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A Molecular Model for the Triplicated A Domains of Human Factor VIII
Based on the Crystal Structure of Human Ceruloplasmin
By S. Pemberton, P. Lindley, V. Zaitsev, G. Card, E.G.D. Tuddenham, and G. Kemball-Cook
The hemophilia A mutation database lists more than 160
missense mutations: each represents a molecular defect in
the FVIII molecule, resulting in the X-linked bleeding disorder hemophilia A with a clinical presentation varying from
mild to severe. Without a three-dimensional FVIII structure
it is in most cases impossible to explain biological dysfunction in terms of the underlying molecular pathology. However, recently the crystal structure of the homologous human plasma copper-binding protein ceruloplasmin (hCp) has
been solved, and the A domains of FVIII share approximately
34% sequence identity with hCp. This advance has enabled
the building of a molecular model of the A domains of FVIII
based on the sequence identity between the two
proteins. The model allows exploration of predictions regarding the general features of the FVIII molecule, such as
the binding-sites for factor IXa and activated protein C; it
has also allowed the mapping of more than 30 selected mutations with known phenotype from the database, and the
prediction of hypothetical links to dysfunction in all but a
few cases. A computer-generated molecular model such as
that reported here cannot substitute for a crystal structure.
However, until such a structure for FVIII becomes available,
the model represents a significant advance in modeling FVIII;
it should prove a useful tool for exploiting the increasing
amount of information in the hemophilia A mutation database, and for selecting appropriate targets for investigation
of the structure-function relationships via mutagenesis and
expression in vitro.
q 1997 by The American Society of Hematology.
F
of a3.A3.C1.C2.5 During coagulation thrombin cleaves the
heterodimers at three points to form FVIIIa: after R372 near
the N-terminus of A2, R740 at the N-terminus of B, and
R1689 at the N-terminus of A3.6 Critical for FVIIIa activity,
fragment A2.a2 remains noncovalently associated with
the A1.a1 and A3.C1.C2 polypeptides, while the variable B
fragments and the short a3 peptide are lost; thus, FVIIIa
is a heterotrimer that may be designated A1.a1*A2.a2
*A3.C1.C2.
FVIII may also be cleaved by FXa after residues R336,
R372, R740, R1689, and R1721,6 and by FIXa after residues
R336 and R1719.7 All these specific cleavages either promote or degrade the functional activity of FVIII/FVIIIa, thus,
its activity in coagulation is closely regulated by limited
proteolysis. APC mediates negative feedback destruction of
FVIIIa activity by cleavage after R336 and R562.8
Various interaction sites have been defined on FVIII. Two
FIXa binding-sites have been localized to the vicinity of
residues S558-Q565 in A2 and E1811-K1818 in A3.9,10 Recent work has shown that the A3 site binds to the FIXa
EGF1 domain,11 whereas the A2 site binds to the serine
protease domain.12 In addition, an APC binding-site has been
described in A3 within H2007-V2016.13 FVIII(a) binds to
phospholipid surfaces via sequence(s) in the C2 domain, and
to its carrier vWF via the a3 peptide, and possibly also
ACTOR VIII (FVIII) plays an essential role as a cofactor
in blood coagulation1: absence of functional FVIII is
responsible for the commonest bleeding disorder, hemophilia
A. The extensive database of FVIII mutations correlating
with clinically-defined bleeding underlines the central regulatory role of FVIII in the hemostatic network.2 FVIII is
synthesized in hepatocytes as a mature single-chain polypeptide of 2,332 amino acids (predicted Mr Å 264,763), but
circulates in plasma as a population of noncovalently linked
heterodimers in a procofactor form bound to von Willebrand
factor (vWF). During coagulation, FVIII is proteolytically
activated to FVIIIa by trace amounts of thrombin or factor
Xa (FXa) and greatly accelerates the proteolysis of factor X
to FXa by factor IXa (FIXa) on the activated platelet surface.
