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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy
disrupt protein-protein interactions within the triplicated A domains of
thrombin-activated factor VIIIa
Steven W. Pipe, Evgueni L. Saenko, Angela N. Eickhorst, Geoffrey Kemball-Cook, and Randal J. Kaufman
Thrombin-activated factor VIII (FVIIIa) is a
heterotrimer with the A2 subunit (amino
acid residues 373-740) in a weak ionic
interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. Patients with hemophilia A have been
described whose plasmas display a discrepancy between their FVIII activities,
where the 1-stage activity assay displays
greater activity than the 2-stage activity
assay. The molecular basis for one of
these mutations, ARG531HIS, is an increased rate of A2 subunit dissociation.
Examination of a homology model of the
A domains of FVIII predicted ARG531 to lie
at the interface of the A1 and A2 subunits
and stabilize their interaction. Indeed, patients with mutations either directly contacting ARG531 (ALA284GLU, ALA284PRO) or
closely adjacent to the A1-A2 interface in
the tightly packed hydrophobic core
(SER289LEU) have the same phenotype of
1-stage/2-stage discrepancy. The ALA284GLU
and SER289LEU mutations in FVIII were
produced by transfection of COS-1 monkey cells. Compared to FVIII wild-type
both mutants had reduced specific activity by 1-stage clotting activity and at least
a 2-fold lower activity by 2-stage analysis
(COAMATIC), similar to the reported clinical data. Analysis of immunoaffinity purified ALA284GLU and SER289LEU proteins in
an optical biosensor demonstrated that
A2 dissociation was 3-fold faster for both
FVIIIa mutants compared to FVIIIa wildtype. Therefore, these mutations within
the A1 subunit of FVIIIa introduce a similar destabilization of the FVIIIa heterotrimer compared to the ARG531HIS mutation
within the A2 subunit and support that
these residues stabilize the A domain interface of FVIIIa. (Blood. 2001;97:685-691)
© 2001 by The American Society of Hematology
Introduction
Effective hemostasis is mediated by the regulated and sequential
activation of serine proteases in the coagulation cascade. Factor
VIII (FVIII) functions in the intrinsic pathway of blood coagulation
as a cofactor to accelerate by 100 000-fold the activation of factor
X by factor IXa that occurs on a phospholipid surface in the
presence of calcium ions.1 A quantitative or qualitative deficiency
of FVIII leads to the phenotype of the bleeding disorder hemophilia
A. The FVIII amino acid sequence, deduced from the cloned
complementary DNA (cDNA), identified that the molecule is
synthesized as a single-chain polypeptide having the domain
structure A1-A2-B-A3-C1-C2.2,3 On secretion from the cell, FVIII
is processed to a heterodimer consisting of a carboxy terminalderived light chain (LC) of 80 kd in a metal ion-dependent
association with a variably proteolyzed (90-200 kd) amino terminalderived heavy chain (HC) fragment. The FVIII A domains share
almost 40% amino acid identity with each other and to the factor V
(FV) A domains, as well as with the A domains in the copperbinding protein ceruloplasmin,4 suggesting the A domains may be
involved in copper ion binding. Indeed, studies have detected 1
mole copper ion per mole of both FV and FVIII protein.5-7
In vivo, FVIII activity is regulated by proteolytic activation. On
thrombin cleavage of FVIII there is a rapid 50-fold increase and
subsequent first-order decay of procoagulant activity. The activa-
tion coincides with proteolysis of both the HC and LC of FVIII.8
Thrombin activates FVIII through proteolytic cleavage after ARG740,
ARG1689, and ARG372 generating an FVIIIa heterotrimer consisting
of the A1 subunit (50 kd) in a copper ion–dependent association
with the thrombin-cleaved LC (73 kd) and a free A2 subunit
associated with the A1 subunit through a weak ionic interaction.
The FVIIIa heterotrimer exhibits a pH-dependent dissociation of
the A2 subunit from the A1/A3-C1-C2 heterodimer that correlates
with loss of procoagulant activity.9-11 Porcine FVIIIa exhibits an
increased affinity for its A2 subunit compared to human FVIIIa and
accordingly demonstrates an increased specific activity.10,12 In
addition, a novel genetically engineered FVIII molecule, in which
the A2 subunit remains covalently linked to the LC even after
thrombin activation, has increased specific activity in vitro and
abrogates the first-order decay of procoagulant activity as observed
for native FVIII.13 These observations highlight the role of A2
dissociation in limiting FVIIIa activity in vitro. However, it is not
yet known whether spontaneous A2 subunit dissociation or further
proteolysis limits FVIIIa procoagulant activity in vivo. Insight into
this question has been gained by the recent characterization of an
unusual clinical phenotype of hemophilia A.
