfunction. J Thromb Haemost 2011; 9 (Suppl. 1): 26–34.
Summary. Serpins have been studied as a distinct protein
superfamily since the early 80s. In spite of the poor sequence
homology between family members, serpins share a highly
conserved core structure that is critical for their functioning as
serine protease inhibitors. Therefore, discoveries made about
one serpin can be related to the others. In this short review, I
introduce the serpin structure and general mechanism of
protease inhibition, and illustrate, using recent crystallographic
and biochemical data on antithrombin (AT), how serpin
activity can be modulated by cofactors. The ability of the
serpins to undergo conformational change is critical for their
function, but it also renders them uniquely susceptible to
mutations that perturb their folding, leading to deficiency and
disease. A recent crystal structure of an AT dimer revealed that
serpins can participate in large-scale domain-swaps to form
stable polymers, and that such a mechanism may explain the
accumulation of misfolded serpins within secretory cells.
Serpins play important roles in haemostasis and fibrinolysis,
and although each will have some elements specifically tailored
for its individual function, the mechanisms described here
provide a general conceptual framework.
Keywords: antithrombin, crystallography, haemostasis, hepa-
rin, polymerisation.
Meet the serpin superfamily
In a paper published in 1980, Hunt and Dayhoff [1] noted the
sequence similarity between two apparently unrelated proteins,
chicken ovalbumin (OVA) and human antithrombin III (AT),
and suggested a provisional name of the Ôovalbumin-anti-
thrombin superfamilyÕ. Previous work had found sequence
homology between AT and another human protease inhibitor,
a1-antitrypsin (a1AT, also known as a1-proteinase inhibitor)
[2]. In a seminal review of this new family and its members in
1985, Carrell [3] coined the now-widely used descriptive
Correspondence: James A. Huntington, Department of Haematology,
Cambridge Institute for Medical Research, University of Cambridge,
Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY,
UK.
Tel.: +44 1223 763230; fax: +44 1223 336827.
E-mail: jah52@cam.ac.uk
DOI: 10.1111/j.1538-7836.2011.04360.
acronym SERPIN, since most members of this family were
serine protease inhibitors. There are now well over 1500 serpin
sequences identified in the genomes of organisms representing
all kingdoms of life, and 36 confirmed human serpins [4]. Most
family members are indeed serine protease inhibitors, but
several have additional cross-class inhibition functions and
inhibit cysteine protease family members such as the caspases
and cathepsins, and others, such as OVA, are incapable of
protease inhibition and serve other functions. In plants and
other basic organisms small ÔcanonicalÕ inhibitors (such as
Kunitz and Kazal type inhibitors) predominate [5]. In humans,
serpins have been selected to control processes that require
tight regulation, such as blood coagulation, inflammation and
fibrinolysis. Much has been learnt since 1980 about the
function of individual serpins in human health and disease,
but a single question continues to define the field: Why has
evolution settled on a serpin to fulfil a particular function? The
answer lies in the unique serpin structure, the conserved
inhibitory mechanism, and the regulatory mechanisms that
govern serpin activity.
Serpin structure and metastability
Since serpins were known to be inhibitors of serine proteases,
it was clear that their structure would present a loop to
interact with the protease active site cleft. In other families of
serine protease inhibitors, these Ôreactive centre loopsÕ (RCL)
are short and are maintained in a rigid conformation ideal
for protease docking. In this case, inhibition depends on the
inability of the ends of the scissile bond (P1–P1¢, as per the
nomenclature of Schechter and Berger [6]) to separate after
proteolytic attack. The inhibitor thus binds tightly, but
reversibly to a protease, and neither protein undergoes
conformational change. In contrast, serpin RCLs are long
(typically 20–24 residues) and flexible, resembling a substrate
loop. The first crystal structure of a native serpin was that of
OVA [7], and its RCL was found to form a stable helix.
Subsequent structures of inhibitory serpins showed a range of
RCL conformations and a high degree of conformational
flexibility (often reflected by high B-factors or lack of electron
density). The native serpin is composed of approximately 400
amino acids that fold into an N-terminal helical domain and
a C-terminal b-barrel domain (Fig. 1A). The two main
features related to function are the RCL and the five-
 2011 International Society on Thrombosis and Haemostasis

stranded central b-sheet (b-sheet A). In the classic orienta-
tion, the RCL is on top and sheet A is facing (Fig. 1B).
The unusual, if not unique, aspect of the serpins is that their
native fold is not their most stable. Serpins are able to
incorporate the RCL into b-sheet A as the central fourth strand
(s4A), and in so doing, become hyperstable [8,9]. This confor-
mational/topological change can occur upon proteolytic cleav-
age within the RCL (Fig. 1C), or spontaneously to form the
intact ÔlatentÕ conformation (Fig. 1D). It was clear from early
A B P1
P1′
C D
3
6 5 4
2
1
Fig. 1. Structures of native and stable serpins. (A) A ribbon diagram of
the native form of prototypical serpin a1AT (1QLP, [81]) is shown col-
oured from its N-to-C terminus (blue to red), illustrating the domain
structure of serpins, with the N-terminal helical region (predominantly
blue and green) and the C-terminal b-sheet region (mostly yellow,
orange and red). The RCL is the orange loop on the ÔtopÕ of the mole-
cule and the scissile bond is illustrated by showing rods for the P1 and
P1¢ side chains (Met and Ser). (B) The same structure as in (A) is col-
oured to highlight the main structural features of a serpin, with the RCL in
yellow and b-sheet A in red. The P1 and P1¢ residues are indicated, and the
N-terminal portion (hinge region) of the RCL is indicated by the oval. (C)
When cleaved at the P1–P1¢ bond, inhibitory serpins such as a1AT un-
dergo a rapid conformational change where the RCL inserts as the fourth
strand in b-sheet A (strands are numbered, 8API, [17]). RCL residues that
participate in the antiparallel b-sheet interactions include P15-P3 for most
serpins. This conformation is hyper-stable. (D) A similar increase in sta-
bility can be achieved without proteolytic cleavage to form the ÔlatentÕ
conformation (here of the serpin plasminogen-activator inhibitor 1, 1LJ5).
