Download Severe factor XI deficiency caused by a Gly555 to Glu mutation

Journal of Thrombosis and Haemostasis, 2: 1782–1789
ORIGINAL ARTICLE
Severe factor XI deficiency caused by a Gly555 to Glu mutation
(factor XI–Glu555): a cross-reactive material positive variant
1 defective in factor IX activation
A . Z I V E L I N , * T . O G A W A , S . B U L V I K , * M . L A N D A U , à * J . R . T O O M E Y , – J . L A N E , U . S E L I G S O H N * and
D. GAILANI *Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel Hashomer and Sackler School of Medicine;
àDepartment of Biochemistry, George Wise Faculty of Life Science, Tel Aviv University, Israel; The Departments of Pathology and Medicine,
Vanderbilt University, Nashville, TN; and –the Cardiovascular and Urogenital Diseases Center of Excellence GlaxoSmithKline, King-of-Prussia,
PA, USA
To cite this article: Zivelin A, Ogawa T, Bulvik S, Landau M, Toomey JR, Lane J, Seligsohn U, Gailani D. Severe factor XI deficiency caused by a Gly555
to Glu mutation (factor XI–Glu555): a cross-reactive material positive variant defective in factor IX activation. J Thromb Haemost 2004; 2: 1782–9.
Introduction
Summary. During normal hemostasis, the coagulation protease factor (F)XIa activates FIX. Hereditary deficiency of the
FXIa precursor, FXI, is usually associated with reduced FXI
protein in plasma, and circulating dysfunctional FXI variants
are rare. We identified a patient with < 1% normal plasma FXI
activity and normal levels of FXI antigen, who is homozygous
for a FXI Gly555 to Glu substitution. Gly555 is two amino
acids N-terminal to the protease active site serine residue, and is
highly conserved among serine proteases. Recombinant FXIGlu555 is activated normally by FXIIa and thrombin, and
FXIa-Glu555 binds activated factor IX similarly to wild type
FXIa (FXIaWT). When compared with FXIaWT, FXIa-Glu555
activates factor IX at a greatly reduced rate (400-fold), and is
resistant to inhibition by antithrombin. Interestingly, FXIaWT
and FXIa-Glu555 cleave the small tripeptide substrate S-2366
with comparable kcats. Modeling indicates that the side chain of
Glu555 significantly alters the electrostatic charge around the
active site, and would sterically interfere with the interaction
between the FXIa S2¢ site and the P2¢ residues on factor IX and
antithrombin. FXI-Glu555 is the first reported example of a
naturally occurring FXI variant with a significant defect in FIX
activation.
Keywords: bleeding disorder, factor IX, factor XIa.
Correspondence: David Gailani, Hematology/Oncology Division,
Vanderbilt University, 777 Preston Research Building, 2220 Pierce
Avenue, Nashville, TN 37232–6307, USA.
Tel.: (615) 9361505; fax: (615) 9363853: e-mail: dave.gailani@
vanderbilt.edu
Received 23 March 2004, accepted 23 April 2004
The plasma glycoprotein factor XI (FXI) is the precursor of
the serine protease FXIa, which contributes to blood
coagulation through proteolytic activation of factor IX [1].
Hereditary FXI deficiency is typically an autosomal recessive
bleeding disorder associated with injury or surgery-associated
hemorrhage [2]. Of the 80 FXI gene mutations reported in
FXI-deficient patients, most are associated with proportional
decreases in plasma FXI activity and antigen (cross-reactive
material negative [CRM –] defects), rather than circulating
dysfunctional FXI variants (CRM + defects) [3]. Exceptional CRM + cases of FXI deficiency have been reported
[4], but there are no descriptions of a FXI variant with a
clearly defined defect in FIX activation.
