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Review article
Structure and function of factor XI
Jonas Emsley,1 Paul A. McEwan,1 and David Gailani2
1School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom; and 2Departments of Pathology and Medicine,
Vanderbilt University, Nashville, TN
Factor XI (FXI) is the zymogen of an enzyme
(FXIa) that contributes to hemostasis by
activating factor IX. Although bleeding associated with FXI deficiency is relatively mild,
there has been resurgence of interest in FXI
because of studies indicating it makes contributions to thrombosis and other processes associated with dysregulated coagulation. FXI is an unusual dimeric protease,
with structural features that distinguish it
from vitamin K–dependent coagulation pro-
teases. The recent availability of crystal
structures for zymogen FXI and the FXIa
catalytic domain have enhanced our understanding of structure-function relationships
for this molecule. FXI contains 4 “apple domains” that form a disk structure with extensive interfaces at the base of the catalytic
domain. The characterization of the apple
disk structure, and its relationship to the
catalytic domain, have provided new insight
into the mechanism of FXI activation, the
interaction of FXIa with the substrate factor
IX, and the binding of FXI to platelets. Analyses of missense mutations associated with
FXI deficiency have provided additional
clues to localization of ligand-binding sites
on the protein surface. Together, these data
will facilitate efforts to understand the physiology and pathology of this unusual protease, and development of therapeutics to
treat thrombotic disorders. (Blood. 2010;
115(13):2569-2577)
Introduction
Factor XI (FXI) is the zymogen of a blood coagulation protease,
factor XIa (FXIa), that contributes to hemostasis through
activation of factor IX (FIX; Figure 1).1-3 The protein is a
160-kDa disulfide-linked dimer of identical 607 amino acid
subunits, each containing 4 90- or 91-amino acid repeats called
apple domains (A1 to A4 from the N-terminus) and a C-terminal
trypsin-like catalytic domain.3-7 The structure is distinctly different from those of the well-characterized vitamin K–dependent
coagulation proteases.1 FXI circulates in blood as a complex with
high molecular weight kininogen (HK).8 Prekallikrein (PK), the
zymogen of the protease ␣-kallikrein, is a monomeric homolog of
FXI with the same domain structure9,10 that also circulates in
complex with HK.11 A recent analysis of vertebrate genomes
confirmed that FXI and PK are products of a duplication event
during mammalian evolution.12 The ancestral predecessor is also a
protease with 4 apple domains, but its functional properties have
not been studied.12
In the cascade/waterfall models of coagulation that are the basis
for the activated partial thromboplastin time (aPTT) assay, FXI
activation by FXIIa initiates fibrin formation (Figure 1).1 However,
newer schemes do not assign FXI a role in early fibrin generation,
based largely on the observation that FXI deficiency causes
relatively mild bleeding.1-3 FXIa is now postulated to be part of a
feedback loop that sustains thrombin generation through FIX
activation to consolidate coagulation (Figure 1).13-15 This appears
to be particularly important in tissues with robust fibrinolytic
activity, such as the oropharynx and urinary tract, which are
common sites of bleeding in FXI-deficient patients.2,16-23
Congenital FXI deficiency is associated with a variable, mild to
moderate bleeding disorder.2,16,17 Severe deficiency (⬍ 15% of
normal plasma activity) is prevalent in people of Jewish ancestry.2,16,17,24 The carrier rate is approximately 5% in Ashkenazi Jews,
with severe (homozygous) deficiency found in 1 in 450 persons.16,24 Two mutations account for more than 90% of abnormal
alleles in this population.24,25 The severe mutation Glu117Stop
encodes a truncated protein,25 and homozygotes lack plasma FXI
antigen. The more subtle missense mutation Phe283Leu causes a
defect in FXI dimer formation.26-28 More than 180 other FXI gene
mutations associated with FXI deficiency have been reported
(http://www.factorxi.org, http://www.isth.org),29 including more
than 100 single amino acid (missense) substitutions. Congenital
deficiencies of vitamin K–dependent coagulation proteases are
often associated with dysfunctional protein in plasma (crossreactive material positive [CRM⫹] deficiency).30 In contrast, most
cases of FXI deficiency are associated with low plasma levels of
FXI protein (CRM⫺ deficiency).31
There has been renewed interest in FXI as a result of populationbased studies and work with animal models strongly indicating that
this protein makes important contributions to arterial32-36 and
venous37 thrombosis, ischemia-reperfusion injury in the central
nervous system,38 and the pathology of sepsis.39 The observation
that deficiency or inhibition of FXI interferes with platelet accumulation in growing thrombi33,35 has led to a reanalysis of the
mechanisms involved in FXI activation and its interactions with
platelets. In this review, we present a summary of recent structural
data on FXI as it relates to current knowledge of protease function
and to congenital FXI deficiency.
Submitted September 6, 2009; accepted January 4, 2010. Prepublished online
as Blood First Edition paper, January 28, 2010; DOI 10.1182/blood-2009-09199182.
The online version of this article contains a data supplement.