 2011 International Society on Thrombosis and Haemostasis
Serpin structure, function and dysfunction 27
studies into the serpin mechanism of protease inhibition that the
ability to rapidly and stably incorporate the RCL into b-sheet A
distinguished the inhibitory (such as a1AT) from the non-
inhibitory serpins (such as OVA) [10,11]. However, the nature of
the inhibitory complex and the purpose of the conformational
change to a hyperstable state were not known until a crystal
structure of the final serpin-protease complex was solved.
The serpin mechanism
The long, flexible RCL of serpins and their ability to stably
incorporate the RCL into b-sheet A suggested a mechanism
other than the well characterised canonical lock-and-key
mechanism used by other families of protease inhibitors, but
just what that new mechanism might be was unclear. One
proposal was that partial incorporation of an intact RCL
would lock it into a canonical (key-like) conformation that
would inhibit proteases via a non-covalent, reversible mecha-
nism [12]. However, the fact that serpin-protease complexes
could be observed on SDS-PAGE suggested a covalent
complex, somehow stabilised by the full incorporation of the
cleaved RCL into sheet A [13]. Fluorescence energy transfer
studies appeared to support of both mechanisms [14,15]. In
2000, we solved and published the first crystallographic
structure of a final serpin-protease complex [16] (Fig. 2A).
Consistent with the fluorescence studies performed by the
Gettins group [15], we found a fully incorporated RCL with
A B
Lys328
P1
Asp194
Ser195
Fig. 2. The structure of the final serpin-protease complex. (A) A ribbon
diagram of the a1AT-trypsin complex is shown (1EZX, [16]), with a1AT
coloured as previously, and trypsin in green. To illustrate the region of
trypsin that was not observed in the crystal structure, native trypsin is
shown as a semi-transparent cyan cartoon. The P3-P1 region of a1AT and
the catalytic loop of trypsin are shown as rods. (B) A close-up of the boxed
region from panel A illustrates the covalent bond between the serpin P1
Met and Ser195 from trypsin, including original electron density for the
region (blue wire). The arrows indicate the nitrogen atoms that normally
form the oxyanion hole (note their distance from the carboxyl O of the P1
residue, indicated by the star), and the new salt-bridge between Asp194 of
trypsin and Lys328 of a1AT is also indicated.
28 J. A. Huntington
the bond between the P1 and P1¢ residue severed, exactly as in
the first structure of an RCL cleaved serpin [17] (Fig. 1C). The
protease was found at the bottom of the serpin, covalently
linked via an ester bond between the catalytic Ser195 of the
protease and main chain carbonyl carbon of the P1 residue
(Fig. 2B). This linkage represents the acyl-enzyme intermediate
of the serine protease catalytic cycle, after the expulsion of the
P¢ residues from the active site, but before the ingress of water
for deacylation. The catalytic loop of the protease was
distended by the pulling force exerted on it by the limited
length of the RCL and the clash between the body of the
protease and the body of the hyperstable serpin. This resulted
in the destruction of the oxyanion hole, required for stabilisa-
tion of the tetrahedral transition state, thus preventing
deacylation (Fig. 2B). In addition to the conformational
change in the serpin (full-loop insertion) and changes in the
catalytic site of the protease, about 40% of the protease also
appeared to have become disordered, based on lack of electron
density (Fig. 2A). The disordered region included residues 16–
41, 62-84, 110–120, 139–156, 186–190 and 223–224. Of these six
linear stretches, four (underlined) correspond to the serine
protease zymogen activation domain [18]. This domain tran-
sitions from a disordered to ordered state upon conversion
from a zymogen to a protease due to the insertion of the N-
terminus (Ile16 forms a salt-bridge with Asp194) into the
activation pocket (formed by the three C-terminal stretches
underlined above). The pulling force exerted on Ser195 by the
serpin extends the loop 3.5Å from its normal catalytic position,
thus destroying the oxyanion hole (main chain amides of
Gly193 and Ser195, Fig. 2B), and also breaking the salt-bridge
between Ile16 and Asp194, effectively converting the protease
back into a zymogen-like state. Intriguingly, in our structure,
Asp194 makes an alternate salt-bridge with Lys328 of the
serpin at the bottom strand 5A (Fig. 2B).
The extent of structural disorder engendered in the protease
by its complexation with serpins is a matter of some debate.
Early studies revealed that discrete regions of the protease
became accessible to proteolytic attack when in complex with
serpins, and these regions were later mapped to the disordered
region in the serpin-protease complex structure. Similarly,
trypsin was found to lose affinity for Ca2+ when in complex
with a1AT, suggesting disordering of the binding site (the 70s
loop) as seen in the crystal structure, and high Ca2+
concentrations were shown to promote the dissociation of the
complex, presumably by populating the protease conformation
[19]. Thrombin was shown to lose the ability to bind to exosite I
ligands (the 30s and 70s-loops) [20,21], and we recently showed
that both exosites I and II become dysfunctional due to the
disordering induced by serpin complexation [22]. These results
suggest a global disordering of the protease, and are consistent
with NMR data implying a molten-globule-like conformation
for proteases when complexed by serpins [23]. In striking
contrast, no disorder was observed in a recent crystal structure
of porcine pancreatic elastase in complex with a1AT [24].
However, on the weight of the currently available data, we can
conclude that some protease unfolding is induced by serpins,
but that the extent of unfolding may depend on the serpin-
protease pair. Factors that could affect the extent of unfolding
include the length of the RCL, the size and composition of
loops on the ÔbottomÕ of the serpin and of loops flanking the
active site of the protease, and the presence of ligands that
preferentially bind to the protease conformation.