Patients with hemophilia A have been described whose plasmas
display a discrepancy between their FVIII activities, where the
From the Departments of Pediatrics and Biological Chemistry, Howard Hughes
Medical Institute, University of Michigan Medical Center, Ann Arbor, MI; Holland
Laboratory, American Red Cross, Rockville, MD; and Haemostasis Research
Group, MRC Clinical Sciences Centre, Imperial College School of Medicine,
London, United Kingdom.
of Hematology.
Submitted April 17, 2000; accepted October 6, 2000.
Supported by National Institutes of Health grant HL52173 and by NIHCHD
HD28820. S.W.P. and E.L.S. are Faculty Scholars of the American Society
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
Reprints: Randal J. Kaufman, Howard Hughes Medical Institute, University of
Michigan Medical Center, Ann Arbor, MI 48109; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
685
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686
PIPE et al
1-stage (1-st) activity assay displays greater activity than the
2-stage (2-st) activity assay.14-18 Initially, the molecular mechanism
for one of these mutations, a missense mutation, ARG531HIS, was
characterized to result from increased rate of inactivation of FVIIIa
by increased rate of A2 subunit dissociation.19 On thrombin
activation, the ARG531HIS A2 subunit exhibited a 4-fold increased
rate of dissociation from the A1/A3-C1-C2 heterodimer. The
increased instability of the ARG531HIS heterotrimer would reduce its
specific activity in both 1-st and 2-st assays. However, the specific
activity of ARG531HIS is disproportionately reduced in the 2-step
procedure, possibly due to the incubation phase in the first step.
This hemophilia A phenotype therefore supports previous in
vitro studies that have suggested that nonproteolytic regulation
of FVIIIa activity, via spontaneous A2 subunit dissociation, is
important in vivo.
Examination of a homology model of the A domains of FVIII20
identified ARG531 to lie at the interface of the A1 and A2 subunits.
We hypothesize that the ARG to HIS amino acid substitution
destabilizes protein-protein interactions between the A1 and A2
subunits. We also hypothesize that if this region is important for
FVIIIa stability, other mutations within this region would have
similar phenotypes. Interestingly, there are 3 described hemophilia
A mutations either directly contacting ARG531 (ALA284GLU,
ALA284PRO) or closely adjacent to ARG531HIS at the A domain
interface in the tightly packed hydrophobic core (SER289LEU). All
patients with these mutations have the phenotype of 1-st/2-st
discrepancy. FVIII cDNA constructs were prepared containing
either the ALA284GLU or SER289LEU mutations and the proteins were
expressed and purified from transfected COS-1 monkey cells.
FVIII harboring these missense mutations were secreted at similar
levels to FVIII wild-type (WT), had reduced specific activity by
1-st clotting activity, and at least a 2-fold lower activity by 2-st
analysis (COAMATIC), similar to the reported clinical data.
Analysis in an optical biosensor demonstrated that on thrombin
cleavage, A2 dissociation was 3-fold faster for both SER289LEU and
ALA284GLU compared to FVIII WT. Thus, these mutations within
the A1 subunit of FVIIIa demonstrate a similar destabilization of
the A domain interface as the originally characterized ARG531HIS
mutation within the A2 subunit. These results support a role for this
region in stabilizing the A domain interface of FVIIIa and support
the validity of the molecular model of the A domains. The amino
acid residues 281 to 290 within human FVIII are conserved in
porcine, canine, and murine FVIII. Further characterization of the
key protein-protein interactions within this region will advance
genetic engineering strategies aimed at improving the stability of
the FVIIIa heterotrimer.
Materials and methods
Materials
Anti-HC FVIII monoclonal antibody (F-8) conjugated to CL-4B Sepharose
was a gift from Debra Pittman (Genetics Institute, Cambridge, MA).
FVIII-deficient and normal pooled human plasma samples were obtained
from George King Biomedical (Overland Park, KS). Activated partial
thromboplastin (Automated aPTT reagent) and CaCl2 were purchased from
General Diagnostics Organon Teknika (Durham, NC). Anti-LC FVIII
monoclonal antibody ESH-8 was purchased from American Diagnostica
(Greenwich, CT). Dulbecco modified Eagle medium (DMEM) and fetal
bovine serum were purchased from Gibco BRL (Gaithersburg, MD).
COAMATIC was purchased from DiaPharma (West Chester, OH).