We evaluated an 80-year-old woman of Bucharan-Jewish
ancestry with undetectable plasma FXI activity (< 1 U dL)1)
and normal antigen (100 U dL)1) [5]. Her parents were first
cousins. The patient bled excessively after tooth extractions,
childbirth and an abortion. Analysis of her FXI genes revealed
homozygosity for a Gly to Glu substitution at residue 555 in
the catalytic domain (FXI-Glu555). Gly555 corresponds to
Gly193 in chymotrypsin, the prototype used to compare
trypsin-like proteases [6], and is highly conserved among serine
proteases [7–9]. Mutations at the corresponding residues in
FIX [10] and FVII [11] cause CRM + hemophilia B and FVII
deficiency, respectively. The amido nitrogen of Gly193 forms
part of the active site oxyanion hole, and assists catalysis by
stabilizing transition state intermediates through formation of
a hydrogen bond with the carbonyl oxygen atom of the
substrate [9,12]. Therefore, Glu555 might affect catalysis
through distortion of the active site. The large charged side
chain of Glu555 may also cause steric clashes with molecules
that interact with the catalytic site. In this report we present our
analysis of the defect in activated FXI-Glu555 (FXIa-Glu555).
2004 International Society on Thrombosis and Haemostasis
Factor XI-Glu555 1783
Methods
Western blot for plasma FXI
Plasma (10 lL) was size-fractionated on an 8% polyacrylamide
sodium dodecyl sulfate gel, followed by transfer to polyvinylidene difluoride membranes. The primary antibody was a
murine monoclonal antihuman FXI IgG (Haematologic
Industries, Essex Junction, VT, USA). Detection was with an
HRP-conjugated goat-antimouse IgG and chemiluminescence.
Expression and purification of recombinant protein
The human FXI cDNA [13] was altered by sequential PCR,
converting the GGA triplet for Gly555 to GAA (Glu). Wildtype FXI (FXIWT) and FXI-Glu555 cDNAs were ligated into
vector pJVCMV, as described [14]. 293 fibroblasts (50 · 106 –
ATCC CRL 1573) were cotransfected by electroporation
(Electrocell Manipulator 600 BTX, San Diego, CA, USA) with
40 lg of FXI construct and 2 lg of plasmid RSVneo. Cells
were grown in DMEM with 5% fetal bovine serum and
500 lg mL)1 G418. Supernatants from G418-resistant clones
were tested for expression by ELISA using goat antihuman
FXI polyclonal antibodies (Affinity Biologicals, Hamilton,
Ontario, Canada). Expressing clones were expanded in
175 cm2 flasks, and conditioned media was collected every
48 h. Proteins were purified from media on an anti-FXI IgG
1G5.12 affinity column [14]. Protein concentrations were
determined by dye binding assay (Bio-Rad, Hercules, CA,
USA), and purity assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). To prepare
FXIa, FXI (300 lg/mL) was incubated with 5 lg mL)1
FXIIa (Enzyme Research Laboratories, South Bend, IN,
USA) at 37 C. Complete activation was confirmed by SDS–
PAGE.
Coagulation assays
Activated partial thromboplastin time (APTT) assays were
performed as follows. FXI deficient plasma (65 lL – George
King, Overland Park, KS, USA) was mixed with 65 lL FXI
(serial dilutions starting at 5 lg mL)1) in TBS containing
0.1% bovine serum albumin (TBSA), and 65 lL PTT A
reagent (Diagnostica Stago, Asnieres-sur-Seine, France) on a
Dataclot II fibrometer (Helena Laboratories, Beaumont, TX,
USA). After 5 min, 65 lL 25 mM CaCl2 was added, and
time to clot formation determined. Proteins were tested in
triplicate and compared with standard curves prepared with
plasma FXI (Specific activity 200 U mg)1, Enzyme
Research Laboratories). A modified PTT was used to assess
preactivated FXIa. Serial dilutions of FXIa in TBSA (65 lL)
were mixed with 65 lL FXI deficient plasma, and 65 lL
rabbit brain cephalin. Thirty seconds later, 65 lL 25 mM
CaCl2 was added and time to clot formation was determined. Results were compared with plasma FXIa, which
was assigned an activity of 100%.