Heparin modulation of serpin activity
The activity of several serpins is modulated by glycosamino-
glycans such as heparin [25]. In general, heparin serves to
ÔbridgeÕ the serpin to the protease, thus enhancing the rate of
complex formation. This is true for protein C inhibitor with
thrombin and activated protein C [26], for PAI-1 and thrombin
[27], for protease nexin 1 and thrombin [28], for ZPI and fXa
and fXIa [29], and importantly for AT and thrombin [30]. This
bridging effect also contributes to the heparin-acceleration of
thrombin inhibition by HCII [31], and fIXa and fXa inhibition
by AT [32], however, the majority of the rate enhancement in
these cases is provided by conformational change in the serpin
(allostery). In order for allostery to contribute to heparin
acceleration of serpin function, the native state of the serpin
must be in a low activity conformation, and the new heparin-
bound conformation must somehow enable the formation of
productive recognition (Michaelis) complexes with target
proteases. Over the years we and others have studied the
activation of AT by heparin, and have successfully determined
the molecular basis of the relative inactivity of the native form,
how heparin binds, how its binding results in conformational
change, and how the main targets of AT, thrombin, fXa and
fIXa, are recognised in the presence of heparin. AT is thus a
thoroughly described system that provides a paradigm for the
heparin binding serpins, as well as other serpins where binding
to cofactors and proteases is regulated by conformational
change (e.g. HCII [33], and even non-inhibitory serpins such as
thyroxine and corticosteroid binding globulins [34]).
AT conformations
AT circulates in a native state that is a poor inhibitor of its
target proteases, namely thrombin, fXa and fIXa. The work of
Steve Olson and Ingemar Björk (and others) elegantly demon-
strated that AT undergoes a conformational change upon
heparin binding that results in a 500-fold acceleration of fXa
and fIXa inhibition, but that thrombin is insensitive to the AT
conformational change (many papers, but most completely
described in Ref. [32]). The conformational basis for the low
activity of the circulating form of AT was unknown until the
structure of native AT was solved in 1994 [35,36]. It was found
to have the normal serpin fold, but with two important
differences – the RCL was partly incorporated into b-sheet A
(P15-P14 residues Gly379 and Ser380) and the P1 residue was
oriented toward the main body of AT and was thus unavailable
for interaction with a protease (Fig. 3A, centre panel).
Subsequent biochemical and structural work has confirmed
that the native conformation does have the RCL partially
 2011 International Society on Thrombosis and Haemostasis
 Serpin structure, function and dysfunction 29

Fig. 3. The AT native equilibrium and pentasaccharide binding mechanism. (A) AT exists in a low activity native state as an ensemble of conformations.
The left panel is the structure of native AT in the monomeric state (i.e. not crystallised in complex with latent AT, 1T1F, [41]), and shows the hinge
region inserted (P15-P14) into sheet A, resulting in the approximation of the RCL to the body of AT. The P1 residue (sticks) interacts via a salt-
bridge with Glu237 on the surface of AT. The middle panel is the structure of native AT in complex with latent AT (1E04, [82]). It shares hinge
region insertion, but the RCL has moved into a more exposed position, and the P1 residue makes only a weak main-chain hydrogen bond with the ÔtopÕ
of AT. The right panel represents the P1-exposed version of the centre panel, and is poised for reaction with a protease (e.g. thrombin and fXa). (B) Binding
of the heparin pentasaccharide (green rods) to AT (coloured as before) occurs in a three-step mechanism. The first (left) is presumably just the
approximation of the negatively charged heparin with the positively charged region along helix D (cyan), and involves no conformational rearrangement
of native AT (1E04). The second step (centre) involves conformational changes in the N-terminal (lower) portion of AT, and contributes 40% of the
induced-fit binding energy (3EVJ, [37]). The final step (right) involves the expulsion of the hinge and closure of sheet A (2GD4, [55]), followed by the
elongation of helix D. This step contributes 60% of the energy of binding and essentially locks AT into a high-activity state [37]. All representations of AT
in this and other figures exclude the N-terminal 44 residues. (C) AT can be subdivided into four fragments that move as rigid bodies during the heparin
activation steps, and plastic regions that move independently (grey). The largest fragment (blue) is considered rigid, and the green and yellow frag-
ments undergo their conformational change in the first heparin binding step (in panel B, left to centre). The red region, composed of the tops of strands
2 and 3A, slides over to close b-sheet A to the 5-stranded form during the final step of heparin binding (panel B, centre to right). The inherently
flexible regions, including the N-terminus and part of the RCL, were excluded from the analysis.

incorporated, and that expulsion of the hinge is sufficient for low activity state was unclear, due in part to crystal contacts
full allosteric activation [37–40]. However, the orientation of involving the RCL. A structure of AT in another crystal form
the P1 residue and its role in maintaining circulating AT in a [41] again showed the P1 oriented toward the main body of AT,

 2011 International Society on Thrombosis and Haemostasis

30 J. A. Huntington
however, in this case it was making a different, more intimate
contact (Fig. 3A, left panel). The RCL of native AT was thus
capable of at least two conformations that sequestered the
RCL away from an attacking protease, however, the appre-
ciable rate of thrombin inhibition in the absence of heparin
(7000 M)1 s)1) suggested that the P1 residue must be accessible
to some degree (as illustrated in Fig. 3A, right panel). Native
AT can thus be thought of as an ensemble of conformations,
such as those shown in Fig. 3A, in rapid equilibrium.
The effect of heparin binding on the conformation of AT can
be summarised as the expulsion of the RCL from b-sheet A, and
its subsequent closure so that the activated state of AT resembles
the native conformation of most other serpins (such as a1AT,
Fig. 1B, right panel). This conformational change improves
affinity for the specific heparin pentasaccharide by 1000-fold
through an induced-fit mechanism [42]. Recent structural and
biochemical data have revealed that conformational changes in
the lower helical part of the AT precede expulsion of the RCL
and contribute about 40% of the binding energy provided by the
conformational change [37,43]. Thus, the binding of AT to the
specific heparin pentasaccharide occurs through three steps
(Fig. 3B): the first is the non-specific association of the
negatively charged heparin with the basic heparin binding site
on helix D (cyan in figure); the second step is the conformational
rearrangement of the helical domain of AT to accommodate the
pentasaccharide and maximise ionic and hydrogen bonding
interactions; in the third step, the hinge is released from b-sheet
A, and the top portions of strands 2 and 3 slide over to make a
continuous five stranded b-sheet. Careful structural analysis of
these structures reveal a large constant domain, primarily
composed of the C-terminal b-barrel domain (blue in Fig. 3C),
three regions that move as rigid bodies (green, yellow and red),
and loops that move independently (grey) [37]. In a reversal of
the heparin binding mechanism, full incorporation of the RCL
that accompanies formation of the final complex between AT
and a protease results in a 1000-fold decrease in heparin affinity
[43,44]. This analysis also provides a detailed paradigm for the
general mechanism of modulation of serpin activity through
conformational change.