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
Plasmid mutagenesis
Mutagenesis was performed within the mammalian expression vector
pMT221 containing the FVIII cDNA (pMT2VIII). Oligonucleotide-directed
mutagenesis was used to create a SpeI-KpnI polymerase chain reaction
fragment in which codon 284 was mutated from CGC to CAC, predicting
an amino acid substitution of glutamic acid for alanine, and codon 289 was
mutated from TCG to TTG, predicting an amino acid substitution of leucine
for serine. The mutagenized and SpeI-KpnI digested fragments were then
ligated into SpeI-KpnI digested pMT2VIII. The resulting mutant plasmids
were designated ALA284GLU and SER289LEU. The plasmid containing the
wild-type FVIII cDNA sequence was designated FVIII WT. The plasmid
containing a B-domain–deleted (BDD) FVIII was designated BDD-FVIII.
The SpeI-KpnI fragment from SER289LEU was ligated into SpeI-KpnI–
digested BDD-FVIII and the resultant plasmid designated BDD-SER289LEU.
The mutant plasmid ARG531HIS was prepared previously.19 All plasmids
were purified by centrifugation through cesium chloride and characterized
by restriction endonuclease digestion and DNA sequence analysis.
DNA transfection and analysis
Plasmid DNA was transfected into COS-1 cells by the diethylaminoethyldextran method as previously described.22 Conditioned medium was
harvested at 64 hours after transfection in the presence of 10% fetal
bovine serum.
Protein purification
Purified ALA284GLU, SER289LEU, BDD-FVIII, and BDD-SER289LEU proteins
were obtained from 200 mL conditioned medium from transiently transfected COS-1 cells by immunoaffinity chromatography23 yielding 750
to1500 ng per purification. FVIII WT protein was purified in parallel from
stably transfected Chinese hamster ovary cells. The proteins eluted into the
ethylene glycol–containing buffer were dialyzed and concentrated against a
polyethylene glycol (molecular weight, ⬃15 000-20 000)–containing
buffer24 and stored at ⫺70°C.
FVIII activity and antigen assay
FVIII activity was measured by (1) a 1-st aPTT clotting assay on an MLA
Electra 750 fibrinometer (Medical Laboratory Automation, Pleasantville,
NY) by reconstitution of human FVIII-deficient plasma or (2) a modified
2-st assay using the COAMATIC chromogenic assay according to the
manufacturer’s instructions. FVIII antigen was quantified by an anti-FVIII
LC sandwich enzyme-linked immunosorbent assay (ELISA) method using
the IMUBIND commercial kit according to the manufacturer’s instructions
(American Diagnostica).
Kinetic measurements using biosensor technology
The kinetics of the A2 subunit dissociation from thrombin-activated FVIII
WT and mutant proteins were studied by surface plasmon resonance using
the Biacore biosensor (Piscataway, NJ), which measures protein binding
and subsequent dissociation in real time.25 Binding of 1 ng protein/mm2 of
the biosensor chip produces a resonance signal of 200 Arc seconds.
Monoclonal antibody ESH8 (50 ␮g/mL) in 10 mM sodium acetate, pH 5.0,
was covalently coupled at 16.5 ng/mm2 to the activated carboxymethyldextran-coated biosensor cuvette via amino groups using succinimide ester
chemistry.25 The carboxymethyldextran chip and reagents for its activation,
N-ethyl-N⬘-(dimethylaminopropyl)carbodiimide hydrochloride and Nhydroxysuccinimide, and deactivation, ethanolamine, were purchased from
Affinity Sensors (Cambridge, United Kingdom). FVIII WT and mutant
protein binding to ESH8 and their dissociation from the antibody and
activation by thrombin were measured in 200 ␮L 20 mM Hepes, 0.15 NaCl,
5 mM CaCl2, 0.01% Tween 20, pH 7.4. The chip was regenerated by
addition of 0.1 M glycine, pH 3.0, for 3 minutes, resulting in complete
dissociation of FVIII proteins from the capture ESH8. Identical signals for
reference binding of the FVIII proteins to immobilized ESH8 were obtained
before and after regeneration.
The values of the rate constants for dissociation (koff) of FVIII WT or
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BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
mutant proteins from immobilized ESH8 and those for dissociation of the
A2 subunits on thrombin activation of immobilized FVIII proteins were
calculated using the dissociation model with offset described by the
following equation: R ⫽ Ri ⫻ exp(⫺koffA2t) ⫹ RF.26 In the equation R is
the observed surface plasmon resonance signal during A2 dissociation;
koffA2, the kinetic rate constant for the A2 dissociation on thrombin
activation of FVIII WT or mutant proteins; Ri, the initial resonance signal
when thrombin was injected; Rf, the final signal observed after all A2
dissociated (the final plateau achieved). Ri, Rf , and koffA2 were used as the
independent parameters to fit observed resonance signal R versus time (t)
by nonlinear regression analysis using Sigmaplot (Jandel Scientific, San
Raphael, CA).