In complex with another phospholipid-bound cofactor, factor
Va (FVa), FXa cleaves prothrombin to thrombin; FVa is
homologous to FVIII in both structure and function. Finally,
thrombin cleaves fibrinogen to insoluble fibrin to stabilize a
hemostatic clot. Feedback control is mediated by activated
protein C (APC), which inactivates FVIIIa and FVa by further proteolysis.
FVIII is a large multidomain protein containing internal
repeats.3 There are three homologous A-type domains (A1,
A2, and A3) defined approximately by residue positions 1336, 375-719, and 1691-2025, respectively. An acidic peptide a1 spans 337-374 and separates A1 from A2. A second
acidic peptide, a2, (720-740) connects A2 with the large,
heavily-glycosylated B domain, which encompasses approximately residues 741-1648. This leads to a third short acidic
peptide, here termed a3, (1649-1690), which itself connects
with the A3 domain. Finally, there are two homologous Cterminal domains (C1 and C2) each of approximately 155
amino acids. The domain organization of the FVIII polypeptide can thus be designated as A1.a1.A2.a2.B.a3.A3.C1.C2.
The location of seven disulphide bonds within the FVIII
molecule has been reported: there are two in each of the A1,
A2, and A3 domains and one within the C1 domain.4
On or before secretion proteolytic processing occurs
within the B domain to give heterodimers consisting of a
90- to 220-kD heavy chain A1.a1.A2.a2 with variable extensions of the B domain, associated via a divalent cationdependent interaction with an 80-kD light chain consisting
From Haemostasis Research Group, Medical Research Council
Clinical Sciences Centre, London; and CCLRC Daresbury Laboratory, Warrington, UK.
Submitted June 26, 1996; accepted November 5, 1996.
Supported by the Medical Research Council and the Council for
the Central Laboratories of the Research Councils.
Address reprint requests to S. Pemberton, PhD, Haemostasis Research Group, Medical Research Council Clinical Sciences Centre,
Royal Postgraduate Medical School, Du Cane Rd, London W12 0NN
UK.
The publication costs of this article were defrayed in 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.
q 1997 by The American Society of Hematology.
0006-4971/97/8901-0031$3.00/0
Blood, Vol 89, No 7 (April 1), 1997: pp 2413-2421
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Fig 1 (A through D).
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MOLECULAR MODEL OF FACTOR VIII
2415
Fig 1. Amino acid sequence lineups of human FVIII with other A domain proteins. (A through C) Alignment of human FVIII A1 (A), A2 (B),
and A3 (C) domains with hCp, showing SCR and VR placing together with conserved residues. Lowercase, Vrs; uppercase with bar above
sequence, SCRs. Sequence numberings refer to the mature proteins. (D through F) Alignment of A1 (D), A2 (E), and A3 (F) domain sequences
of: human and mouse FVIII (humf8, murf8); human and bovine FV (humfv, bovfv); rat and human Cp (ratcp, humcp), showing extent of
conservation of human FVIII residues with these related proteins. Key: white characters on black background, identical in all six sequences;
white on grey, residues identical to that in human FVIII; black on grey, residue similar to that in human FVIII; black on white, residues dissimilar
to human FVIII.
C2.14,15 A recent report also identified a region of the Cterminal portion of A1 (T291-F309), which may be involved
in binding to the chaperonin BiP; this protein is associated
with inhibition of FVIII secretion.16
FVIII shows clear homology to FV; the latter has a domain
structure A1.A2.B.A3.C1.C2. There is approximately 35%
to 40% sequence identity between all the corresponding domains of FVIII and FV, with the exception of the B domains,
which are apparently unrelated.17 Additionally, FV lacks the
short acidic sequences a1, a2, and a3 found in FVIII. The
blue copper-binding plasma protein ceruloplasmin (Cp) is
also homologous to FVIII and FV.3,18 The three A domains
of Cp show approximately 34% sequence identity with the
FVIII A domains. A crystal structure of human Cp (hCp)
has been solved, showing that the A domains are arranged
in a triangular structure, with each domain made up of two
cupredoxin-type folds.19 We have used the coordinates of
the hCp crystal structure to build a model of the A domains
of FVIII, using homology modeling techniques. The third
edition of the hemophilia A mutation database is now accessible as a World Wide Web site2 (URL: http://europium.
mrc.rpms.ac.uk) and has been used here with the model to
investigate hemophilic mutations; it may also be used to
direct experimental studies, which will shed light on the
molecular pathology of hemophilia A. In addition, modeling
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of the structure with FIXa binding-sites leads to a hypothesis
for the general configuration of the factor X-activating complex.