Heparin activation of AT
Determining the mechanism of heparin activation of AT is
crucial for understanding the mode of action of the numerous
heparin fractionations and synthetic heparins currently on the
market as anticoagulants. In all cases, specificity and high
affinity binding to AT is conferred by the presence of the
pentasaccharide sequence, present in about a third of unfrac-
tionated heparin chains. All such chains will allosterically
activate AT towards fXa and fIXa inhibition. However, chains
must be 18 monosaccharide units in length (5.4 kDa) to
accelerate thrombin inhibition by a bridging mechanism [45],
and about twice as long to bridge fXa and fIXa to AT [46].
Most low molecular weight heparin preparations contain a
distribution of sizes close to that capable of bridging thrombin,
but only unfractionated heparin is likely to bridge factors IXa
and Xa. It has long been understood that thrombin inhibition
is insensitive to the conformation of AT [47], and thus the
recognition complex should not require the expulsion of the
RCL from b-sheet A. The crystal structure of the ternary
complex (solved with a synthetic hexadecasaccharide) showed
why, with thrombin positioned forward and towards the
heparin binding site of AT (Fig. 4A) [48]. The hinge region
could not be fully resolved in electron density due to flexibility,
however, clear evidence for the pre-insertion of P15 was seen,
and the position of thrombin could easily be maintained with
the RCL inserted to the level seen in native AT. When the hinge
region was prevented from being expelled from b-sheet A by an
engineered disulphide bridge, the rate of thrombin inhibition in
the presence of heparin was actually slightly increased [37].
Thus, the position of thrombin found in this structure is equally
valid for heparin-activated and native AT. One important
A B
147-loop
60-loop
C
Fig. 4. Structure of the recognition (Michaelis) complex between AT and
thrombin in the presence of an activating heparin chain. (A) A ribbon
diagram of the ternary complex between AT (coloured as previously),
S195A thrombin (heavy chain in magenta, light chain in light pink), and
a synthetic hexadecasaccharide (green rods) containing the pentasaccha-
ride sequence and a highly sulphated thrombin binding site [48,83]. The
hinge region is in a similar conformation as that seen in native AT,
with P15 Gly making b-strand H-bonds (the rest of the hinge is not
resolved in electron density), due to the forward orientation of throm-
bin on AT. (B) A surface representation of thrombin and AT without
the RCL (coloured and oriented as in panel A) illustrates the intimate
nature of the contact between the body of AT and the 60- and 147-
insertion loops (indicated). (C) A close-up of the RCL is shown to illus-
trate the elongation of the P¢ insert (green) to allow the orientation of
thrombin relative to AT and to accommodate the 60- and 147-insertion
loops. The RCL of pentasaccharide-bound AT (1E03, [84]) is shown in
orange, with the P¢ insert in green.
 2011 International Society on Thrombosis and Haemostasis

feature of thrombin is the depth of the active site cleft due to the
60 and 147-insertion loops, both of which make significant
contacts with the body of AT (Fig. 4B). AT has a unique three-
residue insert on the P¢ side of the RCL (shown in green in
Fig. 4C) that normally forms a tightly H-bonded turn, and that
is seen to unwind in the Michaelis complex to accommodate
the bulk of the 60 and 147-insertion loops and to allow the
forward orientation of thrombin (Fig. 4C). Consistent with
these findings, deletion of the three-residue insert reduces the
rate of thrombin inhibition [49]. Thus, recognition of thrombin
by AT in the absence and presence of heparin is independent of
the extension of the N-terminal portion of the RCL (the hinge
region), but does require the extension of the C-terminal
portion (the P¢ region).
In contrast, recognition of fXa and fIXa by AT is exquisitely
sensitive to the conformation of the hinge region. Disulphide
engineering studies have shown that fXa can form two separate
recognition complexes depending on the state of the hinge
region [37,50]. Presumably when AT is in the native state the
recognition complex would resemble that of thrombin. How-
ever, fIXa can only react with thrombin when the hinge region is
expelled and free to extend away from b-sheet A. It was also
clear from studies by Alireza Rezaie and Steve OlsonÕs groups
that the 147-loop (and Arg150 in particular) made a crucial
exosite contact with the body of AT [51–54]. The structures of
the pentasaccharide-activated recognition complexes between
AT and fXa [55] (Fig. 5A) and fIXa [56] (Fig. 5B) revealed a
rotation in the protease (relative to the position observed for
thrombin) that resulted in an orientation requiring hinge region
extension (Fig. 5C). The rotation allowed the 147-loop of the
protease to engage in extensive contacts with AT, and of
particular importance, the burying of Arg150 in an acidic (and
also hydrophobic) pocket formed by Arg235, Glu237, Met251,
Tyr253, and His319. Intriguingly, this pocket is occupied in the
native monomeric state of AT by the P1 residue in a similar
fashion (Fig. 5D), supporting the idea that heparin binding
ÔexposesÕ the exosite utilised by factors IXa and Xa [57]. The
unique AT P¢ insert is partially unwound to allow fXa to rotate
into the observed position, but the tight H-bonded turn is
preserved in the complex with fIXa and makes several important
interactions to improve the stability of the complex [56]. Thus,
the RCL of AT has unique features that are exploited to allow
for heparin regulation of protease binding and inhibition.