BLOOD, 1 APRIL 2010 䡠 VOLUME 115, NUMBER 13
FXI structure
Crystal structures are available for full-length zymogen FXI,7 and
the isolated active protease domain of FXIa in complex with
© 2010 by The American Society of Hematology
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BLOOD, 1 APRIL 2010 䡠 VOLUME 115, NUMBER 13
EMSLEY et al
Figure 1. Plasma coagulation protease reactions. Scheme for the cascade/
waterfall model of thrombin generation triggered by activation of FXII. Conversion of
FXI to FXIa is shown in red. A mechanism for initiation of coagulation by the FVIIa/TF
complex through FX activation is also shown. Roman numerals indicate unactivated
coagulation factors, and activated factors are indicated by “a.” Nonenzymatic
cofactors are indicated by numerals in black ovals. The dotted line designated “1”
indicates feedback activation of FXI by thrombin, whereas the dotted line indicated by
“2” represents activation of FIX by FVIIa/TF.
natural and synthetic inhibitors.40-42 An NMR structure for the A4
domain dimer is also available.43 The apple domains of FXI and PK
are members of the PAN (plasminogen, apple, nematode) module
family, with homology to the N-terminal domains of hepatocyte
growth factor and plasminogen.44 Each apple domain consists of
7 ␤-strands that fold into a curved antiparallel sheet cradling an
␣-helix (Figure 2A).7 Two disulfide bonds lock the helix onto the
central ␤4 and ␤5 strands, whereas a third connects the N- and
C-termini of the domain. This core topology is equivalent to PAN
domains from hepatocyte growth factor,44 leech antiplatelet protein,45 and microneme antigen EtMIC5.46 The ␤4-␤5 loop and
␤5-␤6 crossover loop generate a small pocket on the opposite side
of the sheet from the ␣-helix (Figure 2A).
A remarkable feature of the FXI structure, which it probably
shares with PK,10 is the intimate linkage of the apple domains into a
A
B
disk-like platform around the base of the catalytic domain (Figure
2B).7,10 The disk has 2 sets of topologically equivalent interfaces.
The side interfaces between A1 and A2 and between A3 and A4 are
substantial, burying surface areas of 441 Å2 and 444 Å2, respectively. In comparison, the end interfaces between A1 and A4 (380
Å2) and between A2 and A3 (284 Å2) are smaller. The FXI subunit,
therefore, is compact compared with the vitamin K–dependent
proteases, whose elongated structures serve to position the catalytic
domains above phospholipid membranes.1 Amino acid substitutions in the apple domains of FXI-deficient patients are shown in
Figure 2C. With few exceptions, these mutations involve residues
that are conserved in FXI, PK, and their ancestral predecessor
(supplemental Table 1, available on the Blood website; see the
Supplemental Materials link at the top of the online article). Many
involve buried residues (green in Figure 2C) that probably affect
apple domain folding or disrupt formation of interdomain interfaces,29 which would explain why the majority of cases of FXI
deficiency are CRM⫺.
FXI dimer
The 2 A4 domains form a substantial interface (886 Å2) between
the dimer subunits, with the ␤-sheets packing against each other
(Figure 2D).7 Cys321, which is located in a finger-like loop in A4,
projects away from the body of the domain and forms the
interchain disulfide bond (Figure 2B,D). However, FXI with
substitutions for Cys321 still form stable dimers.28,47-51 Leu284,
Ile290, and Tyr329, which form the hydrophobic core of the A4
domain interface, are required for dimer formation,7,50,51 as are salt
bridges between Lys331 of one subunit and Glu287 of the other.7,48
The Cys321-Cys321 interchain bond forms poorly in FXI with a
single alanine substitution of Leu284, Ile290, or Tyr329,51 indicating that the interface between A4 ␤-sheets forms before the
disulfide bond. A number of A4 domain mutations associated with
Thrombin
C321
β1
pocket
β2
β6
β7
β4
HK
β5
β3
Heparin
FIX
GpIb
C
D
C321-C321
Y329
L284
I290
Figure 2. Human FXI apple domains. Topology diagrams based
on the FXI zymogen structure. For all diagrams, the first, second,
third, and fourth apple domains (A1, A2, A3, and A4) are shown in
cyan, light blue, orange, and yellow, respectively. (A) The apple
domain consists of a 7-stranded ␤-sheet (blue) with a central
␣-helix positioned on top (red). Disulfide bonds are shown in
orange. (B) Topology diagram showing the disk formed by the
4 apple domains, with the catalytic domain removed. Sites of
residues implicated in ligand binding are red for thrombin, green
for high-molecular-weight kininogen (HK), black for GPIb, blue for
heparin, and orange for FIX. (C) Apple domain disk showing the
positions of mutations identified in FXI-deficient patients that
involve buried residues (green) or surface exposed residues
(red). Mutations Gly155Glu, Arg184Gly, and Ser248Asn (boxed)
are examples of rare CRM⫹ mutations. (D) Two A4 domains
forming the FXI dimer interface. The Cys321 interchain disulfide
bond is shown at the top in orange. Hydrophobic residues
Leu284, Ile290, and Tyr329 are shown in black, and a salt bridge
is formed between Lys331 (blue) and Glu287 (red).