Results
Recombinant-derived ARG531HIS, ALA284GLU, and SER289LEU
proteins demonstrate a similar functional phenotype
to patient plasma FVIII
The synthesis and secretion of FVIII WT and ARG531HIS, ALA284GLU,
and SER289LEU mutants were compared after transient DNA transfection into COS-1 monkey cells. FVIII antigen determinations by
ELISA were performed on conditioned media collected from 24 to
64 hours after transfection and were similar for all mutants
compared to FVIII WT suggesting no significant defect in synthesis
or secretion.
FVIII activity (Figure 1) was measured by a 1-st aPTT clotting
assay or by a modified 2-st method using the COAMATIC
chromogenic assay. Activities were normalized to their expressed
FVIII antigen levels as determined by ELISA and are presented as a
percent of FVIII WT activity. Similar to the patient phenotypes and
data reported previously, ARG531HIS had discrepant activity as
measured by the 1-st aPTT clotting assay (44% ⫾ 15% of FVIII
WT) compared to the 2-st chromogenic assay (COAMATIC;
19% ⫾ 4% of FVIII WT). For the ALA284GLU mutation, 1-st aPTT
(26% ⫾ 6%) and 2-st chromogenic assay (6% ⫾ 4%) activities
showed discrepancy. For the SER289LEUmutation, 1-st aPTT
(32% ⫾ 11%) and 2-st chromogenic assay (13% ⫾ 5%) activities
also showed discrepancy. The activity measurements for the patient
plasmas represent all of the reported data obtained from the
HAMSTeRS hemophilia A mutation database.27 The slightly higher
Figure 1. Recombinant-derived missense mutations in FVIII demonstrate a
similar phenotype to described patient plasmas. (A) One-stage (f) and 2-stage
(䡺) activity assays from plasmas obtained from hemophilia A patients with these
missense mutations as reported in the hemophilia A mutation database. (B)
Full-length cDNAs containing missense mutations generated by oligonucleotide
site-directed mutagenesis were expressed in COS-1 monkey kidney cells. The data
from recombinant-derived protein was obtained from assaying the activity in the
conditioned medium at 64 hours following transfection. One-stage activity (f) was
determined by aPTT-based assay. Two-stage activity (䡺) was determined by
COAMATIC assay. Specific activities for recombinant-derived proteins are presented
as a percent of recombinant FVIII WT specific activity.
FVIII MUTATIONS INCREASE A2 SUBUNIT DISSOCIATION
687
Table 1. Analysis of FVIIIa A2-subunit dissociation by optical biosensor
koff (s⫺1)
t1/2 (In2/koff)
pdFVIII
4.9 (⫾ 0.3) ⫻ 10⫺3
141 ⫾ 6 s
FVIII WT
5.9 (⫾ 0.6) ⫻ 10⫺3
117 ⫾ 9 s
ARG531HIS
2.9 (⫾ 0.2) ⫻ 10⫺2
24 ⫾ 2 s
SER289LEU
1.9 (⫾ 0.1) ⫻ 10⫺2
36 ⫾ 1.4 s
ALA284GLU
2.0 (⫾ 0.1) ⫻ 10⫺2
35 ⫾ 1.6 s
BDD-FVIII
4.2 (⫾ 0.02) ⫻ 10⫺3
164 ⫾ 2.6 s
BDD-SER289LEU
2.3 (⫾ 0.03) ⫻ 10⫺2
30 ⫾ 1.2 s
2-st activity results obtained with the recombinant-derived mutant
proteins are consistent with those obtained from the few reported
patient plasmas where a chromogenic assay rather than the classical
2-st assay was used.18 Following immunoaffinity purification of
FVIII WT and mutant proteins from the conditioned medium,
similar discrepancy in 1-st/2-st activities were obtained (data
not shown).
ARG531HIS, ALA284GLU,
and SER289LEU proteins exhibit increased
rate of A2 subunit dissociation from the thrombin-activated
heterotrimer
The relative rates of A2 dissociation for plasma-derived (pd) FVIII,
FVIII WT, and mutant proteins were determined using an optical
biosensor (Figure 2). An anti-LC antibody, ESH8, was covalently
immobilized on a carboxymethyldextran-coated biosensor chip.
