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The Apple 1 and Apple 4 Domains of Factor XI Act Synergistically to
Promote the Surface-Mediated Activation of Factor XI by Factor XIIa
By Frank A. Baglia, Frances
S.Seaman, and Peter N. Walsh
Binding sites for high molecular weight kininogen (HK) and
for factor Xlla are present in the Apple 1 ( A l l and the A4
domains of factor XI, respectively. To define the roles of
these two sites in surface-mediated factor-XI activation we
prepared conformationally constrained synthetic peptides
and recombinant A I domain (rAl) and determined their effects on the activation of factor XI byfactor Xlla in thepresenceof HK and either kaolin or dextran sulfate. Surfacemediated factor-XI activation by factor Xlla was inhibited by
a conformationally constrained A4 peptide (Ala3’7-Gl$59), by
an A1 peptide (Phew-Se$B), and by rAl (Glu’-Serw). When
used in combination at equimolar concentrations, rAl and
A4 peptide were 10-fold more effective than either one alone
in inhibiting surface-mediated activation of factor XI by fac-
tor Xlla. The A4 peptide was a competitive inhibitor of factor
Xlla amidolytic activity and a noncompetitive inhibitor of
factor-XI activation by factor Xlla, whereas r A l and the A1
peptide did notinhibit factor Xlla. The r A l domain inhibited
factor XI binding to HK, whereas the A4 peptide did not. We
conclude that specific sequences exposed on the surfaces
of the A1 (Valss-Lysm)
and A4 (Ala3’7-Gl$50)domains of factor
Xi act synergistically to promotesurface-mediated factor-XI
activation by factor Xlla in the presence of HK by binding
factor XI to surface-bound HK (A1 domain) and by binding
factor Xlla near the cleavage site (Arg369-lle370)
of factor XI
(A4 domain).
0 1995 by The American Societyof Hematology.
I
the surface-mediated activation of factor XI. Experiments
are presented that support the notion that specific sequences
exposed on the surface of the A1 and A4 domains of factor
XI promote surface-mediated factor XI activation by factor
XIIa.
T IS WELL KNOWN THAT the initiation of the contact
phase of intrinsic coagulation involves a complex interaction of proteins on negatively charged surfaces leading to
the activation of factor IX. Human factor XI participates in
the contact phase of blood coagulation and is involved in
intermolecular interactions with factor XIIa, high molecular
weight kininogen (HK), and factor IX.”I7 Factor XI is a 160
kD homodimer consisting of two identical disulfide-linked
polypeptide chains, each of which can be cleaved at a single
peptide bond (Arg3h9-Ile370)
by factor XIIa to give rise to
factor XIa9. Factor XI circulates in plasma in a noncovalent
complex with HK that is required for the binding of factor
XI to negatively charged surfaces and its proteolytic activation to factor XIa.7~1h
The molecular mechanisms of factor
XI activation and expression of factor Xla enzymatic activity
have been studied in detai1.1~6~10”s.17
From the sequence of a
cDNA insert coding for factor XI, the primary structure has
beenelucidated.’ Four tandem repeat sequences (Al, A2,
A3, and A4 domains) or Apple domains, each encoded by
two exons, are present in each heavy chain.” We have identified a binding site for HK in the first (Al) of the four Apple
domains:.”’ whereas a substrate binding site for factor IX
was localized to the A2 domain4and a binding site for factor
XIIa was localized to the A4 domain.l9 The present study
was undertaken to determine which domains are involved in
From The Sol Sherry Thrombosis Research Center, Departments
of Medicine and Biochemistry, Temple University School of Medicine, Philadelphia, PA.
Submitted April 15, 1994; accepted December 2, 1994.
Supported byresearchgrantsfrom
the National Institutes <~
Health (HL.46213, HL45486, and HL25661) and from the W.W.
Smith Charitable Trust.
Address reprint requests to Peter N . Walsh, MD, PhD,Sol Sherry
Thrombosis Research Center,Temple University School of Medicine,
3400 N Broad St, Philadelphia, PA 19140.
The publication costsof this article were defrayedin part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8508-0025$3.00/0
2078
MATERIALS AND METHODS
PuriJication of coagulationproteins. FactorXI, purified from
human plasma by immunoaffinity chromatography using a monoclonal antibody to factor XI,’4 had a specific activity of 250 U/mg
protein. HK (specific activity, 15 U/mg) was purified by the method
of Kerbiriou and Griffin.” Human a-factor XIIa was prepared by
the method of Shore” and was a generous gift of Drs Joseph D.