Serpin dysfunction
The advantages of the serpin mechanism over the standard lock-
and-key mechanism are manifold, and include: the ability to
regulate activity by altering the conformation or accessibility of
the RCL (AT activation by heparin is a good example); the
covalent complex is irreversible; the change in serpin and
protease structure releases complexes from cofactors and
substrates (e.g. thrombin loses the ability to interact with exosite
I and exosite II ligands [22], and AT loses the ability to bind with
high affinity to heparin [58]); and change in serpin and/or
protease conformation can result in receptor binding and
 2011 International Society on Thrombosis and Haemostasis
Serpin structure, function and dysfunction 31
A B
C D
Fig. 5. Crystal structures of the pentasaccharide-activated complexes be-
tween AT and factors Xa and IXa. (A) A ribbon diagram of the ternary
complex between AT (coloured as previously), fXa (epidermal growth-
factor [EGF] domain 2 [light blue] plus S195A variant of the catalytic
domain [slate blue]) and the pentasaccharide (green sticks) (2GD4) reveals
exosite contacts that necessitate the expulsion and extension of the hinge
region of the RCL. The critical contact involves residue Arg150 of the
protease (sticks). (B) The same exosite contact (Arg150 in stick represen-
tation) was observed in the crystal structure of the AT, fIXa (magenta),
pentasaccharide complex (3KCG), although the fIXa was rotated by
about 40. (C) The requirement for hinge region expulsion and extension is
best visualised by comparing the position of thrombin on AT to that of
fIXa on AT. The heavy chain of fIXa is shown coloured from N-to-C
terminus from blue-to-red, and is placed in the position found in its
complex with AT (opaque) and in the position of thrombin (semitrans-
parent). The stereo representation helps to appreciate the radical move-
ment of the protease domain, involving a 16Å translation and a 104
rotation. (D) To illustrate the exosite interaction involving Arg150 (cyan
for fXa and magenta for fIXa), the surface of AT (1T1F, without the
RCL) is shown coloured according to electrostatics. The P1 Arg (yellow)
of AT competes for the same pocket when in the native form.
signalling [59]. However, the inherent dependence of the serpin
mechanism on conformational mobility and a metastable native
fold render serpins highly susceptible to mutations that perturb
function [60,61]. There are many things that can go wrong with
serpins that lead to loss or gain of function (for reviews see Refs
[60–63] and others). Classic examples are mutations in the RCL
that either change specificity (such as the Pittsburgh variant of
a1AT that turns it into an inhibitor of thrombin [64]) or knock
out inhibitory function by slowing RCL incorporation into
sheet A (such as the Cambridge II mutation in AT [65]). Another
common cause of serpin deficiency is the failure to secrete active
protein due to mutations that affect the ability of serpins to fold
into the metastable native state. These mutations can occur
almost anywhere in the serpin, and are normally associated with
the formation and accumulation of stable polymers within the
endoplasmic reticulum of secretory cells (for reviews see Refs
32 J. A. Huntington

[66,67] and others). Polymerisation always leads to lower levels A B

of secreted serpin, and in cases where the serpin concentration is
critical, heterozygous deficiency leads to disease (such as
thrombosis for AT). In other cases, only the homozygous
carriers of mutations develop a loss-of-function disease (such as
emphysema for a1AT). In rare cases, the accumulation of serpin
polymers is toxic to cells and leads to tissue damage through an
unknown gain-of-function mechanism [68]. Several homozy-
gous mutations in a1AT have been associated with liver disease,
including cirrhosis, and several heterozygous mutations in
neuroserpin have been described that lead to dementia and C
death. Although the incidence of disease caused by serpin
polymerisation (through both loss- and gain-of-function mech-
anisms) is quite rare, there has been a great deal of interest in the
phenomenon, due in part to its classification as a Ôconforma-
tional diseaseÕ, along with AlzheimerÕs, HuntingtonÕs and
ParkinsonÕs diseases, and the prion encephalopathies [69].

The mechanism of serpin polymerisation

Consistent with the polymerisation of a folding intermediate in
cells, serpins are capable of polymerising in vitro when
incubated under mildly denaturing conditions (heat, low pH
or chaotropic agents, or in combination) [70]. It has been
demonstrated for a1AT and for neuroserpin that unfolding
and folding proceed via an intermediate that is prone to
polymerisation [71–75]. For wild-type protein this intermediate
is short-lived and will thus form only a small amount of
polymers upon renaturation, but mutations that slow the
conversion from intermediate to native state result predomi-
nantly in polymeric serpin. Polymers obtained from cells or by
partial denaturation have similar properties and are thought to
share a common intermolecular linkage. Indeed, it has long
been believed that the molecular basis of polymerisation is the
same for all serpins, whether induced by mutations or by mild
denaturation, and that the mechanism involved the incorpo-
ration of the RCL of one monomer into b-sheet A of another
(the Ôloop-sheetÕ mechanism). However, there is no direct
evidence for the formation of loop-sheet polymers by partial
denaturation or in vivo, and several experiments contradicting
the loop-sheet hypothesis have been reported [76–78]. An
alternative mechanism of polymerisation was suggested by a
recent crystal structure of a stable AT dimer formed by
incubating native AT at pH 5.7, 37 C [79]. AT was found to
have undergone a large domain swap that included both the
RCL (strand 4A) and strand 5A, along with other regions
(Fig. 6A). The structure could readily be extended to form an
open dimer with a flexible linker region (Fig. 6B), and
suggested a polymerigenic folding intermediate (or unfolding
intermediate) that had strand 5A as well as the RCL exposed
(Fig. 6C). Several experiments have been conducted to deter-
mine if this mechanism is general for the serpins, and
preliminary data suggest that it may account for AT, a1-AT
and neuroserpin polymerisation under certain conditions
[72,79,80]. More work is required to determine if this or other
domain swaps are responsible for polymerisation of serpins
Fig. 6. Structure and models related to the Ôdomain-swapÕ mode of serpin
polymerisation. (A) A crystal structure of a dimer of AT (2ZNH, [79])
revealed that the basis of intermolecular linkage was the swapping of
strands 4 and 5 of b-sheet A from one monomer (yellow) into another
(green). (B) The closed dimer could be opened by simply uncoiling part of
the region following strand 6A, to form a head-to-tail dimer that could be
elongated indefinitely. (C) The dimer structure showed that strand 5A
extraction from its position in the native state is required to form the
polymeric linkage, therefore suggesting a polymerigenic folding interme-
diate such as the model shown here (coloured as before).
within cells, and if the regions that participate in domain
swapping depend on the serpin and the mutations involved.
Conclusions
Serpins are found in all branches of life and appear time-and-
again controlling proteolytic pathways related to human health
and disease. It was perhaps serendipity that the most important
anticoagulant serpin AT was a founding member of the
superfamily, but nevertheless, AT provides a useful and well-
characterised model for understanding the possible range of
serpin structure and how it relates to serpin function and
dysfunction.
Acknowledgements
JAH is a Senior Medical Research Council (MRC) Non-
clinical Fellow.