MATERIALS AND METHODS
A model of the A1, A2, and A3 domains of FVIII was built using
the Homology module (version 2.3) of the Insight II modeling suite
(Biosym/MSI, San Diego, CA). The X-ray coordinates of hCp were
imported and displayed with its associated linear amino-acid sequence. The sequences of the A domains of FVIII were imported
as a single polypeptide, and aligned with the hCp sequence as previously reported.3
The set of primary-structure conserved regions (SCRs) were defined using this alignment (Fig 1, A through C), and the coordinates
of the hCp crystal structure were then imposed on the FVIII sequence. Insertions and deletions were contained within variable regions (VRs). A search of the Brookhaven Protein Database generated
a selection of peptide loops in which the distance between the Nand C-termini was similar to that in each of the VRs. These candidate
loops varied in the amount of overlap between their N-and C-termini
and those of the corresponding VR, thus each potential loop was
scrutinized for the best fit within the model. Loops with the closest
overlap which also lay on the surface of the FVIII model were
chosen.
The coordinates of these loops were then superimposed to give
an a-carbon backbone for each VR, and the sidechains of each
residue were then varied to correspond with the FVIII sequence
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PEMBERTON ET AL
Fig 2.
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MOLECULAR MODEL OF FACTOR VIII
2417
within the VR. Finally, coordinates from the Insight II amino acid
library were imposed on the short N- and C-termini which lay beyond
the first and last SCR, respectively.
Each domain was then examined for serious steric overlaps. These
were relieved as far as possible by selecting lower-energy conformations from a library of rotamers within the program. The molecule
was then energy-minimized in stages. The splice points between
each VR and SCR were the most strained regions of the model, so
these were minimized first as a group using the standard steepest
descents algorithm to give an acceptably low energy derivative of
around 10 kcal.mol01. The rest of the molecule was then minimized
in the order: N- and C-termini, VRs, and finally SCRs.
Finally the molecule was energy-minimized, one domain at a time,
with the Discover module (Biosym/MSI, version 3.1) of Insight II,
using the steepest descents algorithm until a low energy derivative
of around 1 kcal.mole01 had been reached; at this point the calculated
total energy of the molecule was consistent with a relaxed and stable
conformation.
To examine the possible effects on the model of selected missense
mutations reported in the hemophilia A database, residue sidechains
were altered using the Biopolymer module of Insight II and, where
appropriate, the variant molecules were reminimized using Discover.
Alignments of A domains of human20 and mouse FVIII,21 human,22
and bovine FV,23 and finally human,18 and rat Cp24 were made using
PILEUP from the Genetics Computer Group (GCG)25 package.
These were exported as multiple sequence files (MSF) and displayed
using the program BOXSHADE.
RESULTS AND DISCUSSION
A homology-based molecular model of the A domains of
FVIII based on the crystal structure of the copper-binding
protein nitrite reductase (NIR) has recently been proposed.26
NIR, which is much more distantly related to FVIII than is
Cp, crystalizes as a triangular homotrimer whose three identical A domains each consist of a two-subdomain d1.d2
structure (whose d-type subdomain sequences are themselves related). Although useful in terms of prediction of the
hypothetical general shape of FVIII, this previous model
possesses two serious defects. Firstly, the low level of identity between NIR and FVIII A domains (varying between
only 11% and a maximum of 28%) renders modeling of
other than broad features very suspect; secondly, the arrangement of subdomains is almost certainly incorrect, with the
C-terminal d subdomains of each FVIII A domain pointing
outwards toward the top of the molecule rather than inwards
as predicted by comparison with Cp,19 because of the alignment of these subdomains’ sequences with the N-terminal
d1 subunit of NIR rather than the C-terminal d2 subunit.