Shore and M. Margarida Bemardo (Division
of Biochemical Research, Henry Ford Hospital, Detroit,
MI). The protein migrated at an
apparent molecular weight(Mr) of 80 kD by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in the absence
of
reducingagents,whereas,
in thepresence of reducingagents, it
migrated as a heavy chain of 50 kD Mr and a light chain of 30 kD
Mr. The factor XIIa concentration in solution was estimated from
mol of thechromoitsamidolyticactivity.Ithydrolyzed 3.78 X
Ortho Diaggenic substrate S-2302 (D-Pro-Phe-Arg-p-nitroanilide;
nostics, Raritan, NJ) per minute per microgram of protein at pH 8.0
and 37°C. Amidolytic activity was assayed using 0.12
m m o K S2302 in 0.1 m o l L Tris, 0.15 m o l L NaC1, pH 8.0. The change in
absorbence at 405 nm was measured during incubation at 37°C. All
purified proteins appeared homogeneous by SDS-PAGE.
Preparation of recombinant AI ( r A l ) domain. Details of the
expression of the rAl domain are reported elsewhere.*’” Briefly, the
AI domain of factor XI was prepared inEscherichia coli, following
amplification using the polymerase chain reaction with primers correspondingtothe
A1 domainandhumanfactorXIcDNA.This
factor XI cDNA, a 2. l-kb EcoRI fragment containing the complete
factor XI cDNA coding sequence, is a gift from Drs Dominic W.
Chung, Kazuo Fujikawa and Earl W. Davie (Department of Biochemistry, University of Washington, Seattle, WA). The resulting
polymerase chain reaction amplification product, which was found
to contain the correct nucleotide sequence, was ligated
into the cloning site of the pQE-9 vector (Qiagen Inc, Chatsworth, CA) and was
expressed and purified according to the protocols contained in the
QIAexpress kit manual. The expressed andpurified recombinant A1
domain contained an NH2 terminal amino acid sequence identical
to that of factor XI, showed a single band on SDS-PAGE, and was
refolded according to the method recorded below.
Peptide synthesis. Peptides were synthesized on an Applied Bio-
Blood, Vol 85, No 8 (April 15), 1995: pp 2078-2083
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2079
FACTOR XI APPLE DOMAINS IN CONTACTACTIVATION
Table 1. Effects of Recombinant and Synthetic Peptides onthe
SurfaceMediated Activation of Factor XI by Factor Xlla
Peptide of Heavy
Chain (domains)
Reference
No. Where
Amino Acid
Sequence
Is Given
9
Glu’-Se$o( A l )
5
Phes-Sep6 ( A l l
4
Ala’34-Ala’76 (A21
5
A ~ n ’ ~ ~ - A r(A3)
g’~
Kaolin 19
Ala3’7-Gly3” (A4)
A ~ p ’ ~ ~ - L(A4)
yKaolin
s ~ ~ 19
Glu’-SePo( A l )
PhP-SeP (Al)
Ala’34”A1a’75
(A2)
A~n’”-Arg’~~
(A3)
Ala3’7-Gly3” (A41
A~p’”-Lys’~~
(A4)
9
5
4
5
Dextran
19
Dextran
19
Surface
Concentration (moll
L) of Peptide to
Inhibit SurfaceMediated FXI
Activation by FXlla
50%
Kaolin
Kaolin
Kaolin
Kaolin
5 X 10-~
1 x 10-6
1 x 10“
5 X 10-5
5 X 10-7
6.5 X 10-5
Dextran sulfate
Dextran sulfate
Dextran sulfate
Dextran sulfate
sulfate
sulfate
5x
5X
1X
5X
7.5 x
7.5 x
Abbreviations: FXlla, factor Xlla; FXI, factor XI.
