A novel factor XI missense mutation (Val371Ile) in the
activation loop is responsible for a case of mild type II
factor XI deficiency
Cristina Bozzao
1
*, Valeria Rimoldi
1
*, Rosanna Asselta
1
, Meytal Landau
2
, Rossella Ghiotto
3
,
Maria L. Tenchini
1
, Raimondo De Cristofaro
4
, Giancarlo Castaman
3
and Stefano Duga
1
1 Department of Biology and Genetics for Medical Sciences, University of Milan, Italy
2 Department of Biochemistry, George S. Wise Faculty of Life Science, Tel Aviv University, Israel
3 Department of Hematology and Hemophilia and Thrombosis Center, San Bortolo Hospital, Vicenza, Italy
4 Haemostasis Research Centre, Catholic University School of Medicine, Rome, Italy
Coagulation factor XI (FXI) is the precursor of a tryp-
sin-like serine protease that catalyzes, upon activation,
the conversion of factor IX (FIX) to activated FIX
(FIXa) [1,2]. Human FXI, primarily produced by
hepatocytes, is a glycoprotein of 160 kDa circulating

(Received 21 May 2007, revised 11 Septem-
ber 2007, accepted 8 October 2007)
doi:10.1111/j.1742-4658.2007.06134.x
Coagulation factor XI (FXI) is the zymogen of a serine protease that,
when converted to its active form, contributes to blood coagulation
through proteolytic activation of factor IX. FXI deficiency is typically an
autosomal recessive disorder, characterized by bleeding symptoms mainly
associated with injury or surgery. Of the more than 100 FXI gene muta-
tions reported in FXI-deficient patients, most are associated with a propor-
tional decrease in FXI functional and immunologic levels (type I defects),
whereas only a few mutations leading to the presence of dysfunctional
molecules in plasma have been molecularly analyzed to date (type II defi-
ciencies). We report the functional and molecular characterization of a
missense mutation (Val371Ile) identified, in the heterozygous state, in a
25-year-old Italian male with mild FXI deficiency. Laboratory analysis
revealed reduced functional FXI levels (34%), but normal antigen levels
(102%), distinctive of a type II defect. Given the proximity of Val371 to
the FXI activation site, a possible interference with zymogen activation
was postulated. Expression experiments of the FXI–Val371Ile recombinant
protein, followed by activation assays, showed both a different time course
in FXI activation and a slight delay in factor IX activation by thrombin-
activated FXI.
Abbreviations
FIX, coagulation factor IX; FIXa, activated factor IX; FXI, coagulation factor XI; FXIa, activated factor XI; FXI:Ag, antigen FXI level;
FXI:C, functional FXI level; FXIIa, activated factor XII.
6128 FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS
light chain, containing the catalytic domain [10]. These
two chains are held together by three disulfide bonds
and both are essential for FIX activation [5]. FXI acti-
vation generates a new N-terminus of the catalytic

Ashkenazi Jewish patients, two prevalent mutations
(Glu117stop and Phe283Leu, also called type II and
type III mutations) account for 95% of cases of FXI
deficiency [12]. However, in patients belonging to other
ethnic groups a significantly higher level of allelic het-
erogeneity has been reported. Remarkable exceptions
are represented by French Basques, French patients
from Nantes, and English patients, in whom different
prevalent ancestral mutations were found [18–20]. An
unusual dominant transmission of FXI deficiency has
been described in some families, in which four different
missense mutations exert a dominant-negative effect on
wild-type FXI secretion through intracellular hetero-
dimer formation [21,22].
The aim of this study was the molecular character-
ization of the F11 germline missense mutation
Val371Ile identified in the heterozygous state in an
Italian patient affected by mild FXI deficiency, who
had normal immunologic FXI levels associated with a
reduced activity of the factor, distinctive of a type II
defect.
Results
Patient data
The propositus was a male born in 1981, who was
referred in 2001 for the evaluation of a prolonged par-
tial thromboplastin time discovered prior to a surgical
procedure. An appendectomy, adenoidectomy, and
right-knee arthroscopy carried out previously had been
without mishap. Laboratory analysis revealed a
reduced functional FXI level (FXI:C ¼ 34%), although