2004 International Society on Thrombosis and Haemostasis
Activation of FXI
FXI (100 lg mL)1) and FXIIa (2 lg mL)1) were incubated in
TBS at 37 C. At various time points, samples were removed
into reducing SDS sample buffer and size-fractionated on 12%
polyacrylamide SDS gels, followed by staining with GelCode
blue (Pierce, Rockford, IL, USA). FXI (2 lg mL)1) was
incubated with 1 U mL)1 human thrombin (Diagnostica
Stago) in TBSA containing 1 lg mL)1 dextran sulfate
(500 000 Da) at 37 C. Aliquots were removed into reducing
sample buffer, and run on 8% PAGE–SDS. Proteins were
transferred to PVDF membranes and analyzed by Western
blotting, using polyclonal sheep antihuman FXI IgG (The
Binding Site, Birmingham, UK). Detection was with HRPconjugated antisheep IgG and chemiluminescence. To study
autoactivation, FXI (100 lg mL)1) was incubated in TBS with
2.5 lg mL)1 dextran sulfate at 37 C. Samples were mixed
with reducing SDS-sample buffer, run on 8% PAGE–SDS and
stained.
Activation of FIX by FXIa studied by Western blotting
FXIa (2 nM) and FIX (300 nM; Enzyme Research Laboratories) were incubated in TBSA with 2.5 mM CaCl2 at 37 C.
Aliquots were removed into non-reducing SDS-sample buffer,
and size fractionated on 8% PAGE–SDS. Proteins were
transferred to polyvinylidene difluoride membranes and analyzed by chemiluminescent Western blot. The primary antibody was a polyclonal goat antihuman FIX IgG (Enzyme
Research Laboratories).
Chromogenic assay for FIX activation
Factor IX (200 nM) and FXIa (1.0 nM) were incubated in
TBSA with 2.5 mM CaCl2 at 37 C. At various time points,
50 lL aliquots were removed and mixed with aprotinin and
EDTA (final concentrations 100 lg mL)1 and 20 mM, respectively). Reactions were added to equal volumes of 1 mM S299
(Spectrozyme FIXa, American Diagnostica, Greenwich, CT,
2 USA) in 66% ethylene glycol/TBSA. Changes in absorbance at
405 nm were measured on a SpectraMax 340 microplate reader
(Molecular Devices, Sunnyvale, CA, USA), and compared
with standard curves prepared with purified FIXa. Additional
experiments were run at concentrations of FIX up to 2 lM and
FXIa-Glu555 up to 10 nM.
FXIa cleavage of S-2366 and effect of antithrombin
FXIa was diluted to 3 nM in TBSA containing 250–2000 lM
S-2366
(L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanaline,
Diapharma, Franklin, OH, USA). Changes in OD 405 nm
were followed on the SpectraMax 340 microplate reader.
Michaelis–Menten constants (Km and Vmax) were determined
by standard methods using averages from six experiments. kcat
was calculated from the ratio of Vmax to enzyme concentration.
FXIaWT (3 nM) or FXIa-Glu555 (10 nM) in TBSA was mixed
1784 A. Zivelin et al.
with various concentrations of antithrombin (Dr Paul Bock,
Vanderbilt University), and incubated for 12.5 min at RT.
Reactions were diluted 1 : 5 in TBSA containing 500 lM
S-2366, and rate of substrate cleavage was followed by
measuring changes in OD 405 nm.
default parameters, and atoms assigned atomic radii and full
charges [21]. Predicted interactions between P2¢ residues on
antithrombin or FIX and FXIa were determined by
superimposing FXIa on the crystal structure of FVIIa in
complex with a bovine pancreatic trypsin inhibitor (BPTI)
analog [22]. The P4-P4¢ sites of BPTI were substituted with
those of antithrombin or FIX, and side chain conformations
were predicted by SCAP. Models were visualized using
MolScript [23] and Raster 3D [24].