10-8
10-5
10-3
lo-’
Mass spectrometry was used to assess homogeneity and molecular
mass of each synthesized peptide. MALDI was performed as previously described.” The spectra of synthesized peptides showed
masses almost identical to calculated values (ie, within 0.5%). with a
single ion species detected in each case. The calculated and observed
masses of the synthesized peptides were as follows: Phes6-SerS6,
3404.5 and 3408.9; Ala’M-Ala’76,
4777.2 and 4787.7; AsnZ3’-ArgZM,
3425.8and 3413.5; Ala”7-Gly3s0,3457.2 and 3466.8; andAspZsoLysZ8’, 1187.0 and 1182.2.
Preparation and characterization of factor Xla. Purified factor
XI was activated by incubation at 37°C with human factor XIIa as
previously describedi4 to be used as a reference standard in assays
of factor XIa generation. Gel electrophoresis in the presence of
NaDodSO,of this preparation under reducing conditions showed
two major bands of Mr 50 and 30 k D .
Factor XIIa assays. Factor XIIa was assayed using the chromogenic substrate S-2302 according to the procedure previously described?‘
Assay of factor XI activation. The rate of factor XI activation
by factor XIIa was determined as previously reported.’ Human factor
XI (0.17 pmol/L) was incubated with factor XIIa (0.017 pmol/L) in
the presence of HK (0.07 pmoVL) and
dextran sulfate (2 pglmL)
or kaolin (0.5 mglmL) for 15 minutes at 37”C, and the rate of factor
XIa generation was measured using the substrate S-2366 at 405 nm.
The amidolytic assay of factor XIa was performed by a modification
of the method of Scott et al.27Factor XIa generation, in the experiments reported here, was quantitated from a standard curve prepared
using purified factor XIa.
Effect of peptides on the rate of activation of factor X I by factor
XIIa. The assay procedure was the same as described above, except
factor XIIa and HK were incubated for 3 minutes at 37°C with either
peptide or buffer solution before the addition of other components
of the assay mixture.
systems 430A peptide synthesizer (Applied Biosystems, Foster City,
CA) by a modification of the procedure described by Kent andClarkLewis” as previously de~cribed.’~The sequences of the synthetic
peptides used in this study and referred to in Table 1 are given in
previous p u b l i c a t i ~ n s ? ~ ~ ~ ~ ’ ~
Refolding and reduction and alkylation of peptides. To refold
peptides containing cysteine residues, the peptide was dissolved in
deionized water as a 0.1 mg/mL solution in a flask containing a stir
bar. The pH was adjusted to 8.5 with NH,OH, and the solution was
allowed to stir at5°C for at least 3 days. After stirring, all the
RESULTS
peptide was apparently still in solution. The resulting solution was
lyophilized. Allthe lyophilized peptide was able to redissolve in
Effects of heavy chain-derived peptides on the activation
high performance liquid chromatography (HPLC) grade water, and
XI by factor XIIa in thepresence of HK and kaolin.
of
factor
there was no particulate material apparent. Alternatively, peptides
It is well documented that factor XI circulates inplasma
were reduced with dithiothreitol and alkylated with iodoacetamide
in a noncovalent complex with HK, and this interaction is
as previously described.,
necessary for the binding of factor XI to a negatively charged
HPLC. The HPLC system usedwas from Waters (Waters 600
surface and for its efficient proteolytic activation to factor
Gradient Module, Model 740 Data Module, Model 46K Universal
~ 1 ~ . l O . I l . l 6 Because a binding site for HK is present in the
Injector, and Lambda-Max Model 481 Detector; Milford, MA). The
reverse phase chromatography described here was performed using a
A1 domain of factor XI335and a binding site for factor XIIa
Waters C8 pBondapak Column, whereas gel filtration was performed
is present in the A4 domain,” we examined the contributions
using a Waters Protein-Pak 60 Column as previously de~cribed.~.’.’~ of these two sites to the surface-mediated activation of factor
Characterization of synthetic peptides. All the peptides used in
XI by factor XIIa. Conformationally constrained synthetic
this study were examined by HPLC (both reverse-phase and gelpeptides of the A1 and A4 domains of factor XI were tested