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BLOOD, 1 APRIL 2010 䡠 VOLUME 115, NUMBER 13
FACTOR XI STRUCTURE AND FUNCTION
FXI deficiency interfere, or are predicted to interfere, with dimerization. The best characterized are Phe283Leu,26-28 which causes a
partial dimerization defect, and Gly350Glu,27 which prevents
dimer formation. Riley et al showed that the Phe283Leu substitution causes increased dimer dissociation and stabilizes the monomer through altered side-chain packing that is unfavorable for
dimer formation.28
The dimeric structure of FXI, unique among coagulation proteases,
has implications for inheritance of FXI deficiency.Although Glu117Stop
and Phe283Leu are associated with a bleeding diathesis inherited as an
autosomal recessive disorder (mutations in both alleles required for
symptomatic deficiency),2,27 significant deficiency can occur in heterozygotes for some types of mutations, as follows. An amino acid substitution could prevent protein secretion (CRM⫺ mutation) but not interfere
with intracellular dimer formation. A FXI subunit containing such a
mutation would exert a dominant-negative effect on normal FXI
subunits by forming nonsecretable heterodimers with 1 normal and
1 abnormal subunit. The mutant subunit, in effect, traps wild-type
subunits within the cell. This mechanism has been demonstrated in
deficiencies of multimeric proteins, such as von Willebrand factor52 and
fibrinogen.53 The FXI missense mutations Ser225Phe, Cys398Tyr,
Gly400Val, and Trp569Ser exhibit a dominant negative effect when
coexpressed with wild-type FXI and are associated with FXI deficiency
that may be inherited in a dominant manner.27,54 It is probable that these
mutations do not perturb apple domain disk structure significantly,
allowing dimerization to occur. In this regard, it is interesting to note that
3 of 4 mutations known to operate by a dominant negative mechanism
involve residues in the catalytic domain.
The structure of the FXI dimer is shown in Figure 3 with
highlights on residues implicated in zymogen activation, substrate
recognition, and receptor binding (also shown in Figure 2B). Many
AL
AS
C321
H2
AL
T
H1
FIX
GpIb
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of the complex protein-protein interactions involving FXI and
FXIa are mediated by its apple domains. Binding sites for
HK,10,55,56 thrombin,7,57 heparin,58-61 FIX,62,63 and platelet receptors, such as glycoprotein Ib (GPIb),64 will be discussed in the
following sections.
FXI and PK binding to HK
HK is a 120-kDa multifunctional plasma protein with 6 domains,
designated D1 to D6.65 Almost all FXI8 and 75% to 90% of PK11,66
circulate in a complex with HK. The D6 domain mediates binding
to both proteins.65 The physiologic importance of the PK-HK
interaction seems clear as cleavage of HK by ␣-kallikrein
(activated PK) liberates the vasoactive peptide bradykinin.65
The importance of the FXI-HK interaction is not as evident, as
HK deficiency is not associated with a bleeding disorder.30 In the
presence of zinc ions, HK is required for optimal FXI binding to
GP1b on activated platelets in suspension, enhancing the
binding stoichiometry by approximately 2-fold.67-69 However,
FXI binding to platelets assayed under flow (discussed in “FXI
interactions with platelet receptors”) is not enhanced by HK.70
The sites on FXI and PK that bind HK are probably similar. Studies
using FXI/PK chimeras, individual apple domains, and short peptide
sequences derived from apple domains indicate the A2 domain is
required for HK binding, with additional contributions from A1 and
A4.55,56,71 Detailed mutagenesis to localize the binding site has not been
performed; however, analysis of the natural FXI mutation Gly155Glu
provides some information. Gly155Glu is a rare example of a mutation
causing CRM⫹ FXI deficiency.72,73 Gly155 is located at the center of a
loop connecting the ␤5 and ␤6 strands of A2. The Gly155 main chain
nitrogen forms a hydrogen bond with the carbonyl group of Thr152 in a
type I ␤-turn (Figure 4A). Gly155 points toward the center of a cavity in
the apple domain disk and lies in a positively charged channel on the
surface of A2 pointing away from the protease domain (Figure 4B).10
Recombinant FXI-Glu155 binds HK with approximately 10-fold lower
affinity than wild-type FXI in a solid phase assay (D.G., unpublished
observation, June 2008). The loop containing Gly155 forms hydrogen
bonds with a second loop spanning residues 103 to 105 (Figure 4A) that
contains the FXI mutation Gly104Asp and borders a pocket in the A2
domain at the end of the charged channel (Figure 4B). A Gly104Arg
mutation in the PK A2 domain associated with CRM⫹ PK deficiency
also causes decreased HK binding.74
HK
FXI zymogen activation
Figure 3. The structure of zymogen FXI. Topology diagram of dimeric FXI viewed
from 2 perspectives rotated 90 degrees. The catalytic domain is in white. Sites of
residues implicated in ligand binding are red for thrombin (T), green for HK, black for
GPIb, blue for heparin sites (H1 and H2), and orange for FIX. Positions for the
activation loop (AL) cleavage site (Arg360-Ile370) residue Ile370 and active site (AS)
residues Ser557, Asp462, and His413 are shown in purple.