Disclosure of Conflict of Interests
The author states that he has no conflict of interest.

 2011 International Society on Thrombosis and Haemostasis


References
1 Hunt LT, Dayhoff MO. A surprising new protein superfamily con-
taining ovalbumin, antithrombin-III, and alpha 1-proteinase inhibitor.
Biochem Biophys Res Commun 1980; 95: 864–71.
2 Borsodi AD, Bradshaw RA, Rughani IK, Bruce RM. Structural and
functional relationships of human antithrombin III and alpha-anti-
trypsin. Adv Exp Med Biol 1975; 52: 249–53.
3 Carrell R, Travis J. [alpha]1-Antitrypsin and the serpins: variation and
countervariation. Trends Biochem Sci 1985; 10: 20–4.
4 Law RH, Zhang Q, McGowan S, Buckle AM, Silverman GA, Wong
W, Rosado CJ, Langendorf CG, Pike RN, Bird PI, Whisstock JC. An
overview of the serpin superfamily. Genome Biol 2006; 7: 216.
5 Rawlings ND, Tolle DP, Barrett AJ. Evolutionary families of pepti-
dase inhibitors. Biochem J 2004; 378: 705–16.
6 Schechter I, Berger A. On the size of the active site in proteases. I.
Papain. Biochem Biophys Res Commun 1967; 27: 157–62.
7 Stein PE, Leslie AG, Finch JT, Turnell WG, McLaughlin PJ, Carrell
RW. Crystal structure of ovalbumin as a model for the reactive centre
of serpins. Nature 1990; 347: 99–102.
8 Im H, Ahn HY, Yu MH. Bypassing the kinetic trap of serpin protein
folding by loop extension. Protein Sci 2000; 9: 1497–502.
9 Kaslik G, Kardos J, Szabo E, Szilagyi L, Zavodszky P, Westler WM,
Markley JL, Graf L. Effects of serpin binding on the target proteinase:
global stabilization, localized increased structural flexibility, and con-
served hydrogen bonding at the active site. Biochemistry 1997; 36:
5455–64.
10 Stein PE, Tewkesbury DA, Carrell RW. Ovalbumin and angiotensi-
nogen lack serpin S-R conformational change. Biochem J 1989; 262:
103–7.
11 Hood DB, Huntington JA, Gettins PG. Alpha 1-proteinase inhibitor
variant T345R. Influence of P14 residue on substrate and inhibitory
pathways. Biochemistry 1994; 33: 8538–47.
12 Engh RA, Huber R, Bode W, Schulze AJ. Divining the serpin inhi-
bition mechanism: a suicide substrate ÔspringeÕ? Trends Biotechnol
1995; 13: 503–10.
13 Lawrence DA, Ginsburg D, Day DE, Berkenpas MB, Verhamme IM,
Kvassman JO, Shore JD. Serpin-protease complexes are
trapped as stable acyl-enzyme intermediates. J Biol Chem 1995; 270:
25309–12.
14 Wilczynska M, Fa M, Karolin J, Ohlsson PI, Johansson LB, Ny T.
Structural insights into serpin-protease complexes reveal the inhibitory
mechanism of serpins. Nat Struct Biol 1997; 4: 354–7.
15 Stratikos E, Gettins PG. Formation of the covalent serpin-proteinase
complex involves translocation of the proteinase by more than 70 A
and full insertion of the reactive center loop into beta-sheet A. Proc
Natl Acad Sci U S A 1999; 96: 4808–13.
16 Huntington JA, Read RJ, Carrell RW. Structure of a serpin-
protease complex shows inhibition by deformation. Nature 2000; 407:
923–6.
17 Loebermann H, Tokuoka R, Deisenhofer J, Huber R. Human alpha 1-
proteinase inhibitor. Crystal structure analysis of two crystal modifi-
cations, molecular model and preliminary analysis of the implications
for function. J Mol Biol 1984; 177: 531–57.
18 Huber R, Bode W. Structural basis of the activation and action of
trypsin. Acc Chem Res 1978; 11: 114–22.
19 Calugaru SV, Swanson R, Olson ST. The pH dependence of serpin-
proteinase complex dissociation reveals a mechanism of complex sta-
bilization involving inactive and active conformational states of the
proteinase which are perturbable by calcium. J Biol Chem 2001; 276:
32446–55.
20 Fredenburgh JC, Stafford AR, Weitz JI. Conformational changes in
thrombin when complexed by serpins. J Biol Chem 2001; 276: 44828–
34.
21 Bock PE, Olson ST, Bjork I. Inactivation of thrombin by antithrombin
is accompanied by inactivation of regulatory exosite I. J Biol Chem
1997; 272: 19837–45.
 2011 International Society on Thrombosis and Haemostasis
Serpin structure, function and dysfunction 33
22 Li W, Johnson DJ, Adams TE, Pozzi N, De Filippis V, Huntington
JA. Thrombin inhibition by serpins disrupts exosite II. J Biol Chem
2010; 285: 38621–9.
23 Peterson FC, Gettins PG. Insight into the mechanism of serpin-pro-
teinase inhibition from 2D [1H-15N] NMR studies of the 69 kDa
alpha 1-proteinase inhibitor Pittsburgh-trypsin covalent complex.
Biochemistry 2001; 40: 6284–92.
24 Dementiev A, Dobo J, Gettins PG. Active site distortion is sufficient
for proteinase inhibition by serpins: structure of the covalent complex
of alpha1-proteinase inhibitor with porcine pancreatic elastase. J Biol
Chem 2006; 281: 3452–7.
25 Rau JC, Beaulieu LM, Huntington JA, Church FC. Serpins in
thrombosis, hemostasis and fibrinolysis. J Thromb Haemost 2007; 5
(Suppl. 1): 102–15.
26 Li W, Huntington JA. The heparin binding site of protein C inhibitor is
protease-dependent. J Biol Chem 2008; 283: 36039–45.
27 Ehrlich HJ, Keijer J, Preissner KT, Gebbink RK, Pannekoek H.
Functional interaction of plasminogen activator inhibitor type 1 (PAI-
1) and heparin. Biochemistry 1991; 30: 1021–8.