filtration), and all showed a single homogeneous peak (data not
for their effects on the activation of factor XI by factor XIIa
shown). When the refolded peptides were examined by HPLC (both
in the presence of HK and kaolin. The results are presented
reverse-phase and gel-filtration), single homogeneous peaks with
in Fig 1 and summarized in Table 1. Peptide Ala3’7-Gly350
identical retention times to the original mixtures were observed. This
shows the presence of a single homogeneous mixture of refolded
from the A4 domain inhibited factor XI activation by factor
peptides and not a mixed population of diverse polymers. The results
XIIa by 50% at a concentration of 5 X
m o m , whereas
were the same after reduction and alkylation of these same peptides;
peptide Phe56-Sep6from the A1 domain inhibited factor XI
ie, single peaks with identical retention times to the original mixtures
activation by 50% at a concentration of l X
m o m (Fig
were observed by both reverse-phase and gel-filtration HPLC. In
1). Moreover, the recombinant A1 domain (Glu’-Sergo)was
addition, all reduced and alkylated or refolded peptides were examrequired at a concentration of 5 X
mom to achieve
ined for free SH groups using the Ellman reagent (5,5‘-dithiobis[250% inhibition. In contrast, a peptide derived from the A2
nitrobenzoic acid]). It was determinedz4that there was less than 0.02
domain (Ala’34-Ala’76)
required 200-fold higher concentramol of free SH per mol of peptide, which further verifies that these
tions than the A1 and A4 peptides to produce 50% inhibition
refolded peptides were homogeneous preparations consisting of inof factor XI activation, ie, 1 X
m o m . Moreover, peptramolecular disulfide-bonded peptide.
Matrix-assisted laser desorption flash ionization (MALDI).
tides from the A3 domain ( A ~ n ~ ” - A r and
g ~ ~the
~ )other half
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2080
BAGLIA, SEAMAN, AND WALSH
Effect of A I and A4 peptides on the amidolytic activity
of factor Xlla in the presence of dextran sulfate and HE(.
Because we previously showed that the A4 peptide (Ala3I7Gly350)is a competitive inhibitor of factor XIIa amidolytic
activity suggesting that it binds near the active site of factor
XIIa,I9 we determined whether the A4 peptide has a similar
effect on surface-mediated factor XI activation by factor
XIIa. We have shown" that the A4 peptide (Al~17-Gly7so)
inhibits (by 50%) the amidolytic activity of factor XIIa at a
concentration of 5 X
mom inthe presence of dextran
sulfate (2 pg/mL) and HK (0.7 pmol/L; data not shown).
However, the AI domain peptide (Glu'-Serg")did not inhibit
the amidolytic activity of factor XIIa at a concentration of
up to
m o m in the presence of dextran sulfate (2 pgl
10'
lo"
104
101
lo"
10"
10'2
CONCENTRATION OF PEPTIDE (W)
mL) and HK (0.7 pmol/L; data not shown).
Effects of heavy chain-derived peptides on the activation
Fig 1. Effects of factor XI heavy-chain recombinant and synthetic
of factor X I by factor XIla in the presence of dextran sulfate.
paptides on theactivation of factor XI by
factor Xlla in the presence
of kaolin. The effects of various recombinant and synthetic peptides Because of the effect of kaolin on the amidolytic activity of
factor XIIa, we reexamined the effect of heavy chain-derived
on the activation of factor XI by factor
Xlla in the presence of kaolin
were examined including Glu'-SeP' (0-1, Ala3'7-Gl$s I- A -1, Phe"factor XI peptides using the negatively charged surface, dexS e p 1- 0 -1, AsnP'-Argm (- V -1, Aspzso-Lysz" l- 0 -1, and Ala'3'tran sulfate, which does not affect the amidolytic activity
Ala"' (- A -1. HK (0.07 pmol/L) and kaolin (0.5 rng/mL) were incubated for3 minutes at 37% either with the designated peptide at the of factor XIIa. The results are presented in Fig 3 and are
summarized in Table 1. Peptide Ala317-Glf50from the A4
concentration shown or with buffer solution. The factor XI (0.17
pmol/L) wasadded with factor Xlla(0.017 prnol/L), the mixture was
domain inhibited factor XI activation by factor XIIa by 50%
incubated for 15 minutes, and the rate of factor Xla formation was
at a concentration of 7.5 X
mol/L, whereas the recombidetermined as described under Materialsand Methods.