Similar amounts (1.12 ng/mm2) of immunoaffinity purified FVIII
WT or mutant proteins were bound to ESH8 antibody. Unbound
material was removed by washing with buffer and free (nonproteolytic) dissociation from antibody was measured. The values of
dissociation rate constants, koff ⫽ 8.9 ⫾ 0.23 ⫻ 10⫺5/s, were similar for FVIII WT and mutant proteins. Subsequently, thrombin was
added to a final concentration of 1 U/mL. Because the FVIII
preparations are bound to an ESH8-coated chip via the C2 domain
of their LCs, this interaction is not disturbed by thrombin cleavage
and the A1 subunit remains associated with the LC through the
copper ion–dependent linkage between the A1 and A3 domains.
Thus, the thrombin-induced release of the A2 subunit from the
heterotrimer can be measured as a dissociation curve registered by
the optical biosensor. Control experiments with thrombin concentrations ranging from 0.05 to 2.25 U/mL demonstrated that
maximal thrombin cleavage under these experimental conditions
occurs at a thrombin concentration of 0.75 U/mL (data not shown).
The koff values and half-lives of A2 dissociation for the A2 subunits
are presented in Table 1. Results for ARG531HIS have been reported
previously19 and are presented for comparison purposes. Both the
ALA284GLU and SER289LEU mutants displayed a 3-fold greater rate of
A2 dissociation compared to pdFVIII WT. Because A2 dissociation
correlates with inactivation, the mutant thrombin-activated heterotrimers would be expected to inactivate approximately 3-fold faster
compared to plasma-derived FVIII or FVIII WT. The single
exponential model used by us to describe dissociation of the A2
subunit observed for FVIII WT in this study is similar to that
previously reported for FVIIIa decay.12,24 The minor difference in
thrombin-mediated A2 dissociation rates for pdFVIII compared to
recombinant FVIII WT did not reach statistical significance
(P ⫽ .24, n ⫽ 5).
Because these mutations were expressed within the FVIII
WT construct, they contain a B domain. The B domain is also
released on thrombin activation and contributes to the loss of
mass observed by the optical biosensor. Although the release of
B-domain fragment(s) is immediate on thrombin activation we
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688
PIPE et al
prepared BDD forms of FVIII and the SER289LEU mutant so that
only A2 subunit dissociation would be measured by the optical
biosensor. Both BDD-FVIII and BDD-SER289LEU were expressed from COS cells by transient transfection and the purified
proteins subjected to the same analysis as the full-length
proteins. BDD-SER289LEU exhibited similar 1-st/2-st discrepancy
as SER289LEU in activity assays (data not shown). Purified
BDD-SER289LEU was then subjected to optical biosensor analysis
and displayed 5-fold greater rate of A2 dissociation compared to
the BDD-FVIII control. Because the amount of each immobilized FVIII was approximately 310 RU when thrombin was
added, the reduction of the resonance signal on dissociation of
A2 is 28% and 27.6% for BDD-FVIII and BDD-SER289LEU,
respectively. This is identical to the expected reduction of the
resonance signal (27.6%) on complete dissociation of the A2
subunit calculated using molecular weights of 170 kd and 123
kd for BDD-FVIII and its A1/A3-C1-C2 dimer. In contrast, B
domain–containing samples in panels A and B of Figure 2
demonstrated an initial rapid loss of resonance signal immediately following addition of thrombin (consistent with loss of
B-domain fragments) that was not observed for the BDD
samples in panel C. However, all samples subsequently exhibited a relatively slower loss of resonance signal (consistent with
A2 subunit dissociation) that was used to calculate the koff for
dissociation.
Figure 2. Determination of the kinetic parameters for nonproteolytic and
thrombin-mediated dissociation of FVIII and variants from monoclonal antibody ESH8. The monoclonal antibody ESH8 was covalently immobilized to a
biosensor chip at 20 ng/mm2. FVIII WT, SER289LEU, and pdFVIII (A); FVIII WT and
ALA284GLU (B); or BDD-FVIII and BDD-SER289LEU (C) (2.5 nM) were bound to ESH8 at
1.12 ng/mm2. A resonance response of 200 Arc seconds corresponds to 1 ng protein
bound per mm2 of the biosensor chip surface. The kinetics of plasma-derived FVIII,
FVIII WT, SER289LEU, ALA284GLU, BDD-FVIII, or BDD-SER289LEU nonproteolytic dissociation from ESH8 was recorded after replacement of the ligand by dissociation buffer
(at arrow). At the second arrow, thrombin (1 U/mL) was added and thrombinmediated dissociation of the A2 subunit from immobilized dimers was followed. The
koff values for nonproteolytic and thrombin-mediated dissociation were derived from
dissociation kinetic curves.