General arrangement. The crystal structure of hCp consists of three A domains in a triangular array showing a clear
pseudo-threefold axis.19 Each A domain is formed from two
related d subdomains with d1 at the N-terminus of hCp and
d6 at the C-terminus: each d subdomain resembles the fold
of a typical cupredoxin-like molecule and consists of a bbarrel structure.27 The first and second b-strands of each
subdomain are linked by a large loop lying at the ‘top’
of the domain, with the loop conformations appearing very
different for the odd and even d subdomains. The odd-numbered subdomains d1, d3, and d5 all point outwards towards
the ‘top’ of the pseudo-threefold axis, whereas the evennumbered subdomains d2, d4 and d6, which contain the
mononuclear type-I copper atoms, point inwards.19 In NIR
the mononuclear type-I copper atoms are sited in the Nterminal d1 domains so that they are near the outside of the
molecule. This is consistent with NIR participating in electron transfer with pseudoazurin, a reasonably substantial
molecule of some 130 amino acids. However, the location
of the copper atoms in the even d subdomain of hCP (and
therefore towards the inside of the molecule) implies that
interactions with hCP are mediated by small molecules.
The overall structure of the FVIII A domain model we
have constructed by homology with hCp also shows these
broad features (Figs 2 and 3): the A1, A2, and A3 domains
correspond to d1.d2, d3.d4, and d5.d6 in hCp and the ‘top’
of the molecule is covered by the loops which connect the
first two b-strands in each of the six d subdomains. The d2,
d4, and d6 subdomains point inwards toward the top of the
molecule, unlike in the NIR-based model of FVIII where
these subdomains point outwards.26
Disulphide links. Of the six disulphide links identified
in the FVIII A domains,4 five (A1: 153-179, 248-329; A2:
528-554, 630-711; A3: 1832-1858) are homologous to those
in hCp and can be placed at the ‘bottom’ of the model. The
sixth A domain disulphide (A3: 1899-1903) is nonhomolo-
R
Fig 2. Homology model of the triplicated A domains of human FVIII. (Left) FVIII a-carbon ribbon viewed from ’top’ of the molecule down
the pseudo threefold axis. A1 (red), A2 (blue), and A3 (green); this color coding is consistent throughout this and subsequent figures. Cysteine
residues predicted to participate in disulphide links are highlighted in yellow. (Right) View perpendicular to the axis, with A1 to the front and
the ’top’ of the molecule bearing the loops anchoring the first and second b-strands of subdomains corresponding to the d subdomains of
hCp; the A3 C terminus is marked to the left. Also marked are the location of insertion points of acidic peptides in FVIII; peptide a1 may be
inserted on the surface between A1 and A2, whereas peptide a2, B domain and third acidic peptide a3 may be inserted on surface between
A2 and A3.
Fig 3. Secondary structure of FVIII A domains. Viewed perpendicular to threefold axis with ‘top’ of molecule to top of figure. a-Helices
(red), b-sheets (yellow), turns (blue), and random coil (magenta).
Fig 4. The copper atom in FVIII and its ligands. A single copper atom (gold) is shown with ligands H99 and H1957, whereas an oxygen
heteroatom (not shown) of water or hydroxide acts as a third ligand and is hydrogen bonded to the A100 main chain carbonyl and the sidechain
hydroxyl of Y105. H161 is too distant to act as a copper ligand. Neighboring residues are displayed as a-carbon ribbons: A1 (red) and A3
(green).
Fig 5. Predicted binding-sites for FIXa and BiP on the FVIII A domain model. FIXa: two loops, in A2 (S558-Q565) and A3 (E1811-K1818) are
shown in pink on the surface of a CPK FVIII image: view perpendicular to the threefold axis with ’top’ and ’bottom’ of the molecule marked.