Binding of FIXa to FXIa studied by surface plasmon resonance
(SPR)
Binding of FIXa to FXIa was studied on a dual flow cell
Biacore X device (Biacore, Inc. Uppsala, Sweden) as described
[15]. FXIa was immobilized on carboxymethyl dextran (CM5)
flow cells. Plasma kallikrein (PKa), a protein homologous to
FXIa [13], was immobilized on a separate cell as a control for
non-specific binding. FIXa (25–2000 nM) was injected across
the flow cell in 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005%
polysorbate 20, 2.0 mM CaCl2 (flow rate 35 lL min)1). Twominute association and 3-min dissociation times were used.
Data were corrected for non-specific binding by subtracting the
signal for binding to PKa. The BIAcore equilibrium analysis
method was employed, using software from the manufacturer.
The response at equilibrium (Rueq) was determined for each
concentration of analyte. Non-linear regression was used to
determine Kd using a 1 : 1 interaction steady state affinity
model, Rueq ¼ C · Rmax/C + Kd (C ¼ analyte concentration, Rmax ¼ maximal binding capacity).
Results
Clotting assays
The Western blot shown in Fig. 1(a) demonstrates that FXI
in the plasma of the patient homozygous for the Gly555Glu
substitution is of similar molecular mass (160 000 Da) to
normal FXI. On SDS–PAGE, recombinant FXIWT and
FXI-Glu555 run as 160 kDa proteins unreduced and
80 kDa reduced (Fig. 1b), consistent with the homodimeric
structure of plasma FXI [1]. Plasma FXI and FXIWT have
comparable specific activities in aPTT assays (200 U mg)1
and 180 U mg)1, respectively); however, FXI-Glu555 lacks
detectable activity (< 2 U mg)1). This could be caused by
poor activation during the contact stage of the assay, failure
of FXIa-Glu555 to activate FIX, or a combination of both
defects. FXIaWT and FXIa-Glu555 activated by FXIIa
appear similar on reducing SDS-polyacrylamide gels
(Fig. 1c), and are activated by FXIIa at similar rates
(Fig. 2a,b). FXIIa is predicted to bind to the FXI A4
domain [25], and cleaves FXI between Arg369 and Ile370
[13]. These areas are unlikely to be affected by the Gly555
mutation. FXIa was studied in a modified PTT assay that
tests the capacity of the protease to initiate coagulation
through FIX activation. FXIaWT and plasma FXIa have
similar activity (arbitrarily assigned a value of 100%), while
FXIa-Glu555 did not exhibit activity (< 1% plasma FXIa
activity). The data demonstrate that the Glu555 mutation is
the cause of the patient’s FXI deficiency, and indicate a
significant defect in FIX activation.
Modeling of the FXIa catalytic domain
The FXIaWT catalytic domain structure was modeled with
3 the program NEST [16], using the structure of FVIIa
inhibited by Glu-Gly-deoxy-methyl-Arg [17]. FXIa-Glu555
was modeled using NEST and SCAP [18] for prediction of
side chain conformation. FXIa models were superimposed
on structures for FVIIa, mouse glandular kallikrein-13 [9],
human brain trypsin IV in complex with benzamidine [19],
and snake venom plasminogen activator in complex with a
chloromethylketone [8] using the C-Alpha Match program
[20] and INSIGHT-II (Accelrys Inc). Maps of surface
electrostatic potential were generated using GRASP with
(a)
1
2
3
(b)
1
250
200
200
100
75
116
2
3
4
1
2
3
200
D
116
97
66
M
66
50
(c)
45
33
Z
HC
LC
Fig. 1. Plasma and recombinant proteins. (a) Western immunoblot of plasma using an anti-FXI monoclonal antibody. Lane 1, normal; 2,
homozygote for Glu117Stop (CRM –), and 3, homozygote for Gly555Glu. (b) FXIWT (lanes 1 and 3, and FXI-Glu555 (lanes 2 and 4) run under
non-reducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions on a 10% polyacrylamide-SDS gel. (c) FXIWT (1), FXIaWT (2) and FXIa-Glu555
(3) run on a reducing 12% polyacrylamide-SDS gel. Gels in (b) and (c) were stained with GelCode Blue. Positions of molecular mass markers in kDa
are indicated at the left of each panel. D, dimer; M, monomer; Z, zymogen; HC, FXIa heavy chain; and LC, FXIa light chain.