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
Figure 3. ARG531HIS, ALA284GLU, and SER289LEU proteins exhibit unstable activity
during the first phase of the 2-st chromogenic assay. Conditioned medium from
COS cells expressing FVIII WT or mutant protein was collected 64 hours after
transfection and analyzed in a modified 2-stage activity assay (COAMATIC). The first
stage of the assay was modified such that each protein was subjected to increasing
duration of the first stage. FVIII, 䡺; ARG531HIS, E; SER289LEU, ‚; ALA284GLU, 䉫.
ARG531HIS, ALA284GLU,
and SER289LEU proteins exhibit unstable
activity during the first phase of the 2-st chromogenic assay
To address the effect of the increased rate of A2 dissociation on the
discrepant activity of the mutants in the 2-st assay, FVIII WT,
ARG531HIS, ALA284GLU, and SER289LEU proteins were assayed in the
chromogenic assay under conditions of increased duration of the
first stage (Figure 3). Conditioned media samples collected at 64
hours following transfection were used for the assay. Antigen
concentrations were similar for FVIII WT and the mutants and
ranged between 10 and 20 ng/mL. The manufacturer’s protocol was
modified whereby duplicated samples were incubated in the first
stage for 2, 4, 8, and 16 minutes in parallel with FVIII WT. The first
stage was initiated for the 16-minute samples first, followed by the
8-, 4-, and 2-minute incubation samples. The second stage was then
initiated for all samples simultaneously and then measured together. During the first-stage incubation, the FVIII samples are
activated due to the presence of thrombin in the reagent mix. The
resultant FVIIIa is then available to exert cofactor activity with
factor IXa to generate factor Xa from unactivated factor X. The
total amount of factor Xa generated in the first stage is then
quantified at the second stage by the amount of chromophore
released from the chromogenic reagent by factor Xa. A standard
curve was performed in parallel with dilutions of pooled normal
plasma and incubated for 2 minutes during the first stage. The
y-axis of Figure 3 is thus the relative FVIII activity of the samples
following the various one-stage incubations as compared to this
standard curve. We hypothesized that both FVIII WT and the
mutants with increased rate of A2 dissociation would become fully
activated in the first-stage incubation and exhibit FVIII activity as
determined by the total amount of factor Xa generated. However,
with increased rate of inactivation due to A2 dissociation, the
mutants would be unable to sustain FVIII activity and the total
amount of factor Xa generated would not increase at the same rate
as FVIII WT. Indeed, FVIII WT was observed to have continued
cofactor activity (as demonstrated by increasing factor Xa generation) throughout even the longest first-stage incubation of 16
minutes. In contrast, each of the mutants, though showing continued cofactor activity for up to 4 minutes of first-stage incubation
fail to contribute to any further factor Xa generation beyond 4
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BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
minutes. This is consistent with complete spontaneous inactivation
of the mutants after 4 minutes. Thus the relative amount of factor
Xa generation decreases with increasing duration of first-stage
incubation compared to FVIII WT.
Discussion
Despite the 4 decades of availability of both the 1-st and 2-st assays
for measuring FVIII activity, there is still no consensus on which
method most accurately represents FVIII cofactor function in vivo.
Although there is little assay discrepancy between the methods
when comparing human plasmas or various FVIII-containing
concentrates, considerable variability has been described when not
comparing FVIII molecules with similar molecular composition
and structure. For instance, recombinant FVIII shows a reduced
potency in the 1-st assay compared to the 2-st assay (chromogenic)
when plasma FVIII is used as the standard.28 Similar discrepancy is
observed following infusion of recombinant FVIII into patients
with hemophilia, prompting recommendations that recombinant
FVIII be used to prepare the standards for this type of analysis.
In addition, a naturally occurring patient mutation was described whereby the 1-st assay reports reduced activity compared to
the 2-st assay.29 The mutation GLU720LYS within FVIII results in a
mild hemophilia A phenotype with detectable circulating FVIII
antigen. Patients with this mutation exhibit normal levels of FVIII
cofactor activity as measured by the chromogenic assay method but
have reduced levels of procoagulant activity when measured by
1-st assay. Recombinant GLU720LYS was purified and found to
require higher concentrations of FIXa for full cofactor activity as
measured by FXa generation, whereas lower concentrations of
FIXa (as would be expected in a 1-st assay) resulted in 2- to 3-fold
reduced activity. It was proposed that this mutation at residue 720,
adjacent to one of 3 putative FIXa binding sites within the A2
domain (residues 698-712), could result in reduced affinity for
FIXa and therefore contribute to the higher FIXa concentration
required for optimal cofactor activity.