Also shown is the partly exposed putative BiP binding site (T291-F309) in gold.
Fig 6. Predicted activated protein C (APC) and thrombin interaction sites. APC binding loop (H2007-V2016) on the A3 surface is shown in
white: postulated thrombin interaction via sulphated tyrosine residues Y718 and Y719 (deep blue) in FVIII A2 domain.
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2418
PEMBERTON ET AL
gous and is predicted to be further towards the top of the
model.
Proteolytic cleavage sites. The FVIII acidic peptides a1,
a2, a3, and the large B domain all contain function-related
cleavage sites at one or both of their N- or C-termini, and
thus these sites would be predicted to be accessible to serine
proteases such as thrombin and APC. Although these peptide
sequences are far too large to be modeled realistically, the
FVIII A domain model can predict the location of the termini
of these loops, which must be surface-exposed. Thus, the
FVIII model predicts that the N- and C-termini of the first
acidic region a1 (330-379) lie on the surface of the molecule
at the interface between A1 and A2 (Fig 2). Similarly, the
interface between A2 and A3 (into which the second acidic
region a2, the B domain, and the third acidic region a3 must
be inserted) also lies on the surface (Fig 2). Predictions such
as these and others described below help to support the
functional significance of the model.
The copper atom in FVIII. Six integral copper atoms
have been positively identified in the hCp crystal structure.19
Three are type-I blue copper atoms, found in homologous
positions in subdomains d2, d4, and d6. The remaining three
copper atoms are located in a trinuclear cluster at the interface between subdomains d1 and d6, that is, between A1
and A3: two of these are type-III copper atoms, each coordinated to three histidine residues (to H103 and H161 in A1,
and H1022 in A3; to H163 in A1, and to H980 and H1020
in A3). The third atom in the cluster is type-II and is coordinated to H101 in A1 and H978 in A3. On close examination
of the electron density of the trinuclear cluster in hCp, a
similarity is seen to the layout of the homologous trinuclear
centre in the crystal structure of ascorbate oxidase (AO); in
AO, one oxygen heteroatom bridges the type-III copper
atoms and a second coordinates to the type-II copper atom.28
Thus (based on analogy with AO) it is likely that in the
crystal structure of hCp, this latter oxygen heteroatom can
form hydrogen bonds with the sidechain hydroxyl group of
Y107 and the main chain carbonyl oxygen of S102.
FVIII has been shown to contain one copper atom per
molecule,29 and therefore it is likely that the position of this
copper atom in the molecule will correspond to one of the
six copper atoms identified in the crystal structure of hCp.
Because purified FVIII protein shows none of the spectral
characteristics typical of a type-I copper-binding protein, this
rules out occupancy of either of the conserved type-I sites
in subdomains d2 and d6. Of the six histidine ligands that
coordinate the two type-III coppers in the trinuclear cluster
of hCp, only one (H163 in hCp) is conserved in FVIII as
H161. However, the two histidine residues (H101, H978)
that coordinate to the type-II copper in the hCp trinuclear
cluster are conserved as H99 and H1957 in FVIII, as is the
aforementioned tyrosine (Y107) residue as Y106 in FVIII.
We suggest that the single copper atom in FVIII lies at the
interface between domains A1 and A3 (between subdomains
d1 and d6) homologous to the position of the type-II copper
in hCp, coordinated by H99 in A1 and H1957 in A3. H161
in A1 (postulated in the earlier study26 to be a third histidine
ligand), is too far displaced from the others to act as a copper
ligand in our model. It is possible that the third ligand may
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(by analogy with the hCp structure) be an oxygen atom of
a water molecule, which is hydrogen-bonded both to the
sidechain hydroxyl of Y106 and to the main chain carbonyl
oxygen of A100 (Fig 4). The interdomain contact mediated
by the copper atom may contribute to the association of the
heavy and light chains of FVIII through a divalent cationdependent interaction involving A1 and A3.