2004 International Society on Thrombosis and Haemostasis
Factor XI-Glu555 1785
(a)
0
1
2
3
4
6
(b)
8
Z
HC
Z
HC
LC
LC
(c)
Time (min)
0 2.5 5 7.5 10 20 30 60 60*
(d)
0
1
2
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4
6
8
1
2
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HC
Z
Z
HC
LC
HC
Fig. 2. Activation of FXI and FXI-Glu555. (a and b) Activation by XIIa. SDS–PAGE of FXI (100 lg mL)1) incubated with FXIIa (2 lg mL)1). At
various time points, indicated at the tops of the panels in hours, samples were removed into sample buffer and processed as described under Methods.
(c) Activation by thrombin. Western blots of FXIWT or FXI-Glu555 (2 lg mL)1) incubated with thrombin (1 U mL)1) and dextran sulfate (1 lg mL)1).
At various times (top of panel), aliquots were removed into sample buffer, and processed as described under Methods. The FXIWT sample in the separate
box at the right (60*) contains dextran sulfate in the absence of thrombin, and demonstrates no activation after 60 min. (d) Autoactivation. SDSpolyacrylamide gel of FXI (100 lg mL)1) incubated with dextran sulfate (2.5 lg mL)1). Lanes 1–3, FXIWT at 0, 30, and 60 min; lanes 4–6, FXI-Glu555 at
0, 30, and 60 min; lane 7, FXIa control. Z, zymogen FXI; HC, FXIa heavy chain; LC, FXIa light chain.
FXI is activated by thrombin on the surface of platelets [26] or
in the presence of polyanions such as dextran sulfate [27].
Thrombin activates FXI and FXI-Glu555 at similar rates in the
presence of dextran sulfate (Fig. 2c). Thrombin is thought to
bind to the FXI apple 1 domain [28], and it is unlikely that the
Glu555 substitution would interfere with this interaction. FXI
can undergo autoactivation in the presence of dextran sulfate
under certain conditions [27]; however, FXI-Glu555 does not
undergo autoactivation (Fig. 2d).
incubation of FIX with FXIaWT results in complete activation in
30 min, while FXIa-Glu555 does not cleave FIX appreciably. In
Fig. 3(b) a time course of FIX activation measured by
chromogenic assay clearly demonstrates the defect in FXIaGlu555. The rate of activation is so low that attempts to establish
a Km and kcat were not possible. Using high concentrations of
FIX (2 lM) and FXIa-Glu555 (10 nM) it was estimated that
FIX activation by FXIa-Glu555 is 400-fold slower than for
FXIaWT. The data agree with the clotting assay results, and
suggest that the Glu555 substitution, given its proximity to the
active site serine (Ser557), causes a significant catalytic defect.
Activation of FIX by FXIa
Cleavage of S-2366 by FXIa
FIX is cleaved by FXIa at two sites, releasing an activation
peptide, and producing the protease FIXa [1,29]. In Fig. 3(a),
To further evaluate the catalytic activity of FXIa-Glu555, the
ability of the protease to cleave the chromogenic substrate
Activation of FXI by thrombin and autoactivation
(b)
Wild type fXI
fXI Glu555
fIX
fIXaβ
0 5 10 15 20 30 0 30 60
Time (min)
Vmax (mOD405/min)
(a)
30
25
20
15
10
5
0
0 10 20 30 40 50 60
Time (min)
Fig. 3. Activation of FIX. (a) Western blots of FIX (300 nM) incubated with FXIaWT or FXIa-Glu555 (2 nM) in the presence of 2.5 mM CaCl2. At time
points indicated across the bottom of the panel, aliquots were removed into non-reducing sample buffer and processed as described under Methods.