In vitro, known plasma activators of FXI include FXIIa,4 ␣-thrombin,13,14,75 the prothrombin activation intermediate meizothrombin,76 and FXIa (autoactivation),13,15 all of which cleave FXI at the
Arg369-Ile370 bond.5,6 Although FXIIa activates FXI in the
cascade/waterfall model (Figure 1), the absence of a bleeding
disorder in FXII-deficient patients indicates that alternative mechanisms for FXI activation exist. This is demonstrated clearly by the
work of Spronk et al using mice lacking FIX, FXI, or FXII on a
background of low tissue factor (TF) expression.77 Low TF mice
have a severe bleeding disorder but are viable.78 Superimposing
FIX or FXI deficiency on the low TF background results in death in
utero from bleeding, implying FIX activation by FXIa is required
for viability in the setting of a hemostatic system crippled by low
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EMSLEY et al
A
B
G155E
HK
G155
T152
G104D
G104
A2
Figure 4. FXI-binding site for HK. (A) The relative positions of Gly104 and Gly155 in
the A2 domain are shown. Gly155 is involved in a tight ␤-turn forming a hydrogen
bond with Thr152. The compact hydrogen bond network extends through Thr152 and
Thr158 to form contacts to the main chain through residues Lys103 and Ile105.
Changes at Gly104 or Gly155 will probably disrupt this network. (B) Charged surface
(blue represents positive; red, negative) representation of the underside of the FXI
apple domain disk, showing positions of Gly104 and Gly155. These residues are
required for proper formation of a charged channel that is a probable binding site for
HK. The dotted line represents the predicted binding site of HK that terminates in a
pocket at the base of the A2 domain.
TF levels. Mice with deficiency of FXII and low TF, on the other
hand, are viable and phenotypically similar to low TF mice with
normal FXII expression. Although these data confirm the impression that FXIIa is not required for FXI activation, they should not
be interpreted as indicating that FXIIa does not activate FXI in
vivo. Indeed, FXIIa-mediated activation of FXI appears to play a
central role in formation of pathologic thrombi in murine thrombosis models.33,38
Naito and Fujikawa13 and von dem Borne et al15 were the first to
present results indicating that FXI is activated by thrombin in
plasma. In purified protein systems, FXI activation by thrombin is
enhanced by charged substances, such as dextran sulfate or
heparin,13,14 but the relevance to activation in vivo is not clear.
Thrombin interacts with most of its substrates through 2 electropositive anion-binding exosites (ABE I and ABE II).79 Using a panel of
thrombins with site-specific mutations, Yun et al showed that
ABE I and ABE II are required for efficient FXI activation in the
presence of dextran sulfate.80 Here ABE II interacts with dextran
sulfate, whereas ABE I probably binds FXI. Recent experiments in
plasma, however, did not identify a role for ABE I in FXI
activation, as wild-type thrombin and thrombin with mutations in
ABE I initiated FXI-dependent thrombin generation similarly.81
Available information indicates that thrombin binds to the A1
domain of FXI, with residues Glu66, Lys83, and Gln84 forming
part of the binding site.7,57,75 These residues cluster near the
interface with the A4 domain (Figures 2B, 3A) and are suitably
positioned in proximity to the activation loop containing the
Arg369-Ile370 cleavage site. The FXI residues involved in binding
FXIIa have not been definitively determined, and the interaction
may extend beyond a single apple domain surface. For example,
although studies using peptide mimicry point to a binding site for
FXIIa on the A4 domain,82 a monoclonal antibody that recognizes
A2 selectively interferes with FXI activation by FXIIa.76,81
A role for the FXI dimer in promoting zymogen activation by
thrombin in trans has been proposed.7 Here the activating protease
would bind one subunit of the dimer and cleave Arg369-Ile370 on the
other. Consistent with this, Wu et al used a series of recombinant
monomeric FXI proteins in a purified system to show that the dimer is
required for optimal FXI activation by thrombin, FXIIa, and FXIa.50
Some of these studies used dextran sulfate to facilitate FXI activation,
which may not suitably model the physiologic environment. A second
study with similar FXI monomers gave somewhat contradictory results,
with monomers proving more susceptible than dimers to spontaneous
autoactivation by FXIa, but here experiments were conducted in
conditioned media (unpurified system).51
The term FXIa describes the protease form of FXI that has been
cleaved at the Arg369-Ile370 peptide bonds on both subunits of the
dimer. Recently, FXI activation by thrombin or FXIIa was shown to
proceed through an intermediate with 1 activated subunit, referred
to as 1/2-FXIa.3,83 The intermediate can be observed on nonreducing sodium dodecyl sulfate-polyacryalmide gels because FXI,
1/2-FXIa, and FXIa migrate at slightly different rates (Figure 5).
1/2-FXIa can be detected in plasma induced to undergo contact
activation.83 Given that conversion of FXI to fully activated FXIa is a
slow process, 1/2-FXIa may be a major species of activated FXI. The
functional properties of this protease are discussed further in “Recognition and cleavage of FIX by FXIa.”