28 Cunningham DD, Wagner SL, Farrell DH. Regulation of protease
nexin-1 activity by heparin and heparan sulfate. Adv Exp Med Biol
1992; 313: 297–306.
29 Huang X, Rezaie AR, Broze GJ Jr, Olson ST. Heparin is a major
activator of the anticoagulant serpin, protein Z-dependent protease
inhibitor (ZPI). J Biol Chem 2011; 286: 8740–51.
30 Olson ST, Bjork I. Predominant contribution of surface approxima-
tion to the mechanism of heparin acceleration of the antithrombin-
thrombin reaction. Elucidation from salt concentration effects. J Biol
Chem 1991; 266: 6353–64.
31 Verhamme IM, Bock PE, Jackson CM. The preferred pathway of
glycosaminoglycan-accelerated inactivation of thrombin by heparin
cofactor II. J Biol Chem 2004; 279: 9785–95.
32 Olson ST, Swanson R, Raub-Segall E, Bedsted T, Sadri M, Petitou M,
Herault JP, Herbert JM, Bjork I. Accelerating ability of synthetic ol-
igosaccharides on antithrombin inhibition of proteinases of the clotting
and fibrinolytic systems. Comparison with heparin and low-molecular-
weight heparin. Thromb Haemost 2004; 92: 929–39.
33 Baglin TP, Carrell RW, Church FC, Esmon CT, Huntington JA.
Crystal structures of native and thrombin-complexed heparin cofactor
II reveal a multistep allosteric mechanism. Proc Natl Acad Sci U S A
2002; 99: 11079–84.
34 Qi X, Loiseau F, Chan WL, Yan Y, Wei Z, Milroy LG, Myers RM,
Ley SV, Read RJ, Carrell RW, Zhou A. Allosteric modulation of
hormone release from thyroxine and corticosteroid binding-globulins.
J Biol Chem 2011; 286: 16163–73.
35 Schreuder HA, de Boer B, Dijkema R, Mulders J, Theunissen HJ,
Grootenhuis PD, Hol WG. The intact and cleaved human anti-
thrombin III complex as a model for serpin-proteinase interactions.
Nat Struct Biol 1994; 1: 48–54.
36 Carrell RW, Stein PE, Fermi G, Wardell MR. Biological implications
of a 3 A structure of dimeric antithrombin. Structure 1994; 2: 257–70.
37 Langdown J, Belzar KJ, Savory WJ, Baglin TP, Huntington JA. The
critical role of hinge-region expulsion in the induced-fit heparin binding
mechanism of antithrombin. J Mol Biol 2009; 386: 1278–89.
38 Futamura A, Gettins PG. Serine 380 (P14) fi glutamate mutation
activates antithrombin as an inhibitor of factor Xa. J Biol Chem 2000;
275: 4092–8.
39 Huntington JA, Gettins PG. Conformational conversion of anti-
thrombin to a fully activated substrate of factor Xa without need for
heparin. Biochemistry 1998; 37: 3272–7.
40 Huntington JA, Olson ST, Fan B, Gettins PG. Mechanism of heparin
activation of antithrombin. Evidence for reactive center loop prein-
sertion with expulsion upon heparin binding. Biochemistry 1996; 35:
8495–503.
41 Johnson DJ, Langdown J, Li W, Luis SA, Baglin TP, Huntington JA.
Crystal structure of monomeric native antithrombin reveals a novel
reactive center loop conformation. J Biol Chem 2006; 281: 35478–86.
34 J. A. Huntington
42 Olson ST, Srinivasan KR, Bjork I, Shore JD. Binding of high affinity
heparin to antithrombin III. Stopped flow kinetic studies of the
binding interaction. J Biol Chem 1981; 256: 11073–9.
43 Schedin-Weiss S, Richard B, Olson ST. Kinetic evidence that allosteric
activation of antithrombin by heparin is mediated by two sequential
conformational changes. Arch Biochem Biophys 2010; 504: 169–76.
44 Skinner R, Chang WS, Jin L, Pei X, Huntington JA, Abrahams JP,
Carrell RW, Lomas DA. Implications for function and therapy of a 2.9
A structure of binary-complexed antithrombin. J Mol Biol 1998; 283:
9–14.
45 Bray B, Lane DA, Freyssinet JM, Pejler G, Lindahl U. Anti-thrombin
activities of heparin. Effect of saccharide chain length on thrombin
inhibition by heparin cofactor II and by antithrombin. Biochem J 1989;
262: 225–32.
46 Rezaie AR. 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 1998; 273: 16824–7.
47 Olson ST, Bjork I. Role of protein conformational changes, surface
approximation and protein cofactors in heparin-accelerated anti-
thrombin-proteinase reactions. Adv Exp Med Biol 1992; 313: 155–65.
48 Li W, Johnson DJ, Esmon CT, Huntington JA. Structure of the
antithrombin-thrombin-heparin ternary complex reveals the anti-
thrombotic mechanism of heparin. Nat Struct Mol Biol 2004; 11: 857–
62.
49 Rezaie AR. Partial activation of antithrombin without heparin
through deletion of a unique sequence on the reactive site loop of the
serpin. J Biol Chem 2002; 277: 1235–9.
50 Langdown J, Johnson DJ, Baglin TP, Huntington JA. Allosteric
activation of antithrombin critically depends upon hinge region
extension. J Biol Chem 2004; 279: 47288–97.
51 Yang L, Manithody C, Olson ST, Rezaie AR. Contribution of basic
residues of the autolysis loop to the substrate and inhibitor specificity
of factor IXa. J Biol Chem 2003; 278: 25032–8.
52 Manithody C, Yang L, Rezaie AR. Role of basic residues of the
autolysis loop in the catalytic function of factor Xa. Biochemistry 2002;
41: 6780–8.
53 Izaguirre G, Olson ST. Residues Tyr253 and Glu255 in strand 3 of
beta-sheet C of antithrombin are key determinants of an exosite made
accessible by heparin activation to promote rapid inhibition of factors
Xa and IXa. J Biol Chem 2006; 281: 13424–32.
54 Izaguirre G, Zhang W, Swanson R, Bedsted T, Olson ST. Localization
of an antithrombin exosite that promotes rapid inhibition of factors Xa
and IXa dependent on heparin activation of the serpin. J Biol Chem
2003; 278: 51433–40.