recombinant FXI in COS-1 cells
To evaluate the pathogenic role of the Val371Ile muta-
tion, both the wild-type and mutant protein were
expressed in COS-1 cells. To this end, mutagenesis
was performed on the pCDNA3 ⁄ FXI plasmid to
produce the pCDNA3 ⁄ FXI–Val371Ile vector as des-
cribed in Experimental procedures. Following tran-
sient transfection with either pCDNA3 ⁄ FXI or
C. Bozzao et al. FXI–Val317Ile – a novel factor XI type II defect
FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS 6129
pCDNA3 ⁄ FXI–Val371Ile or with equimolar amounts
of both expression plasmids (to mimic the heterozy-
gous condition), serum-free conditioned media and cell
extracts were analyzed for the presence of FXI antigen
using ELISA. FXI antigen levels, measured in both
conditioned media and lysates of cells expressing the
mutant protein (in either the heterozygous or homozy-
gous state), were not significantly different from those
measured in wild-type samples (Fig. 1A). In particular,
in media conditioned by cells expressing either wild-
type or mutant FXI, antigen levels ranged from 300 to
500 ngÆmL
)1
, whereas, levels of immunoreactive FXI
were between 20 and 40 ngÆmL
)1
in the corresponding
lysates.
FXI specific activity was measured in conditioned
media as the ratio between FXI:C and FXI:Ag levels.

1
02
0
3
04
05
06
07
08
0
9
001
0
1
1
0
21
031
sn
sn
dn
dn
sn
s
n
CAIDEMDENOITIDNOCSETASYLLLE
% of wild-type
YTIVITCA
CIF
ICEPS

0
7
08
09
001
011
***
*
**
dn
A
I
D
E
MD
ENOITIDNOC
% of wild-type
Fig. 1. Transient expression of wild-type and mutant FXI protein in COS-1 cells. pCDNA3 ⁄ FXI, pCDNA3 ⁄ FXI–Val371Ile or equimolar amounts
of both plasmids (heterozygous condition) were transiently transfected in COS-1 cells. Equal numbers of cells and equal amounts of plas-
mids were used in transfection experiments, as described in Experimental procedures. (A) Antigen levels of recombinant FXI were measured
in both conditioned media and the corresponding cell lysates using an ELISA assay. Bars represent relative concentrations of protein in
media and cell lysates compared with the mean antigen level measured in the wild-type. Results are given as mean ± SD. (B) The specific
activities of recombinant proteins were determined by calculating the ratio between FXI activity (measured using a one-stage method based
on a modified partial thromboplastin time) and FXI antigen levels. Bars represent mean ± SD of four independent experiments, each per-
formed in duplicate. The mean value of wild-type FXI was set as 100%. The results were analyzed by unpaired t-test (*P<0.05;
**P<0.01; ***P<0.001), ns, not significant; nd, not determined.
FXI–Val317Ile – a novel factor XI type II defect C. Bozzao et al.
6130 FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS
about half of the total mutated protein remains uncut,
while the wild-type FXI is almost entirely activated

for wild-
type and FXI–Val371Ile, respectively. These findings
showed that the Val371Ile mutation reduces by
approximately twofold the specificity of thrombin
interaction with the FXI–Val371Ile.
Activation of FIX by FXIa
The functional properties of activated FXI–Val371Ile
were explored both by a proteolytic assay using a com-
mercially available FIX and by measuring Michaelis
parameters of S-2366 hydrolysis. To this purpose,
wild-type FXIa and FXIa–Val371Ile, completely acti-
vated by thrombin (as described in Experimental pro-
cedures) were incubated for different periods with
commercial FIX. Upon FXI activation, FIX is cleaved
at two sites, releasing an activation peptide, and pro-
ducing the protease FIXa [10,24]. As shown in Fig. 4,
incubation of FIX with wild-type FXIa results in
almost complete activation after 30 min, whereas
FXIa–Val371Ile causes a dramatic reduction in the
uncleaved FIX form only after 60 min of incubation.
A possible effect of dextran sulfate on FIX activation
was ruled out by performing the same experiment in
the absence of FXI. No activation of FIX was detect-
able after 60 min of incubation (data not shown).
The observed delay in FIX activation may be due to
a decrease in the catalytic activity of mutant FXIa,
possibly caused by a perturbed conformational state of
FXIa linked to the Val371Ile mutation. A moderate
but significant reduction in the catalytic competence of
A