Protein interaction sites. Amino acid residues implicated in the two binding sites identified recently for FIXa9,10
lie on the surface of the model, as would be expected (Fig
5). Also shown in this figure is the putative BiP binding-site
within T291-F309,16 which appears as a partly exposed region rich in hydrophobic residues. The sequence within
which the suggested binding site for APC lies is toward the
C-terminus of the A3 domain, and is partly buried in the
model (Fig 6).13 Finally, two tyrosine residues in the model
which are known to be posttranslationally sulphated30 (Y718
and Y719) lie close to the surface at the interface between
A2 and A3 (Fig 6). These residues may play a role in thrombin binding via its anion-binding exosite, which is known
to interact strongly with sulphated tyrosine (eg, in hirudin).
It should be noted that our model of the FVIII A2 domain
terminates at Y719 and that the predicted structure here
should be regarded with particular caution.
Interpretation of mutations implicated in clinical disease.
The possible effects on the FVIII A domain model of 36
single missense mutations from the hemophilia A database
have been examined (Table 1). These were only those mutations for which circulating FVIII antigen (FVIII:Ag) assay
levels are reported and, in the absence of three-dimensional
structural information, they may be conveniently divided
into two main classes. Firstly, cross-reacting material positive (CRM/): circulating FVIII:Ag levels normal (ú50%)
with biological activity (FVIII:C) reduced (õ50%). These
are dysfunctional variants - the mutation may affect a functional region but still allows the molecule to fold, be secreted,
and circulate in a stable form. Study of these dysfunctional
mutants allows mapping of functional regions on the model,
and experimentally verifiable predictions can be made. Secondly, cross-reacting material negative (CRM0): both
FVIII:Ag and FVIII:C levels are sharply reduced, indicating
that these mutations are severe enough to interfere with the
secretion of the molecule or its circulation in a stable form;
such molecules may or may not be functionally deficient in
addition.
Three mutations with plausible mechanisms for clinical
disease based on the FVIII A domain model are briefly discussed below: to allow inspection of the level of conservation
of all A domain residues in human FVIII with murine FVIII,
human and bovine FV, and human and rat Cp, multiple
sequence lineups for the three A domains are presented in
Fig 1 (D through F).
M1772T (FVIII:C õ1 %, FVIII:Ag 72%; CRM/) is an
example of a mutation affecting a predicted FIXa interaction site. Replacement of M1772 by Thr creates a new
glycosylation consensus sequence (N-X-S/T): the N1770
sidechain is surface-exposed (Fig 7) and thus potentially
available for glycosylation. The model indicates that
N1770 lies close to the proposed binding-site for FIXa
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MOLECULAR MODEL OF FACTOR VIII
2419
Table 1. Table of 36 Missense Mutations Studied, With FVIII:C and FVIII:Ag Levels, Clinical Severity, Level of Conservation and Comments
on Possible Molecular Pathology
Mutation
ID*
FVIII:C
(%)
FVIII:Ag
(%)
Clinical
Severity
Conservation†
Hypothetical Pathology
Y114C
T1181
V162M
K166T
S170L
D203V
E272G
T275I
R282H
S289L
T295A
C329S
L412F
K425R
A469G
I475T
G479R
D525N
R527W
R531H
D542G
S558F
I566T
V634A
A644V
F658L
A704T
G1750R
L1756V
M1772T
R1781H
P1825S
H1848R
R1941Q
G1948D
H1961Y
HP19
HP20
HP25
JH139
LKC
HP28
JH20
HP29
JH86
JH152
HP32
Lisboa2
JH131
JH74
HP38
HP39
Porto1
MS
DG
HP46
JH63
JH151
FW
JH156
JH136
HP49
HP51
HP77
HP83
JH116
HP84
HP88
HP89
JH33
Porto2
HP91
6.3
2.0
5.3
8.0
3.5
2.0
2.0
4.8
õ1
37
9.5
2.6
7.0
õ1
2.3