nant A1 domain peptide (Glu'-Serg0)required a concentration of 5 X lo-' m o m for a similar effect (Fig 3). When
used in combination at equimolar concentrations, these two
of the A4 domain (A~p**o-Gl~*xx)
also required concentrapeptides proved to be an order of magnitude more effective
tions more than 100-fold higher than the A1 and A4 domain
(Ki = 7.5 X
m o m ) than either one alone in inhibiting
peptides for similar effects. The inhibitory effects of these
the surface-mediated activation of factor XI by factor XIIa.
peptides are not unexpected given the 23% to 34% identity
Moreover, peptides from the A? ( A ~ n ' ~ ~ - A r g A2
' ~ ) ,(Ala'34of these tandem repeats.' However, we have previously
shown that the A2 domain peptide (Ala1"-Ala'76) inhibits
factor IX activation by factor XIa with an inhibition constant
120
(Ki) of 1 X
moL4
zi
ii 100
The effects of negatively charged surface on amidolytic
x
activity of factor XIIa. We have previously shown that the
80
A4 domain of factor XI (peptide Ala3'7-Gly350)
binds factor
N
2
XIIa and is a competitive inhibitor of factor XIIa amidolytic
2 60
activity (Ki = 3.8 pmoVL)." To better understand the surface-mediated activation of factor XI and to interpret the
g 40
L
experiment presented in Fig 1, we examined the effect of
20
the recombinant A1 peptide (Glu'-Serg0) on the amidolytic
activity of factor XIIa both in the presence and absence of
g
d
o
kaolin and HK (Fig 2). Kaolin (0.5 mg/mL) was shown to
P
inhibit (by -35%) the amidolytic activity of factor XIIa
f -20
using the substrate S2302 in the absence of the recombinant
0
2
4
6
8
10
12
A1 peptide (Gl~l-Ser'~).
In the presence of recombinant AI
CONCENTRATION OF Glul-Ser9O (pH)
peptide, HK, and kaolin, an even greater inhibition (85%)
Fig 2. The amidolytic activity of factor Xlla in the presence and
of amidolytic activity was observed. However, in the presabsence of kaolin andvariousconcentrations of A1 domain
recombience of HK and peptide in the absence of kaolin, no inhibinant peptide Glu'-SeP. Factor Xlla (0.017 pmol/L) was incubated
tion of amidolytic activity was observed. Because of this
with various concentrations of thepeptide for 3 minutes at 37°C in
effect of kaolin on factor XIIa, we investigated another negathe presence and absence of kaolin (0.5 mg/mL). The amidolytic
assay was performedaccording t o S C O as
W described under Materitively charged surface, dextran sulfate. Dextran sulfate (2
als and Methods. The graph shows the effects of the A1 (Glu'-SeP)
mg/mL), either in the presence or the absence of HK, did
peptide on the amidolytic activity of factor Xlla in the presence of
not affect the amidolytic activity of factor XIIa (data not
kaolin and HK (- A -1, in the presence of kaolin but theabsence of
shown). Therefore, we used dextran sulfate in all our subseHK I- -), in the absence of kaolin but the
presence of HK t- 0 -1,
and in the absence of kaolin and HK 1- 0 -1.
quent experiments on surface-mediated factor XI activation.
-
4
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2081
FACTOR XI APPLE DOMAINS IN CONTACT ACTIVATION
(Glu'-Ser9'; Ki = 6 X
m o m ; see Fig 3); (2) whenused
in combination at equimolar concentrations, A1 (Glul-Sergo)
and A4 (Ala317-Gly350)
peptides were an order of magnitude
more effective (Ki = 6 X
m o m ) than either one alone
in inhibiting surface-mediated activation of factor XI by factor XIIa (Fig 3); (3) the A4 peptide (Ala317-Gly350)
was a
competitive inhibitor of factor XIIa amidolytic activity (Ki
= 3.8 X
m o m ) and a noncompetitive inhibitor of factor
XI activation by factor XIIa (Ki = 3.75 X
m0yL)l9
whereas the A1 peptide (Glul-SergO)did not inhibit factor
XIIa (data not shown); and (4) the A1 (Glul-Sergo)domain
-20
.