)1
and 739 ± 100 lm for FXI–Val371Ile,
with k
cat
⁄ K
m
¼ 6.09 · 10
4
m
)1
Æs
)1
. The reduction in
the k
cat
⁄ K
m
value for S-2366 hydrolysis was significant,
but the effect of the mutation on FIX activation was
even more evident, as shown in Fig. 4. This suggests
that the mutation may alter molecular recognition
between FXIa and FIX, which necessarily involves, in
addition to the catalytic residues, a more extended sur-
face area of FXIa. The observed increase in K
m
for
S-2366 of the mutant FXI may arise from allosteric
effects, and thus may be generated from structural per-
turbations located far from the catalytic pocket.
Discussion

XIF
06035150
IXFelI173laVIXFepyt-dliw
)nim()nim(06035150
aXIF
XIF
aXIF
Fig. 4. Time course of FIX activation. Commercially available FIX (12.5 ng) was activated with 1.5 ng of recombinant FXI, either wild-type or
FXI–Val371Ile, both in turn activated by thrombin (0.5 U for 135 min; complete activation was assessed by western blot analysis). At differ-
ent time points (indicated at the top of each panel) digestions were stopped and proteins were resolved by Laemmli SDS ⁄ PAGE using 12%
(w ⁄ v) acrylamide gels.
021
00
1080604
0
20
01
8
6
4
2
0
emiT(nim)
FXIa (n
M
)
IXF-TW
IXF-I173V
Fig. 3. FXI activation by thrombin. Purified wild-type (d) and FXI–
Val371Ile (s) (10 n

an isoleucine, a b-branched amino acid that is not flex-
ible. In the active conformation, Val371 forms contacts
with neighboring residues that are important for stabi-
lizing the active state (e.g. Asp189, which is part of the
S1 pocket responsible for the binding specificity of the
substrate) [26]. Consequently, substitution of Val371 to
isoleucine might prevent the full development of the
active conformation. This hypothesis is further con-
firmed by the results of FIX proteolytic assays, which
showed a slight delay in FIX activation by FXIa acti-
vated by thrombin (Fig. 4); moreover the k
cat
and K
m
values of S-2366 hydrolysis showed that the Val371Ile
mutation has only minor conformational effects on the
geometry of the catalytic site of the enzyme (Fig. 5).
In contrast to the activated FXI, in the structure of
the FXI zymogen, Val371 is located on a loop region,
exposed to solvent, and does not form many contacts
with other residues (Fig. 6). Therefore, the additional
methyl in the Val371Ile mutant probably does not
disturb the structure and the domain rearrangement
in the zymogen FXI. Nevertheless, recombinant FXI–
Val371Ile activation was slower than that of the wild-
type protein (Fig. 2) suggesting a small activation
defect. This might be explained by the proximity of the
mutation to the cleavage site, probably resulting in a
small interference with the binding of the activator to
the FXI zymogen.

cat
¼ 49.8 ± 3 s
)1
,
K
m
¼ 595 ± 63 lM;(s) k
cat
¼ 45 ± 4 s
)1
, K
m
¼ 739 ± 100 lM.
Error bars indicate SEM.
Fig. 6. Structural consequences of the Val371Ile substitution. Rib-
bon representation of the superimposition between the structures
of the catalytic serine protease domain of the zymogen (red) and
activated (green) FXI. The Ile371 residue, in both structures, is dis-
played by space-filled atoms. The catalytic triad (blue space-filled
atoms) is also shown. The conformational movements of Ile371,
located in the activation loop at the N-terminus of the catalytic
domain, are notable. In the zymogen FXI, Ile371 is exposed to the
solvent, while in the activated FXI it is inserted into the protein.
The picture was drawn with
PYMOL (DeLano Scientific, San Carlos,
CA; http://www.pymol.org).
C. Bozzao et al. FXI–Val317Ile – a novel factor XI type II defect
FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS 6133
by altering the functional properties of FIXa or
by delaying its activation by FXIa. In particular,