The 1-st/2-st discrepancy described above relates to reduced
FVIII activity in the 1-st compared to the 2-st assay. However, the
hemophilia A phenotype analyzed in this report relates to FVIII
activity that is reduced in the 2-st assay compared to the 1-st assay.
Early reports characterizing this clinical phenotype were attributed
to type 2N von Willebrand disease (vWD) where a mutation in the
FVIII binding site within von Willebrand factor leads to reduced
affinity for FVIII in plasma.30,31 The reduced FVIII activity in the
2-st assay was due to increased adsorption of FVIII to Al(OH)3
during the pretreatment phase of the 2-st assay. This has been
subsequently confirmed in an additional recent report of type 2N
vWD.32 This type of 1-st/2-st activity discrepancy was first
characterized in hemophilia A pedigrees in 198333 suggesting it
was related to specific FVIII mutations. Subsequently, several
specific mutations were described.18 Biochemical analysis of a
recombinant-derived form of one of these mutations, ARG531HIS,
led to the observation that the thrombin-activated mutant protein
had an increased rate of spontaneous inactivation secondary to an
increased rate of A2 subunit dissociation.19 This suggested a role
for this particular residue in contributing to the stabilization of the
FVIIIa heterotrimer. An additional report describes analysis of
patient plasma from a hemophilia A patient with the ARG531HIS
mutation.34 Using a FVIII ELISA specific for heterodimeric FVIII,
this patient had reduced amounts of heterodimeric FVIII, suggesting the mutation may also reduce the stability of the triplicated A
FVIII MUTATIONS INCREASE A2 SUBUNIT DISSOCIATION
689
domains even before thrombin activation, further highlighting the
role of protein-protein interactions within the A-domain interface.
Molecular modeling of the environment of the ARG531 residue,
using the homology-derived FVIII A domain model20 predicted that
the ARG531 arginine side chain in A2 participates in an H-bond with
the main chain carbonyl oxygen of ARG282 in A1, providing both a
plausible explanation for the molecular pathology of the hemophilic FVIII variant and support for the accuracy of the model.
However, the model also permitted predictions concerning the
hemophilic variants ALA284PRO and ALA284GLU, which also show
the 1-st/2-st discrepancy18,35: here the small hydrophobic side chain
of ALA284 packs closely against the ARG531 side chain at the A1-A2
boundary, very close to the ARG531-ARG282 H-bond (Figure 4A).
Replacement of the small alanine side chain with that of either
proline or glutamic acid (as in the hemophilic FVIII variants) may
be predicted to disrupt the stabilizing ARG531-ARG282 H-bond. We
have tested this prediction by expression and characterization of
the ALA284GLU variant.
In addition we have expressed and studied SER289LEU, another
hemophilia mutation in A1 with a similar 1-st/2-st discrepancy,
placed close in the model to the A1-A2 domain interface. In this
Figure 4. Predicted destabilization of interdomain interaction in the ALA284ARG531-ARG282 region. (A) Close-up view of a portion of the A1-A2 interdomain
boundary in the FVIII A domain model, showing the putative interaction of the ARG531
side chain with the main chain carbonyl of ARG282, together with the close packing of
the ALA284 side chain against that of ARG531. Replacement of the Ala side chain by the
bulky polar Glu side chain (as in the hemophilia A variant ALA284GLU) is predicted to
disturb the ARG282-ARG531 interaction. Side chains are colored by atom (C, green; O,
red; N, blue; H, white). (B) Predicted destabilization of interdomain interaction in the
SER289-TYR1979 region. Close-up view of a portion of the A1-A3 interdomain
boundary, showing the putative interaction of the SER289 side chain with the side
chain of TYR1979. Replacement of the Ser side chain by the bulky hydrophobic Leu
side chain (as in the hemophilia A variant SER289LEU) is predicted to disturb the
SER289-TYR1979 interaction and packing of surrounding residues. Side chains are
colored by atom (C, green; O, red; N, blue; H, white).
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
690
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
PIPE et al
case, inspection of the FVIII A domain model predicts an interdomain H-bond between the A1 and A3 domains, between the side
chains of SER289 and TYR1979 (Figure 4B). Replacement by the
bulky leucine side chain is predicted to abolish this interdomain
H-bond but also to disrupt packing very close to the interface
between all 3 A domains at the pseudo-threefold axis of the model.