Markers to the right of the panel indicate positions of FIX (fIX) and FIXa (fIXaß). (b) Chromogenic substrate assay of FIX (200 nM) activated by 1 nM
wild FXIaWT (s) or FXIa-Glu555 (d) in the presence of 2.5 mM CaCl2.
2004 International Society on Thrombosis and Haemostasis
1786 A. Zivelin et al.
Binding of FIX to FXIa studied by surface plasmon resonance
(SPR)
Using SPR, we previously demonstrated that factors IX and
IXa bind to immobilized FXIa with Kds of 100–150 nM [15]. A
problem with measuring binding of substrate to enzyme by this
technique is that the substrate may be converted to product.
This was previously addressed by preparing FXIa with the
active site serine changed to alanine (FXIa-Ala557) [15]. We
did not prepare an Ala557 version of FXIa-Glu555 because
two amino acid substitutions in close proximity to each other
might cause unpredictable structural perturbations. Instead, we
used FXIa-Glu555 and confined our analysis to the interaction
with FIXa. Figure 4(a) demonstrates that FIXa rapidly
associates with and dissociates from FXIa-Glu555 in a manner
Factor IXaβ bound
(pg/mm2)
(a)
250
200
150
100
50
0
50
100
150
Time (seconds)
0
Factor IXaβ bound
(pg/mm2)
(b)
200
200
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120
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40
0
0.0
0.5
1.0
IXaβ (µM)
1.5
2.0
Fig. 4. Surface plasmon resonance (SPR) studies of fIXa binding to
FXIa-Glu555. (a) SPR tracings of FIXa (0, 4, 8, 16, 32, 64, 125, 250, 500,
1000, and 2000 nM, bottom to top curves) binding to FXIa-Glu555 in the
presence of 2.0 mM CaCl2. (b) Plot of FIXa bound to FXIa-Glu555 as a
function of FIXa concentration. Data represents means ± SEM for three
separate experiments.
similar to that reported for plasma FXIa [15]. The Kd for
binding of FIXa to FXIa-Glu555 is 136 ± 38 nM (Fig. 4b); a
result in good agreement with binding of FIX and FIXa to
plasma FXIa and FXIa-Ala557 [15].
Inhibition of FXIa cleavage of S-2366 by antithrombin
The serine protease inhibitor antithrombin is an important
regulator of FXIa activity in plasma [30]. Inhibition of FXIaGlu555 by antithrombin was studied by chromogenic substrate
assay (Fig. 5). Under the conditions of the assay, nearly all
FXIaWT activity is inhibited after 12.5-min incubation with
20 lM antithrombin. In contrast, FXIa-Glu555 is resistant to
inhibition, even at an inhibitor to enzyme ratio in excess of
4000 : 1.
Modeling of the Glu555 substitution
We modeled substitutions of glycine by glutamic acid at FVIIa
Gly342 and FXIa Gly555 (both corresponding to chymotrypsin Gly193), and calculated the preferable energy-stable
conformation for the Glu555 side chain in FXIa-Glu555.
Using these methods, there were no significant changes
detected in the backbone conformation of the active site or
the geometry of the oxyanion pocket. The prediction that the
Glu555 side chain is oriented toward the protein surface
5 (Fig. 6a, left panel), is supported by analysis of side chain
orientations in the crystals of mouse glandular kallikrein-13
(Fig. 6a, right panel) [19], human brain trypsin IV [9] and snake
venom plasminogen activator [8] (data not shown). These
proteases are unusual in that residue 193 is Asp, Arg and Phe,
respectively. However, the charge on the Glu555 side chain is
predicted to significantly alter the electrostatic surface in the
vicinity of the active site pocket (Fig. 6b). Modeling of binding
interactions demonstrates that the Glu555 side chain clashes
with the P2¢ residues of antithrombin (leucine), FIX (valine),
and a BPTI analog (leucine) (Fig. 6c). Recent crystal structure
data for the interaction between the P4-P4¢ residues of the
inhibitory domain of protease nexin 2 and the S4-S4¢ sites of
the FXIa support this premise [31].