(Ki =
m o m ) inhibited factor XI binding to HK,
1
2
1
1
2
whereas the A4 peptide (Ala317-Gly350) had such
no
C0NQMIUTK)N OF PEPTIDE (M)
We have previously shown" that the A4 peptide Ala3I7Gly350is a noncompetitive inhibitor of factor-XIIa-mediated
Fig 3. Effects of factor XI heavy-chain synthetic and recombinant
factor XI activation in the fluid phase and is a competitive
peptides onthe ectivation of factor XI by factorXlla in the presence
of dextran sulfate. The effects of various synthetic and recombinant
inhibitor of factor XIIa amidolytic activity (Ki = 3.8 X
peptides on the activation of factor XI by factorXlla in the presence
m o m , using the chromogenic substrate S2302. In the present
of dextran sulfate were examined including Glu'-Se$o
(- A -1, Ala3"study, we examined the effect of this A4 peptide on the
G I P (- 0 -1, Ala3"-Glys60 and G l u ' S # ' ((3-1,
Phe"-SeP (- V -1,
amidolytic activity of factor XIIa in the presence of dextran
AsnP6-Arp (- 0 -), AapUO-Lysm (- A -1, and Ala'"-Ala'76 (- W -1.
HK (0.07 pmol/L) and dextran sulfate (2 pg/mL) were incubated for
sulfate. Because the inhibitory effect of Ala3'7-Gly350(Ki =
3 minutes at 37°C either
with the deaignated peptides at
the concen5X
m o m ) in the presence of dextran sulfate is the
tration shown orwith buffer solution. The factor
XI (0.17 pmol/L) was
same
as
that
in the fluid phase and because dextran sulfate
added with factor Xlla (0.017 pmol/L), the mixture was incubated
for
does not alter the amidolytic activity of factor XIIa, dextran
15 minutes, and the rate of factor Xla formation was determinedas
sulfate is a more suitable surface for the present studies than
described under Materials and Methods.
kaolin, which appears to bind to factor XIIa and inhibit its
amidolytic activity (Fig 2). In contrast to the A4 peptide
the recombinant A1 peptide Glul-Sergohad no
Ala'76),and the other half of the A4 ( A ~ p ~ ~ ~ - Gdomains
l y * ~ ~ ) Ala317-Gly350,
effect on the amidolytic activity of factor XIIa in the presrequired 100-fold higher concentrations for similar effects.
ence of dextran sulfate. This result further confirms the suitDISCUSSION
ability of dextran sulfate as a surface in these studies. It also
supports the notion that the mechanism of action of the A1
Factor XI participates in the contact phase of blood coagudomain peptides is limited to its capacity to inhibit binding
lation and interacts directly with HK, factor IX, and factor
of factor XI to HK.
XIIa. Studies in our laboratory have identified and characterThe results of our studies provide insights into the mechaized the amino acids within the first two Apple domains of
nisms of surface-mediated factor-XI activation by factor
the factor XI heavy chain that form binding sites for HK
XIIa. Factor XI exists in normal human plasma in a noncovaand factor IX.3,4,5Recently, we have determined that a selent complex with HK.7.'6 The binding of this complex to
quence of amino acids in the A4 domain of factor XI comnegatively charged surface^^"^.^"'^ Including the activated
prises a site for interacting with factor x11a.l~ It is well
platelet surface29promotes optimal rates of factor XI activadocumented that negatively charged surfaces accelerate the
tion,
presumably by the formation of a ternary complex of
rate of activation of factor XI by factor XIIa in the presence
of ~ ~ . l . l 1 , 1 6 , 2The
8
precise mechanism by which negatively
HK, factor XI, and factor XIIa.'." As summarized above,
peptides derived from the A1 and A4 domains appear to act
charged surfaces and HK facilitate this interaction i s not
synergistically to inhibit surface-mediated factor XI activaunderstood. Therefore, the present study was undertaken to
tion by factor XIIa, as evidenced by the fact that concentradetermine what domains of factor XI are involved in the
tions of equimolar mixtures of these peptides required to
surface-mediated reactions and to gain insight into the nature
inhibit factor XI activation by factor XIIa in the presence of
of the interactions between factor XI and factor XIIa.