FXIa protease domain structure and
interactions with inhibitors
There are no structures available for full-length FXIa or 1/2-FXIa,
but structures for isolated FXIa catalytic domain in complex with
inhibitors have been reported.40-42 Comparison with the zymogen
structure reveals conformational changes expected with activation
of a serine protease, including a 12Å movement of Ile370 (the
N-terminus of the catalytic domain, Figure 6A) into the protease
domain core. Trypsin-like proteases, including FXIa, cleave their
substrates after basic amino acids (arginine in the case of FXIa) that
are designated the P1 residue. The site on the protease that interacts
with the substrate P1 residue is the S1 site. Changes in the catalytic
domain that occur during FXI activation result in a salt bridge
forming between the Ile370 N-terminal amine and the Asp554 side
chain. This allows formation of the S1 site that will be occupied by
the side chain of the P1 arginine from a substrate or inhibitor.
A distinct conformational change is also observed in loops on the
side of the catalytic domain opposite the active site cleft. This
involves unraveling of a short ␣-helix in the zymogen and a closing
together of the loop containing Arg489 and a ␤-hairpin to fill a
pocket left empty by removal of the A3 domain, which forms an
interface with the base of the catalytic domain in the zymogen
(Figure 6A,C). The potential importance of this conformational
Figure 5. FXI activation. Each FXI subunit is activated by cleavage between Arg369 and
Ile370. FXI (0-hour time point) migrates slightly faster than FXIa (top band, 6 hours) on
nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activation of FXI
(schematic at left) by ␣-thrombin (shown) or FXIIa proceeds through an intermediate with 1
cleaved subunit (center), designated 1/2-FXIa. Subsequent conversion of 1/2-FXIa to fully
activated FXIa (right) appears to be a slower process. In the diagrams, the A1, A2, A3, and
A4 domains are shown in light blue, cyan, orange, and yellow, respectively, and the catalytic
domains (CAT) are in white. Circles represent unactivated catalytic domains; three-fourths
circles, activated catalytic domains. In these diagrams, the catalytic domain moves relative
to the apple domain disk after cleavage of Arg369-Ile370, exposing a surface on A3 thought
to contain a FIX-binding site.
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BLOOD, 1 APRIL 2010 䡠 VOLUME 115, NUMBER 13
A
FACTOR XI STRUCTURE AND FUNCTION
B
ligand
Recognition and cleavage of FIX by FXIa
R489
I370
R184
C
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D
R184
Figure 6. The FXIa protease domain. (A) Topologic diagram showing the superposition of FXI (white) and FXIa (red) protease domain crystal structures. Conformational changes include a 12 Å shift in the position of Ile370 into the protease core after
cleavage of the Arg369-Ile370 bond, and an unraveling of an ␣-helix containing
Arg489 to fill a pocket left empty by removal of the A3 domain and Arg184. The Arg
side chain from the FXIa ligand in panel B is shown in blue. (B) Structure of FXIa in
complex with PN2-KPI domain inhibitor (blue). (C) The side chain of Arg184 is buried
in the FXI zymogen through contacts with the protease domain. This residue is
thought to act as a switch, which is released on zymogen activation to engage
substrate FIX. (D) Stick diagram showing the side chain of Arg184 that forms
3 noncovalent interactions with the side chains of Ser268 (gray) from the A3 domain,
and Asp488 (green) and Asn566 (white) from the protease domain in the FXI
zymogen structure.
change to substrate recognition is discussed in “Recognition and
cleavage of FIX by FXIa.”
Activated FXIa is subject to negative regulation by plasma
serpins, including antithrombin, C1-inhibitor, protease nexin 1, and
protein Z–dependent protease inhibitor.59-61,84-87 Basic amino acids
in the autolysis loop of the catalytic domain (Arg504, Lys505,
Arg507, and Lys509) are determinants of serpin specificity.87
Heparin enhances serpin-mediated FXIa inhibition through a
mechanism involving heparin-binding sites on the A3 (Lys252,
Lys253, and Lys 255; Figures 2B, 3)58,59 and catalytic (Lys529,
Arg530, Arg532, Lys536, and Lys540)61,62 domains (Figure 3). The
A3-binding site facilitates inhibition through a template mechanism in which both protease and serpin bind to heparin.61 In
contrast, heparin binding to the catalytic domain facilitates inhibition in a manner not completely explained by a template mechanism, and probably involves charge neutralization or an allosteric
effect that overcomes repulsive interactions between the serpin and
catalytic domain.61
FXIa is also regulated by protease nexin 2 (PN2),41,88,89 which is
released from activated platelets.88 FXIa inhibition requires the
Kunitz-type protease inhibitor (KPI) domain of PN2. A crystal
structure of the FXIa catalytic domain complex with PN2-KPI
shows that the inhibitor has a characteristic disulfide-stabilized
double-loop structure that fits into the FXIa active site, with the P1
arginine occupying the S1-binding pocket (Figure 6B).41
FXIa’s major substrate, FIX, has an elongated structure that
includes (from the N-terminus) a calcium-binding Gla domain,
2 EGF repeats, a 55-amino acid activation peptide, and a Cterminal serine protease domain (Figure 7A).90 Conversion of FIX
to the protease FIXa␤ requires cleavages of the Arg145-Ala146 and
Arg180-Val181 bonds releasing the activation peptide. When FIX
is activated by the factor VIIa (FVIIa)/TF complex (Figure 1), there
is preference for initial cleavage after Arg145, resulting in accumulation of the intermediate FIX␣, followed by cleavage after Arg180
(Figure 7B).91-93 In contrast, relatively little or no FIX␣ accumulates during FIX activation by FXIa (Figure 7C).50,94-96 More than
1 mechanism is compatible with this observation. Cleavage of the
FIX activation sites by FXIa could be sequential, with the rate of
the second cleavage greater than the first. Indeed, FXIa, like
FVIIa/TF, cleaves FIX initially at the Arg145-Ala146 bond.83,96
Alternatively, the protease may cleave sequentially but not release
the intermediate before the second cleavage. This is consistent with
data showing that cleavage rates of FIX and FIX␣ by FXIa are
roughly similar (ie, Arg180-Val181 is not cleaved more rapidly
than Arg145-Ala146).95 The possibility that FXIa requires its
2 catalytic domains to cleave the FIX activation sites simultaneously has been refuted, as 1/2-FXIa83 and monomeric variants of
FXIa50 activate FIX similarly to dimeric FXIa.