Initial velocity (mOD405/min)
S-2366 was determined. Interestingly, kcat for S-2366 cleavage
by FXIa-Glu555 (107 ± 31 s)1) was comparable to FXIaWT
(174 ± 20 s)1), suggesting active site conformation is minimally altered by the mutation. However, the Km for S-2366
cleavage by FXIa-Glu555 is six-fold greater than for
FXIaWT (3.2 mM and 0.56 mM, respectively), consistent with
an altered protease active site. This may be due to conformational changes in the S1, S2, and S3 sites of the protease that
are involved in binding tripeptide chromogenic substrates, or
steric interference due to the large glutamic acid side chain.
60
40
20
0
0
10
20 30
ATIII (µm)
40
50
Fig. 5. Inhibition of FXIa and FXIa-Glu555 by antithrombin. FXIaWT
(3 nM, s) or FXIa-Glu555 (10 nM, d) was incubated with varying concentrations of antithrombin for 12.5 min in TBSA. Residual FXIa activity
was measured with a chromogenic assay as described under Methods.
2004 International Society on Thrombosis and Haemostasis
Factor XI-Glu555 1787
Fig. 6. Modeling of the catalytic domains of fXIa and fXIa-Glu555. (A) Structural Models. Superimposition of fXIa-Glu555 (blue) and human factor VIIa
(green), and mouse glandular kallikrein-13 (violet). Factor VIIa Gly342 and kallikrein-13 Asp211 correspond to chymotrypsin Gly193. The histidine,
aspartic acid, and serin of the catalytic triad, and amino acid 193 (chymotrypsin) are represented by stick structures. The orientation of the side chain of
Glu555 in fXIa and Asp211 in mouse glandular kallikrein-13 are similar, and both point toward the protein surface. (B) Electrostatic models of the fXIa
catalytic domain. Electrostatic potential was mapped onto the fXIaWT (left) and fXIa-Glu555 (right) catalytic domains using GRASP. Contouring levels of
electrostatic potential are -10 kT/e (red) and 10 kT/e (blue). Note the surface exposed side chain of Glu555 affects the electrostatic surface near the
fXIa catalytic pocket. (C) FXIa interactions with antithrombin, factor IX, and a bovine pancreatic trypsin inhibitor (BPTI) analog. The blue ribbon diagrams
depict the active sites of fXIaWT (left panel) and fXIa-Glu555 (right panel) in complex with the P4-P4¢ sites of antithrombin and factor IX (red ribbon
diagrams) and a BPTI analog (yellow ribbon diagrams). The P4-P4’ sites of antithrombin and factor IX overlap. The histidine, aspartic acid and serine
residues of the fXIa catalytic triad are shown as blue stick structures. Amino acid 555 for fXIa and fXIa-Glu555 are represented by green stick structures.
The P2’ sites of antithrombin (leucine in violet), factor IX (valine in red), and BPTI analog (leucine in yellow) are also represented by stick structures. Note
the Glu555 side chain clashes with the P2’ residue of all three molecules.