55 Johnson DJ, Li W, Adams TE, Huntington JA. Antithrombin-S195A
factor Xa-heparin structure reveals the allosteric mechanism of anti-
thrombin activation. EMBO J 2006; 25: 2029–37.
56 Johnson DJ, Langdown J, Huntington JA. Molecular basis of factor
IXa recognition by heparin-activated antithrombin revealed by a 1.7-A
structure of the ternary complex. Proc Natl Acad Sci U S A 2010; 107:
645–50.
57 Olson ST, Chuang YJ. Heparin activates antithrombin anticoagulant
function by generating new interaction sites (exosites) for blood clot-
ting proteinases. Trends Cardiovasc Med 2002; 12: 331–8.
58 Carlstrom AS, Lieden K, Bjork I. Decreased binding of heparin to
antithrombin following the interaction between antithrombin and
thrombin. Thromb Res 1977; 11: 785–97.
59 Huntington JA. Shape-shifting serpins – advantages of a mobile
mechanism. Trends Biochem Sci 2006; 31: 427–35.
60 Kaiserman D, Whisstock JC, Bird PI. Mechanisms of serpin dys-
function in disease. Expert Rev Mol Med 2006; 8: 1–19.
61 Gooptu B, Lomas DA. Conformational pathology of the serpins:
themes, variations, and therapeutic strategies. Annu Rev Biochem 2009;
78: 147–76.
62 Devlin GL, Bottomley SP. A protein family under ÔstressÕ – serpin
stability, folding and misfolding. Front Biosci 2005; 10: 288–99.
63 Janciauskiene S. Conformational properties of serine proteinase
inhibitors (serpins) confer multiple pathophysiological roles. Biochim
Biophys Acta 2001; 1535: 221–35.
64 Owen MC, Brennan SO, Lewis JH, Carrell RW. Mutation of anti-
trypsin to antithrombin. alpha 1-antitrypsin Pittsburgh (358 Met leads
to Arg), a fatal bleeding disorder. N Engl J Med 1983; 309: 694–8.
65 Mushunje A, Zhou A, Carrell RW, Huntington JA. Heparin-induced
substrate behavior of antithrombin Cambridge II. Blood 2003; 102:
4028–34.
66 Belorgey D, Hagglof P, Karlsson-Li S, Lomas DA. Protein misfolding
and the serpinopathies. Prion 2007; 1: 15–20.
67 Knaupp AS, Bottomley SP. Serpin polymerization and its role in
disease – the molecular basis of alpha1-antitrypsin deficiency. IUBMB
Life 2009; 61: 1–5.
68 Davies MJ, Lomas DA. The molecular aetiology of the serpinopathies.
Int J Biochem Cell Biol 2008; 40: 1273–86.
69 Carrell RW, Lomas DA. Conformational disease. Lancet 1997; 350:
134–8.
70 Belorgey D, Irving JA, Ekeowa UI, Freeke J, Roussel BD, Miranda E,
Perez J, Robinson CV, Marciniak SJ, Crowther DC, Michel CH,
Lomas DA. Characterisation of serpin polymers in vitro and in vivo.
Methods 2010; 53: 255–66.
71 Yu MH, Lee KN, Kim J. The Z type variation of human alpha 1-
antitrypsin causes a protein folding defect. Nat Struct Biol 1995; 2:
363–7.
72 Takehara S, Zhang J, Yang X, Takahashi N, Mikami B, Onda M.
Refolding and polymerization pathways of neuroserpin. J Mol Biol
2010; 403: 751–62.
73 Dafforn TR, Mahadeva R, Elliott PR, Sivasothy P, Lomas DA. A
kinetic mechanism for the polymerization of alpha1-antitrypsin. J Biol
Chem 1999; 274: 9548–55.
74 JamesEL,WhisstockJC,GoreMG,BottomleySP.Probing the unfolding
pathway of alpha1-antitrypsin. J Biol Chem 1999; 274: 9482–8.
75 Devlin GL, Chow MK, Howlett GJ, Bottomley SP. Acid denaturation
of alpha1-antitrypsin: characterization of a novel mechanism of serpin
polymerization. J Mol Biol 2002; 324: 859–70.
76 Huntington JA, Sendall TJ, Yamasaki M. New insight into serpin
polymerization and aggregation. Prion 2009; 3: 12–4.
77 Huntington JA, Whisstock J. Molecular contortionism- on the phys-
ical limits of serpin loop-sheet polymers. Biol Chem 2010; 391: 973–82.
78 Yamasaki M, Sendall TJ, Harris LE, Lewis GM, Huntington JA.
Loop-sheet mechanism of serpin polymerization tested by reactive
center loop mutations. J Biol Chem 2010; 285: 30752–8.
79 Yamasaki M, Li W, Johnson DJ, Huntington JA. Crystal structure of
a stable dimer reveals the molecular basis of serpin polymerization.
Nature 2008; 455: 1255–8.
80 Ordonez A, Martinez-Martinez I, Corrales FJ, Miqueo C, Minano A,
Vicente V, Corral J. Effect of citrullination on the function and con-
formation of antithrombin. FEBS J 2009; 276: 6763–72.
81 Elliott PR, Pei XY, Dafforn TR, Lomas DA. Topography of a 2.0 A
structure of alpha1-antitrypsin reveals targets for rational drug design
to prevent conformational disease. Protein Sci 2000; 9: 1274–81.
82 McCoy AJ, Pei XY, Skinner R, Abrahams JP, Carrell RW. Structure
of beta-antithrombin and the effect of glycosylation on antithrombinÕs
heparin affinity and activity. J Mol Biol 2003; 326: 823–33.
83 Herbert JM, Herault JP, Bernat A, Savi P, Schaeffer P, Driguez PA,
Duchaussoy P, Petitou M. SR123781A, a synthetic heparin mimetic.
Thromb Haemost 2001; 85: 852–60.
84 Jin L, Abrahams JP, Skinner R, Petitou M, Pike RN, Carrell RW. The
anticoagulant activation of antithrombin by heparin. Proc Natl Acad
Sci U S A 1997; 94: 14683–8.
 2011 International Society on Thrombosis and Haemostasis