Initial recognition of FIX involves exosites on FXIa that are
distinct from the catalytic active site.96-98 FIX does not bind FXI,99
indicating that conformational changes during conversion of FXI to
FXIa expose the exosites. An antibody that recognizes an epitope
on the FXIa apple domain disk competitively inhibited FIX
activation by FXIa.98 Subsequent work with recombinant proteins
localized a FIX-binding site to the A3 domain (Figures 2B, 3).62,63
A critical residue for FIX activation is Arg184,63 located near the
N-terminus of A3. An Arg184Gly mutation was recently reported
in a CRM⫹ FXI-deficient patient, and recombinant FXIa with this
mutation activates FIX poorly.100 In the zymogen structure, Arg184
occupies a central location in a loop connecting A2 and A3 that is
buried under the protease domain (Figure 6A,C). The long Arg side
A
B
C
Figure 7. FIX activation. (A) FIX contains (from the N-terminus) a Gla domain
(green), 2 epidermal growth factor (EGF) domains (red), an activation peptide (AP;
gray bar), and a catalytic (CAT) domain (white). FIX is converted to FIXa␤ by
cleavage after Arg145 and Arg180, releasing the AP. FVIIa/TF initially cleaves FIX
after Arg145 to form the intermediate FIX␣, with subsequent cleavage after Arg180 to
form FIXa␤ (bold arrows). White three-fourths circle represents the active catalytic
domain of FIXa␤. During this reaction, FIX␣ accumulates before formation of FIXa␤.
FXIa also cleaves FIX initially after Arg145; however, no FIX␣ accumulates. Initial
cleavage of zymogen FIX after Arg180 to generate the intermediate FIXa␣ appears to
be a minor reaction (thin arrows). (B-C) Reducing Western blots of FIX (100nM)
activated by 1nM FVIIa/TF (B) or FXIa (C) over 30 minutes (time indicated between
panels). Markers at the right indicate migration of standards for zymogen FIX (FIX),
the large fragment of FIX␣ (FIX␣), the heavy chain (HC) of FIXa␤, and the light chain
(LC) of FIX␣ or FIXa␤. No FIX␣ accumulates during activation by FXIa.
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EMSLEY et al
chain extends such that the guanidinium group fills the small
pocket formed by the interface between the apple domains and
catalytic domain. Here, Arg184 interacts with 3 residues: 2 from
the catalytic domain (Asp488, Asn566) and 1 from the A3 domain
(Ser268; Figure 6D).
Arg184 is conserved in FXI across species12 but is not present in
PK, consistent with its role in interactions with FIX. The Arg184
side chain must be exposed in FXIa by conformational changes that
occur after cleavage of the Arg369-Ile370 bond in the activation
loop. Full-length FXI and FXIa have been analyzed by small-angle
x-ray scattering and electron microscopy, which revealed a change
in the shape of the molecule on activation, consistent with a shift
in the position of the apple domain disk relative to the catalytic
domain.43 This could break interactions between Arg184 and
Asp488, Asn566, and Ser268 (Figure 6D). Arg184 may, therefore, be part of a switch that holds FXI in an inactive
conformation in the zymogen and, when the switch is thrown,
facilitates engagement of FIX.
It appears that residues in the FIX Gla domain are required for
binding to FXIa,99 explaining the enhancing effect of calcium ions
on the reaction.90 There is also evidence that it is the Gla domain
that binds to the A3 domain.101 Interestingly, loss of the A3 exosite
has a more deleterious effect on cleavage of FIX at Arg180-Val181
than at Arg145-Ala146.83,96 This suggests that the A3 exosite
primarily facilitates cleavage of FIX␣ to FIXa␤96 and that a distinct
exosite is involved in the initial interaction with FIX that facilitates
conversion to FIX␣. Recent work by Sinha et al indicates that this
exosite is located on the FXIa catalytic domain.96 Interestingly,
unlike binding to the A3 exosite, the interaction with the catalytic
domain exosite does not required calcium, suggesting that the
interaction does not involve the FIX Gla domain.