DNA was extracted from whole blood using a standard
salting-out procedure.
Coagulation studies
Immediately after collection, citrated blood was centrifuged
at 2500 g for 15 min at room temperature. FXI activity was
performed by a one-stage method based on a modified par-
tial thromboplastin time, using FXI-deficient plasma as sub-
strate (Hemoliance, Salt Lake City, UT). FXI antigen was
measured by an ELISA based on a goat anti-human FXI
affinity purified IgG as capture antibody and a goat anti-
human FXI peroxidase-conjugated IgG as detecting anti-
body (Affinity Biological Inc., Hamilton, Ontario, Canada).
FXI levels were expressed in both tests as percentages of
pooled normal plasma from 30 normal male and female
individuals. The detection limits of the FXI functional and
immunologic assays were 1 and 0.1%, respectively.
PCR amplifications and DNA sequencing
PCR were performed on 50–100 ng of genomic DNA in a
25 lL volume, following standard procedures [41]. PCR
and sequencing primers were designed on the basis of the
known genomic sequence of F11 (GenBank accession num-
ber NM_000128). The primer couple used to amplify F11
exon 11 and to identify the Val371Ile mutation was FXI-
ex11-F 5¢-GTCAATTCCATTTTTCATGTGC-3¢ and FXI-
ex11-R 5¢-CGTTTTTTACCACTGAAGCAAT-3¢. All other
primer sequences, as well as the specific PCR condition for
each primer couple, are available on request. Sequencing
reactions were performed on both strands on PCR products
purified by MICROCON 100 columns (Millipore, Bedford,
MA). The BigDye Terminator Cycle Sequencing Kit ver-

UT), antibiotics (100 UÆmL
)1
penicillin and 100 lgÆmL
)1
streptomycin; EuroClone) and glutamine (2 mm; Euro-
Clone), and grown at 37 °C in a humidified atmosphere
FXI–Val317Ile – a novel factor XI type II defect C. Bozzao et al.
6134 FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS
of 5% CO
2
and 95% air, according to standard proce-
dures.
Expression of recombinant proteins
In each transfection experiment an equal number of cells
(400 000) were transiently transfected with the Lipofecta-
mine 2000 reagent (Invitrogen, Carlsbad, CA) in six-well
plates with 4 lg of plasmid DNA (pCDNA3 ⁄ FXI, or
pCDNA3 ⁄ FXI–Val371Ile, or equimolar amounts of both
plasmids), essentially as described by the manufacturer.
Twenty-nine hours after transfection, cells were washed
twice with NaCl ⁄ P
i
and cultured for additional 48 h in
1 mL of serum-free medium supplemented with glutamine,
antibiotics, and 5 mgÆmL
)1
BSA. For each experiment (per-
formed four times in duplicate) a mock sample, with the
empty pCDNA3 plasmid, was set up.
Conditioned media from each well were tested for both

wild-type or mutant, were incubated in NaCl ⁄ Tris at 37 °C
for different periods. Each reaction was carried out in a
final volume of 20 lL. Samples were removed into reducing
SDS sample buffer and size-fractionated on 10% polyacryl-
amide SDS gels.
Because in vitro activation of FXI by thrombin is highly
enhanced in the presence of polyanions such as dextran
sulfate [42], 1.5 ng of recombinant FXI, either wild-type or
mutant, was activated with 0.5 U ($ 5nm) of human
thrombin in NaCl ⁄ TrisA (NaCl ⁄ Tris supplemented with
0.1 mgÆmL
)1
BSA) containing 1 lgÆmL
)1
dextran sulfate
(500 000 Da) at 37 °C for different periods. The concentra-
tion of dextran sulfate (1 lgÆmL
)1
) used in our experiments
was found to be optimal in previous studies [23,42,43].
Each reaction was carried out in a final volume of 20 lL.
Aliquots (each containing 1.5 ng of recombinant FXI) were
stopped by adding 10 lLof3· reducing Laemmli sample
buffer, and run on 10% SDS ⁄ PAGE.
Proteins were then transferred onto 0.45 lm pore-size
nitrocellulose membranes (Schleicher & Schuell, Brentford,
UK) and analyzed by western blotting, using a polyclonal
goat anti-human FXI IgG.
Activation of FIX by FXIa
Recombinant FXI, either wild-type or mutant, was acti-