5.5
17.8
6.0
15.0
23.5
õ1
21
4
5
14
5.1
4.5
22
5.0
õ1
2.0
12
1.5
4.5
7.4
10.5
10.7
10.7
14
9.8
8.7
8.5
3.5
20.2
18
106
11.6
3.2
6.4
5.0
45.3
8.8
31.6
61
126
33.2
5.0
175
200
138
25
50.5
5.5
23.5
1.5
72
4.7
18.1
20.1
20
48.7
7.8
Mild
Moderate
Mild
Mild
Mild
Mild
Moderate
Moderate
Severe
Mild
Mild
Moderate
Mild
Severe
Moderate
Mild
Mild
Moderate
Mild
Mild
Severe
Mild
Moderate
Mild
Mild
Mild
Moderate
Mild
Mild
Severe
Moderate
Mild
Moderate
Mild
Mild
Mild
YYYYYY
TTTTTT
VVEVVI
KKEEKK
SSSSSS
DDDDDD
EENNHH
TTVVAA
RRKKHR
SSVVFF
TTTTDD
CCCCCC
LLLLFF
KKKKKK
AAAAGG
IIIIII
GGGGGG
DDDDDD
RRQQVV
RRRRKK
DDDDDD
SSSSSS
IMRRKK
VVTTSS
AATTNN
FFFFYY
AALLQQ
GGGGRK
LLLLLL
MMQQKK
RRRRRR
PPPPAA
HHHHIY
RRRRNN
GGGGGG
HHQQHH
Free Cys hinders correct folding
Steric clash with D116 in core
No assignable reason for phenotype
Loss of positive charge
Strain in core, possible loss of H-bond with K166 main chain carbonyl
Loss of negative charge
Possible loss of H-bond with L307 main chain carbonyl
Possible loss H-bond with H274 main chain carbonyl
Modification of A1/A2 interaction via D525
Modification of A1/A3 interaction via Y1979
No assignable reason for phenotype
Loss of conserved disulphide link
Steric clash with S409 in core
Steric strain in core
No assignable reason for phenotype
No assignable reason for phenotype
Steric clash with R531 in core
Modification of A1/A2 interaction
Disturbance of FIXa binding-site
Modification of A1/A2 interaction via R282
Loss of negative charge
Steric clash with C554/Y555 round FIXa binding-site
Predicted new N-glycosylation at N564 close to FIXa binding-site
No assignable reason for phenotype
Modification of A1/A2 interaction
Decrease in core volume
No assignable reason for phenotype
Addition of positive charge and bulky sidechain
No assignable reason for phenotype
Predicted new N-glycosylation at N1770 close to FIXa binding-site
No assignable reason for phenotype
Close to FIXa binding-site
Modification of A2/A3 interaction via D696
Close to FIXa binding-site
Close to A2/A3 interface
Increases core energy
* Unique patient identifier: where multiple reports occur in the hemophilia A database, only the first entry with full phenotype is given here.
† This column gives the single letter amino-acid code for the wild-type residues in this position in (L-R): human FVIII, murine FVIII, human
FV, bovine FV, rat Cp, and human Cp.
within E1811-K181810: N1770 is a FVIII-specific residue
further implicating this region in FVIII cofactor function.
G1948D (FVIII:C 7.4%, FVIII:Ag 48.7%; CRM/) is a
mutation postulated to affect interdomain interaction. G1948
lies in the core of the molecule near the interface of the A2
and A3 domains and on a turn between two b-strands, and
is an absolutely conserved residue. Replacement with the
bulky Asp sidechain introduces significant steric strain; this
may disrupt the contact between A2 and A3 (important in
continued association of A2 with the rest of the FVIIIa molecule following activation).
L412F (FVIII:C 7%, FVIII:Ag 6.4%; CRM0) is a mutation resulting in decreased expression or stability. The Leu
residue is conserved in bovine and human FV, but is in fact
replaced by the variant residue Phe in rat and human Cp. It
is predicted to be partly buried with the sidechain oriented
towards the core. When mutated to Phe in FVIII, a steric
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clash occurs with the S409 sidechain (the corresponding
residue at this position in hCp is alanine). Expression or
stability of the variant molecule is clearly affected by this
change as the FVIII:Ag level is grossly reduced; however,
functional activity is concordant with antigen level indicating
that procoagulant function is unaffected in this variant.