Discussion
Inherited deficiencies of factors VII [32], IX [33], and X [34] are
commonly associated with CRM + variants. Studies of
mutant versions of these proteins have provided a wealth of
information on structure-function relationships. While a few
FXI mutations are associated with circulating protein, the
majority of FXI deficient patients are CRM – [3]. FXI 2004 International Society on Thrombosis and Haemostasis
Gln226Arg and FXI-Ser248Asn were identified in a compound
heterozygous individual [35]. FXI-Gln226Arg appears to
function normally and may be a neutral polymorphism. FXISer248Asn has similar activity to FXIWT in plasma assays, but
is defective in platelet binding [36]. Activation of FXI by
thrombin on platelets has been proposed as an important
mechanism in vivo [26], and this mutation may explain bleeding
in the propositus and his family members. Heterozygosity for
1788 A. Zivelin et al.
Pro520Leu was identified in a child with mild bleeding [37].
FXI-Leu520 is expressed by transfected fibroblasts, and has a
slight defect in cleavage of S-2366 and FIX activation.
Recently, Quelin et al. reported two mutations, Glu350Ala
and Thr575Met, associated with discrepant antigen and
activity levels [4], however, the functional consequences of
these mutations are not known.
FXIa-Glu555 is clearly a CRM + variant with a profound
defect in FIX activation. There is compelling evidence that
activation of FX [38] and prothrombin [39] involves distinct
steps. Initial binding of substrate occurs at exosites on the
protease that are remote from the active site. Exosite
interactions appear to be largely responsible for determining
affinity and specificity. Exosite binding is followed by a
docking interaction near the active site, and finally catalysis.
Activation of FIX by FXIa likely follows this model [40].
Factor IX initially binds to the FXIa non-catalytic region [14],
an interaction unlikely to be affected by the Glu555 substitution. Indeed, SPR studies indicate FIX binds normally to
FXIa-Glu555. Rather, the location of the substitution suggests
a defect in docking near the catalytic site, or a conformational
change in the active site affecting catalysis, are more likely.
Modeling studies indicate that a major consequence of the
Glu555 substitution is a steric clash that interferes with the
interaction between the P2¢ residues on macromolecular
substrates/inhibitors and the FXIa S2¢ site. Furthermore, kcat
for cleavage of the tripeptide substrate S-2366 by FXIaGlu555 and FXIaWT are comparable. This suggests that
conformation of the FXIa-Glu555 active site is not affected by
the mutation; a premise supported by modeling studies
comparing FXIa-Glu555 with FVIIa and serine proteases
containing non-glycine residues at position 193. While the
major prediction of modeling is interference with P2¢–S2¢
interactions, this analysis does not rule out abnormalities in
the active site. The Km for cleavage of S-2366 by FXIa-Glu555
is sixfold greater than for FXIaWT. It is possible, that Glu555
causes distortion of the S1-S3 specificity sites or other
components of the active site not predicted by modeling.
4 Bajaj et al. examined a FIX variant with valine at the position
corresponding to FXI Gly555 (FIX-Val363), and predicted
the substitution may involve conformational changes in the
active site [10].
In summary, we have identified and characterized the first
CRM + variant of FXI with a severe defect in FIX activation.
FXIa-Glu555 cleavage of FIX is considerably poorer than its
hydrolysis of S-2366, and it is not inhibited by antithrombin.
Interestingly, FIXa-Val363 (see above) also failed to interact
properly with its substrate (FX) or antithrombin [10], suggesting a defect similar to FXIa-Glu555. Modeling predicts that the
large side chain of Glu555 alters the electrostatic charge around
the active site, and causes steric clashes with the P2¢ sites of
substrates and inhibitors. Human trypsin IV, which has an
arginine at residue 193 (chymotrypsin) is markedly more
resistant to inhibition by trypsin inhibitors than is human
trypsin I [9]. The presence of non-glycine residues at this
position therefore may represent a mechanism for restricting
the spectrum of substrates or inhibitors for some serine
proteases [9].
Acknowledgements
The authors thank Melanie A. Abboud, Mao-Fu Sun, Qiufang
Cheng, Nurit Rosenberg and Rivka Yatuv for expert technical
work, and Jean McClure for preparation of the manuscript.
This work was supported by grant HL58837 from the
National Heart, Lung and Blood Institute. DG is an Established Investigator of the American Heart Association.
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