FXI interactions with platelet receptors
Although platelets do not appear to enhance FXI activation in a static
system,75 there is growing evidence that platelets affect FXI and FXIa
behavior under flow.35,70 When human blood lacking FXII activity is
perfused over collagen at arterial shear rates, platelets deposit in
aggregates and fibrin strands form.70 Addition of an antibody that blocks
FXIa activation of FIX prevents fibrin formation. Interestingly, adding
inhibitors of FVIIa/TF does not affect fibrin formation, suggesting that
platelets may play a role in FXI activation. Platelets may also localize
FXIa to the site of fibrin formation, as fibrin does not form when blood
lacking platelets is perfused over collagen.
FXI lacks the Gla domain that facilitates binding of vitamin
K-dependent coagulation proteases to phospholipid surfaces.5-7
Yet, there is solid evidence that FXI binds to platelets. Greengard et
al measured approximately 1500 binding sites for FXI per activated
platelet.67 Subsequent work determined that platelet GP1b mediates binding and that FXI competes with von Willebrand factor, but
not thrombin, for binding to GP1b.102 Optimal binding of FXI to
platelets requires residues in the FXI A3 domain (Figures 2B,
3),64,68,69 the N-terminal domain of the GP1b␣ chain,102 HK, and
zinc ions.67-69 The A3 residues involved, Ser248 and Arg250, are
near to, but distinct from, the A3 heparin-binding site.58,59 A CRM⫹
missense mutation in the A3 domain, Ser248Asn, was identified in
a family with a bleeding disorder.103 FXI-Asn248 binds platelets
with 5-fold reduced affinity compared with wild-type FXI,103 and
other Ser248 substitutions (Gln or Ala) showed similar defects.104
Figure 8 shows the local environment around Ser248. The overall
structure of the A3 domain is probably not affected significantly by
BLOOD, 1 APRIL 2010 䡠 VOLUME 115, NUMBER 13
R250
S248
Figure 8. Platelet-binding site on FXI. The locations of 2 residues (Ser248 and
Arg250 in black), that probably form a GPIb platelet-binding site on the FXI A3 domain
(orange), are shown relative to residues that form the heparin-binding site (Ly252,
Lys253, and Lys255 in blue). Ser248 forms hydrogen bonds with Asp194 and Thr249,
which are probably disrupted in the hereditary FXI mutation Ser248Gln. The adjacent
A2 domain is shown in light blue. The position of an N-linked glycan moiety attached
to residue Asn108 of the A2 domain is shown in yellow and red.
the Ser248Asn mutation, as FXI-Asn248 is (1) secreted, (2) activated by FXIIa, and (3) activates FIX similar to wild-type FXIa.
Cumulatively, the findings make it probable that Ser248 and
Arg250 are central to a site for interactions with platelet GPIb.
FXIa also binds to platelets (⬃ 250 sites per platelet) but does so by a
mechanism that appears to differ from that of FXI.105 FXIa binding to
platelets does not require the A3 domain or HK, and involves residues in
the catalytic domain between Cys527 and Cys542 in the vicinity of the
heparin-binding site (Figure 3).106 The binding site on platelets for FXIa
has not been identified but appears to be distinct from that of FXI, as FXI
does not compete with FXIa for binding.105
The observation that each platelet contains 1500 FXI-binding
sites was somewhat perplexing, considering that there are approximately 25 000 copies of GP1b per platelet.107 New data from
White-Adams et al may offer a solution to this conundrum; this
group reported that FXI binds to a platelet receptor for apolipoprotein E called ApoER2⬘ or LRP8.70 FXI binds to soluble ApoER2⬘
with an affinity of approximately 60nM. Human and murine
platelets bind and spread on glass coated with FXI or fibrinogen.
Binding to FXI, but not fibrinogen, was abrogated by receptorassociated protein, which blocks ligand binding to members of the
low-density lipoprotein receptor family. Platelet binding to FXI
was also blocked by soluble ApoER2⬘, and platelets from ApoER2deficient mice did not bind FXI. Under conditions of physiologic
shear, soluble ApoER2⬘ or its low-density lipoprotein-binding
domain prevented platelet binding to immobilized FXI.
There are an estimated 2000 copies of ApoER2⬘ per platelet,
which agrees well with the number of FXI-binding sites.67 Furthermore, ApoER2⬘ colocalizes with GP1b on platelets,108 suggesting
that FXI binding involves both receptors. Consistent with this,
antibodies to GP1b block platelet binding to immobilized FXI
under physiologic shear. It is not clear whether the platelet-binding
site on the FXI A3 domain is required for interactions with
ApoER2⬘ or whether a different site is involved.