Hypothetical configuration of the FX-activating complex.
It is plausible to orientate the triplicated A domains of FVIII/
FVIIIa with a phospholipid surface via a specific interaction
involving the C2 domain. Thus, the molecule might be held
with the A3 domain closest to the surface: factors IXa and
X also bind to such a surface via their N-terminal g-carboxyglutamic acid-rich (Gla) domains. The EGF1 domain of FIXa
has been shown to contact the A3 domain binding-site
(E1811-K1818) whereas its protease domain contacts the A2
domain binding-site within S558-Q565; thus, the FIXa
molecule may be aligned with the FVIII structure as in Fig 8.
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2420
PEMBERTON ET AL
Fig 7. Variant M1772T. A3 acarbon backbone ribbon (and
line trace of A1) showing location of A3 FIXa binding site
(E1811-K1818, white CPK) with
new glycosylation site N1770
(black CPK) created in the vicinity by mutation of M1772 (black
CPK) to Thr.
Since binding-site(s) for FX have not yet been identified,
one can only speculate how the complex might interact with
its substrate; however, there are potential surfaces for accommodation of substrate on the A1 domain and parts of A2.
Alternatively, the cofactor function of activated FVIII may
be explicable in terms of the orientation and allosteric activation of FIXa alone.
CONCLUSION
From the point of view of functional analysis of FVIII, it
is unfortunate that circulating FVIII:Ag levels are only reported for a minority of missense mutation cases in the hemophilia A database. Only when these levels are reported is
it possible to differentiate between the two likely causes of
clinical disease resulting from missense mutations; either (1)
reduced secretion/stability (low or absent FVIII:Ag, CRM0)
or (2) dysfunction of a normally-secreted molecule (normal
FVIII:Ag with reduced FVIII:C, CRM/). In the latter CRM/
class, hypotheses based on our model may be tested by performing site-directed mutagenesis studies to investigate, for
example, FIXa interaction, thrombin-activation rate or stability of FVIIIa in recombinant variants.
Molecular models are not a substitute for high-resolution
structures obtained by X-ray diffraction or NMR; however,
in the absence of such structures for any part of FVIII or
FVIIIa, we intend this model to point the way to a better
understanding of the molecular pathology of hemophilia A,
by facilitating the rational selection of experimental approaches including in vitro mutagenesis.
ACKNOWLEDGMENT
The authors thank A.I. Wacey (Thrombosis Research Institute,
London, UK) for valuable initial assistance with construction of the
model. The coordinates of the model in Protein Data Bank format,
together with VRML (Virtual Reality Markup Language) representations, will be available following publication at the World Wide
Fig 8. Hypothetical configuration of the FX-activating complex. A cartoon showing how the triangular array of the FVIII A1, A2, and A3 domains may be
anchored to a phospholipid surface via the C1 and
C2 domains (not to scale). The FIXa serine protease
domain interacts with the loop in A2 (S558-Q565),
whereas the FIXa EGF2 domain binds to the loop
in A3 (E1811-K1818). The FIXa Gla domain is also
anchored to the phospholipid surface via a calciumdependent mechanism. This complex between FVIII
and FIXa activates zymogen FX as a key step in the
amplification of the initial trigger in coagulation.
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MOLECULAR MODEL OF FACTOR VIII
2421
Web site of the Hemophilia A Database. URL: http://europium.
mrc.rpms.ac.uk
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1997 89: 2413-2421
A Molecular Model for the Triplicated A Domains of Human Factor VIII
Based on the Crystal Structure of Human Ceruloplasmin
S. Pemberton, P. Lindley, V. Zaitsev, G. Card, E.G.D. Tuddenham and G. Kemball-Cook
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