Our in vitro characterization of both of these FVIII missense
mutations located within the A1 subunit (at residues 284 and 289)
confirms the hemophilia A clinical phenotype of 1-st/2-st discrepancy and demonstrates the same molecular phenotype of increased
rate of A2 subunit dissociation on thrombin cleavage. These
observations provide evidence that the packing of the triplicated A
domains is critical to FVIII function and that interdomain proteinprotein interactions are required for stabilizing both the unactivated
and activated forms of the molecule. These observations also
provide first experimental evidence that validates the molecular
model of the triplicated A domains based on their homology to
ceruloplasmin.
The insights gained from studying this patient phenotype and
the molecular characterization of these particular mutations has
extended the observations made from analyzing the ARG531HIS
mutation. Two additional reports have added to our observations. A
family pedigree has been described in which all members have 2-st
FVIII activities that are one half of that measured by 1-st assay.36
This pedigree included affected males as well as homozygous
females. Molecular analysis identified a missense mutation at
residue 694 within the A2 subunit where ILE is substituted for
ASN. This mutation predicts loss of intradomain hydrogen bonds
as well as introduces severe side-chain packing constraints within
an area adjacent to the A2-A3 interface. The second report
describes a hemophilia A patient with the same 1-st/2-st discrepancy phenotype attributed to a missense mutation at residue 1954
within the A3 subunit where LEU is substituted for a HIS.37 This
patient’s 1-st activity assay was within normal limits though the
2-st activity assay and chromogenic assay were only 18% and 35%
of normal, respectively. This residue is in close proximity to
TYR1979, and to residues ARG698 and MET1947, both previously
indicated to lie at the A2-A3 interface and associated with the same
1-st/2-st discrepant mild hemophilia phenotype under discussion.18,35 This report is particularly interesting in that the hemophilia phenotype was more accurately reflected by the 2-st assay
rather than the more widely used 1-st assay. Thus, mild hemophilia
A variants can be missed when screened in laboratories that
exclusively use the 1-st aPTT-based activity assay. These observations, as well as those reported here, showing that the 2-st assay
more closely reflects in vivo FVIII clotting activity, support that
FVIII activity is limited by A2 dissociation in vivo.
There are reports of this particular discrepant hemophilia
phenotype associated with predictions of disrupted interactions at
Figure 5. Hemophilic mutations with 1-st/2-st discrepancy are located at or
close to the A domain interfaces. Global view of the FVIII A domain model marking
the positions of ALA284, ARG531, and ARG282 at the A1-A2 interface; SER289 and
TYR1979 at the A1-A3 interface close to the pseudo-threefold axis; and HIS1954,
ASN694, MET1947, and ARG698 at, or adjacent to, the A2-A3 interface.
or close to the A1-A2, A1-A3, and A2-A3 domain interfaces.
Figure 5 shows a global view of the FVIII A domain model viewed
down the pseudo-threefold axis, showing the predicted positions of
the amino acids mentioned above, which are associated with the
1-st/2-st discrepancy in hemophilic subjects. There is a clear
clustering of all these residues at or very close to the interface of the
A2 domain with the A1 and A3 domains.
The proposed pseudo-threefold axis at the hydrophobic core of
the A1-A2-A3 interface is tightly packed and predicts several
interdomain interactions. These lie at the interface of A1-A2,
A2-A3, and A1-A3. Sufficient disruption of these protein-protein
interactions along the pseudo-threefold axis would lead to destabilization and increased propensity for dissociation particularly
after thrombin cleavage whereby covalent bonds flanking the A2
subunit are lost.
The validation of the molecular modeling of the A domains and
the characterization of these putative interdomain protein-protein
interactions now allows the hypothesis that increased stabilization
of this interface would lead to greater stability of FVIII and
increased cofactor activity. There is now opportunity to explore
complementary mutations that might allow better packing and
stabilization of the A domains to test this hypothesis. Novel
bioengineering strategies may ultimately enable strategic interdomain cross-linking to provide further stabilization of FVIIIa.
Further characterization of FVIIIa-FIXa sites of interaction
combined with both molecular modeling and crystallography
observations may ultimately allow the design of a peptidomimetic
for FVIIIa.
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From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
2001 97: 685-691
doi:10.1182/blood.V97.3.685
Hemophilia A mutations associated with 1-stage/2-stage activity
discrepancy disrupt protein-protein interactions within the triplicated A
domains of thrombin-activated factor VIIIa
Steven W. Pipe, Evgueni L. Saenko, Angela N. Eickhorst, Geoffrey Kemball-Cook and Randal J.
Kaufman
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