Future directions
Several important aspects of FXI pathophysiology require additional study. Over the past 50 years, many investigators have
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BLOOD, 1 APRIL 2010 䡠 VOLUME 115, NUMBER 13
FACTOR XI STRUCTURE AND FUNCTION
commented on the weak correlation between bleeding and FXI
activity measured by conventional assays. This is reflected in
2 studies involving 49 FXI-deficient kindreds (242 persons), which
determined that severity of bleeding was similar in patients with
severe and mild deficiency.21,22 In contrast, work with patients
primarily with the Glu117Stop and/or Phe283Leu mutations showed
bleeding risk to be much greater in severe than mild deficiency
(odds ratio ⫽ 13.0 and 2.6, respectively).109 Diverse factors could
be contributing to this disagreement, including differences in
clinical criteria for bleeding, variation in genetic backgrounds or
FXI mutations, or coinheritance of other bleeding disorders. That
being said, some patients with severe FXI deficiency do not bleed
abnormally, even with trauma to tissues that typically cause
problems for FXI-deficient patients. We suspect that the manner in
which we measure FXI activity in clinical laboratories does not
accurately assess the protein’s contribution to hemostasis.
FXI-deficient plasma exhibits a prolonged aPTT clotting time,
and FXI deficiency is currently defined as low activity in an
aPTT-based assay in which patient and FXI-deficient plasma are
mixed. However, the aPTT only requires FXI to function as a
component of contact activation initiated fibrin formation, and does
not assess FXI functions such as feedback activation by thrombin,
inhibition of fibrinolysis, and interactions with platelets. The FXI
Ser248Asn variant illustrates the limitations of the aPTT in
assessing FXI function. This mutation is associated with bleeding
and defective FXI binding to platelets but does not affect the aPTT
assay, which does not contain platelets.103 Assays that capture the
importance of FXI to sustained (rather than initial) thrombin
generation, probably in platelet-rich plasma under flow, will be
required to address the deficiencies of the aPTT. Allen et al made
the interesting observation that the contribution of FXI to thrombin
generation varied substantially when platelets from different normal donors were tested in a cell-based model of TF-initiated
coagulation.110 Platelets, therefore, may turn out to be key for
accurately assessing FXI function.
As presented in this review, FXI has features that distinguish it
from vitamin K–dependent coagulation proteases, making it difficult to extrapolate from the lessons learned from the large body of
work on these enzymes to predict structure-function relationships
for FXI. There remain many important unanswered questions
regarding FXI and FXIa from a structural and enzymologic
perspective. Areas of active investigation include determining the
functional significance of the dimeric structure of FXI/FXIa,
elucidating the manner in which FXI/FXIa engages platelets, and
identifying the functional consequences of platelet binding.
New evidence indicates that the dimeric structure of FXI
facilitates its activation by FXIIa and thrombin.50 However, the
FXI homolog PK (a monomer) is activated rapidly by FXIIa, as is a
monomeric FXI chimera containing the PK A4 domain.62 It will be
2575
important to determine whether dimerization is critical for FXI
activation under physiologic conditions, probably using flow-based
systems, or perhaps in vivo.
It has been proposed that one subunit of a FXIa dimer could
bind to a platelet receptor, leaving the other free to interact with the
substrate FIX.105 An intriguing possibility that remains to be tested
is that 1/2-FXIa binds to GP1b, or perhaps ApoER2, through the A3
domain of its unactivated subunit while binding to FIX through A3
on the activated subunit. Note that in Figure 3B the A3 domains
point in opposite directions from the longitudinal plane of the
molecule. As FXI lacks a Gla domain, the dimeric structure may
represent an alternative solution to the problem of tethering the
protease to a surface at a wound site while allowing interaction
with its substrate. Such a hypothesis is also best tested in
flow-based systems, where the FXI-platelet interaction is probably
important to protease function.
Recent studies associating FXIa activity and pathologic coagulation in humans and animal models have stimulated considerable
interest in this protease as a target for clinical intervention, with
potential advantages over targeting thrombin or FXa in terms of
side effects and therapeutic window. Work on FXI structure is at an
early stage, compared with the vitamin K-dependent coagulation
factors, with crystal structural data becoming available only within
the past 5 years. Structures defining full-length FXIa and ligand
complexes with substrate, receptors, and activating enzymes will
be required to completely understand the molecular basis of
processes such as zymogen activation, platelet binding, and
substrate cleavage. This will be critical to understanding the role of
this protease in disease processes and to the development of
specific inhibitors for clinical purposes.
Acknowledgments
This work was supported by the British Heart Foundation (grants
PG/04/129/18095 and RG/07/002/23132; J.E.) and the National Heart,
Lung, and Blood Institute (awards HL58837 and HL81326; D.G.).
Authorship
Contribution: J.E., P.A.M., and D.G. wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Jonas Emsley, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, United Kingdom; e-mail: jonas.emsley@
nottingham.ac.uk; or David Gailani, Hematology/Oncology Division, Vanderbilt University School of Medicine, Nashville, TN
37232; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2010 115: 2569-2577
doi:10.1182/blood-2009-09-199182 originally published
online January 28, 2010
Structure and function of factor XI
Jonas Emsley, Paul A. McEwan and David Gailani
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