HK are about 10-fold less than those required when the A1
The present study supports the notion that specific seor A4 peptides are used alone. The mechanisms by which
quences exposed on the surfaces of the A1 domain
these two sets of peptides act are different, the A1 peptides
LysS3)and the A4 domain (Ala317-Gly350)
of factor XI act
inhibiting binding of HK to the A1 domain and the A4
alone and synergistically to promote surface-mediated factor
XI activation by factor XIIa in the presence of HK by binding
peptides inhibiting the binding of factor XIIa to the A4 domain. It follows that the A1 and A4 domains promote synerfactor XI to surface-bound HK (A1 domain) and by binding
factor XIIa near the cleavage site of factor XI (A4 domain).
gistic interactions of factor XI with HK and factor XIIa.
The evidence supporting this conclusion (summarized in part
The mechanism of this synergism is a matter of conjecture.
in Table 1) is as follows: (1) surface-mediated factor XI
However, it is possible that the binding of HK to the A1
activation by factor XIIa was inhibited by an A4 peptide
domain induces a conformational alteration in factor XI that
(Ala317-Gly350;
Ki = 7.5 X
mol/L) and by the A1 peptide
renders it a more favorable substrate for factor XIIa. Alterna-
'
...S.
'
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2082
BAGLIA, SEAMAN, AND WALSH
tively, the binding of the HK-factor XI complex to surface
(ie, dextran sulfate) could result in the formation of a surfacebound ternary complex of these proteins with factor XIIa,
with a consequent rate enhancement because of favorable
positioning of the enzyme (factor XIIa) active site near the
substrate (factor XI) cleavage site (Arg’69-Ile370).
Another
possible explanation of this phenomenon is that HK binding
of factor XI to a surface where factor XIIa is bound could
also increase the rate of molecular encounters without regard
to “favorable positioning.” Thus, the rate increase could
result from an alteration of the entropy of activation term in
the rate constant expression.
Because the four repeat domains (Al, A2, A3, and A4)
have a 23% to 34% sequence identity, it was not surprising
to see that the A2 and A3 domain peptides inhibit factor XI
activation at concentrations 100- to 200-fold higher than
those required to obtain similar effects with the A1 and A4
peptides. Because of the similar structural motif of these
domains, the A2andA3 synthetic peptides might disrupt
native protein structures and activities by binding to factor
XI and thereby disrupting intermolecular domain-domain interactions. We might better understand these effects by producing chimeras of the Apple domains and testing their function.
Our previous ~ t u d i e s ~ ’suggest
~ ’ ~ . ’ ~that a common structural motif present at similar positions within the A l , A2,
and A4 domains of factor XI mediates the interactions of
factor XI with its plasma ligands, ie, HK (A1 domain), factor
IX (A2 domain), and factor XIIa (A4 domain). Our computer
modeling studies predict the presence of three loop structures
(antiparallel &strands connected by p-turns) in each of the
Apple domains that present solvent-accessible amino acid
side chains unique to each Apple domain that are used for
these highly specific protein-protein interactions. Our studies
with conformationally constrained synthetic peptides and recombinant A1 domain peptides confirm our predictions from
computer modeling, which tend to support the validity of the
paradigm as a predictor of protein-binding surface structures.
However, detailed information about the secondary and tertiary structures of the Apple domains of factor XI will have
to await the conduction of studies using high-resolution xray crystallography or nuclear magnetic resonance imaging.
ACKNOWLEDGMENT
The authors are grateful to Drs Joseph D. Shore and M. Margarida
Bemardo (Division of Biochemical Research, Henry Ford Hospital,
Detroit, MI) for the gift ofhuman factor XIIa; to Drs Dominic
W. Chung, Kazuo Fujikawa, and Earl W. Davie (Department of
Biochemistry, University of Washington, Seattle, WA) for the gift
of human factor XI cDNA; and to Patricia Pileggi for assistance in
manuscript preparation.
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FACTOR XI APPLE DOMAINSINCONTACTACTIVATION
2la. Seaman 1.3. Haglia F A . G u r r JA. Jamcson B A . Wal\h PN:
Binding of h i g h - r n o l c c u l ~ ~ r - ~kininogen
~ ~ ; ~ ~ s t o the Apple I dom:lin
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22. Kent SBH. Clark-Lewis I: Modern mcthtrtls lor the chemical
in Alitalo K. Partuncn l'.
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1995 85: 2078-2083
The Apple 1 and Apple 4 domains of factor XI act synergistically to
promote the surface-mediated activation of factor XI by factor XIIa
FA Baglia, FS Seaman and PN Walsh
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