Chemioluminescence, SuperSignal West Dura Extended
Duration Substrate (Pierce).
Assay of FXI activation
Before activation by thrombin, supernatants from cells
expressing recombinant FXI were concentrated approxi-
C. Bozzao et al. FXI–Val317Ile – a novel factor XI type II defect
FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS 6135
mately four- to fivefold by means of VivaSpin 30 concen-
trators (Sartorius Ltd., Epsom, UK). Activation of both
wild-type and FXI–Val371Ile (10 nm) by thrombin (3 nm),
purified as previously detailed [44], was measured by a
chromogenic assay, as follows. Incubations were carried
out in 100 lLof50mm Tris, 150 mm NaCl, pH 7.5, with
0.1% poly(ethylene glycol) 6000 at 25 °C. In the FXI acti-
vation by thrombin, dextran sulfate was omitted from the
reaction buffer to avoid any spurious effect on FXI auto-
activation. At various time intervals, 10 lL of recombinant
hirudin (Sigma) at a final concentration of 10 nm were
added to inhibit thrombin activity. Then 50 lL of 500 lm
(final concentration) S-2366 (pyroGlu-Pro-Arg-pNA; Chro-
mogenix, Mo
¨
lndal, Sweden) were added to the solution,
and the amount of free paranitroaniline released by FXIa
was determined by measuring the change in absorbance at
405 nm in a Benchmark II microplate reader (Bio-Rad
Laboratories, Hercules, CA). To eliminate any scattering
contribution, the absorbance at 620 nm was always sub-
tracted from the reading at 405 nm. The initial velocity of
S-2366 hydrolysis obtained at each time point was consid-

of thrombin hydrolysis so
that the rate constant k was proportional to the value of
k
cat
⁄ K
m
of the activation, according to:
k ¼ T k
cat
=K
m
ð1Þ
where T is the thrombin concentration.
Measurement of Michaelis parameters of S-2366
hydrolysis by wild-type and FXI–Val371Ile
After 120 min of FXI activation by thrombin, $ 88%
(8.8 nm) of wild-type FXI and 63% (6.3 nm) of mutant
FXI were activated, according to [45]:
½FXIa
120
¼ V
120
=V
1
à FXI
T
ð2Þ
where V
120
is the velocity of S-2366 hydrolysis at 120 min

the surfv program [46] with a probe sphere of radius
1.4 A
˚
and default parameters. The percentage of the
surface-exposure of each residue in the monomer was
calculated from the total solvent-accessible area on a
Gly-X-Gly tripeptide (where X represents each of the 20
amino acids).
Evolutionary conservation analysis
Evolutionary conservation analysis was carried out using
the ConSurf web-server [47] (http://consurf.tau.ac.il/). The
calculations were performed using the structure of FXIa
(PDB code: 1XX9) [25], based on an alignment of 200 ser-
ine protease sequences collected from the SWISSPROT
database [48] and default parameters.
Acknowledgements
The authors would like to thank Sofia H. Giacomelli
for excellent technical assistance. SD is a recipient of a
Bayer Hemophilia Early Career Investigator Award
2006. The financial support of PRIN (Programmi di
Ricerca Scientifica di Rilevante Interesse Nazionale,
Grant n. 2005058307-002) is gratefully acknowledged.
FXI–Val317Ile – a novel factor XI type II defect C. Bozzao et al.
6136 FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS
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FXI–Val317Ile – a novel factor XI type II defect C. Bozzao et al.
6138 FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS