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Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 538-548
The Molecular Basis for Cross-Reacting Material-Positive Hemophilia
A Due to Missense Mutations Within the A2-Domain of Factor VIII
By
Kagehiro Amano,
Rita Sarkar,
Susan Pemberton,
Geoffrey Kemball-Cook,
Haig H. Kazazian Jr, and
Randal J. Kaufman
From The Howard Hughes Medical Institute and the Department of
Biological Chemistry and University of Michigan Medical Center, Ann
Arbor, MI; the Department of Genetics, University of Pennsylvania
School of Medicine, Philadelphia, PA; the Haemostasis Research Group,
Medical Research Council Clinical Sciences Centre, London, UK; and the
Department of Clinical Pathology, Tokyo Medical College, Tokyo, Japan.
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ABSTRACT |
Factor VIII (FVIII) is the protein defective in the bleeding
disorder hemophilia A. Approximately 5% of hemophilia A patients have
normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of
the genetic defects within the A2-domain for FVIII function we
constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T,
V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the
conditioned medium from transiently transfected COS-1 cells correlated
with published data for the patients plasma-derived FVIII, confirming
the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis analysis of immunoprecipitated FVIII protein
radiolabeled in COS-1 cells showed that all CRM-positive mutant
proteins were synthesized and secreted into the medium at rates similar
to wild-type FVIII. The majority of the DeltaF652/3 mutant was
defective in secretion and was degraded within the cell. All mutant
FVIII proteins were susceptible to thrombin cleavage, and the A2-domain
fragment from the I566T mutant had a reduced mobility because of use of
an introduced potential N-linked glycosylation site that was confirmed
by N-glycanase digestion. To evaluate interaction of FVIII
with factor IXa, we performed an inhibition assay using a synthetic
peptide corresponding to FVIII residues 558 to 565, previously shown to
be a factor IXa interaction site. The concentration of peptide required
for 50% inhibition of FVIII activity (IC50) was reduced for the I566T
(800 µmol/L) and the S558F (960 µmol/L) mutants compared with
wild-type FVIII (>2,000 µmol/L). N-glycanase digestion increased
I566T mutant FVIII activity and increased its IC50 for the peptide
(1,400 µmol/L). In comparison to S558F, a more conservative mutant
(S558A) had a sixfold increased specific activity that also correlated
with an increased IC50 for the peptide. These results provided support
that the defects in the I566T and S558F FVIII molecules are caused by
steric hindrance for interaction with factor IXa.
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INTRODUCTION |
HEMOPHILIA A is caused by a quantitative
or qualitative deficiency of plasma factor VIII (FVIII), a cofactor for
factor IXa in the proteolytic activation of factor X to factor
Xa.1,2 FVIII is synthesized as a single chain polypeptide
of 2,351 amino acids from which a 19-amino acid signal peptide is
cleaved, and has a domain organization of
A1-A2-B-A3-C1-C2.3,4 The A-domains are homologous to the
A-domains of ceruloplasmin,5 a copper binding plasma
protein, suggesting a possible role in metal-ion binding. The C-domains
are homologous to phospholipid-binding proteins, suggesting a role in
phospholipid interaction.6 The B-domain displays no
significant homology to any known protein. FVIII is proteolytically
processed on secretion from the cell to form heterodimers composed of a
series of amino-terminal heavy chain fragments ranging in size from 90 to 220 kD and a carboxy-terminal light chain fragment of 80 kD.
Thrombin activates FVIII by cleaving the 90 kD heavy chain (A1-A2) into
50-kD (A1) and 43-kD (A2) fragments, and the 80-kD light chain to a
73-kD (A3-C1-C2) fragment.7 The active form of FVIII is a
metal-ion linked heterotrimer of A1, A2, and A3-C1-C2.8,9
Approximately 5% of hemophilia A patients have normal levels of
dysfunctional FVIII protein and are termed cross-reacting material
(CRM)-positive. CRM-positive patients have considerable amounts of
FVIII protein in their plasma (at least 30% of the normal amount), but
the protein is nonfunctional; ie, the FVIII activity is much less than
the FVIII plasma protein level. In contrast, FVIII antigen is not
detected in CRM-negative patients. Patients with CRM-reduced hemophilia
A have reduced plasma FVIII antigen levels regardless of activity
levels. Some CRM-reduced patients also display lower activity values
compared with the plasma antigen levels.10,11 Approximately
40% of the CRM-positive and CRM-reduced hemophilia A patients contain
missense mutations within the A2-domain.12 The A2-domain
consists of approximately 330 amino acids or approximately 15% of the
entire amino acid sequence of FVIII, indicating that a selective
clustering of missense mutations occur within this region that result
in hemophilia A. Several reports have supported that the A2-domain
subunit is required for FVIII procoagulant activity. The
thrombin-activated heterotrimer exhibited a pH-dependent dissociation
of the A2 subunit from the complex that correlated with a loss in
procoagulant activity.13 The difference in stability
between porcine and human FVIIIa correlates with increased affinity of
the porcine A2-domain compared with the human A2-domain for the
A1/A3-C1-C2 heterodimer.8,14 Furthermore, in-frame deletion
of the A2-domain in recombinant FVIII yielded a secreted protein that
did not exhibit procoagulant activity. However, either cotransfection
of this A2-domain deletion mutant with an A2-domain expression vector
or addition of purified A2-domain fragment itself restored procoagulant
activity for the A2-domain deletion molecule.15 Although
the A2-domain is essential for procoagulant activity, it is not
understood how the A2 subunit contributes to tenase complex activity.
Recent work using synthetic peptides showed that the amino acid region
558 to 565 within the FVIII A2 subunit represents a factor IXa
interaction site.16,17
Results from site-directed mutagenesis studies of human recombinant
FVIII have contributed to our understanding of its structure-function relationships. For example, missense mutations introduced at the thrombin cleavage sites showed that cleavages at R372 and R1689 are
required for full functional activity18 consistent with the
results from analysis of naturally occurring mutations that result in
hemophilia A.19-24 To date there is only one report
describing the characterization of a missense mutation within the
A2-domain that resulted in a defective protein. In that situation,
plasma FVIII from a patient that contained the missense mutation of
isoleucine to threonine at residue 566, creating a novel N-linked
glycosylation site at asparagine residue 564, was defective in
procoagulant activity.25 In this report, we have studied
the mechanistic basis for missense mutations within A2-domain affecting
molecular interactions required for FVIII function. Site-directed
mutagenesis was used to create six different mutations within the
A2-domain that were previously shown to correlate with defective
circulating FVIII protein in patients with severe to mild hemophilia A. Functional characterization of these mutant proteins, expressed in
transiently transfected COS-1 cells, showed that I566T and S558F FVIII
missense mutations have reduced specific activity that can be
attributed to reduced interaction with factor IXa.
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MATERIALS AND METHODS |
Materials.
FVIII-deficient plasma and normal pooled human plasma were obtained
from George King Biomedical Inc (Overland Park, KS). Monoclonal antibody to the heavy chain of FVIII (F8) coupled to CL4B-sepharose was
a gift from Debra Pittman (Genetics Institute Inc, Cambridge, MA).
ESH-4 and ESH-8 antibodies were purchased from American Diagnostica Inc
(Greenwich, CT). Activated partial thromboplastin (Automated APTT
reagent) was purchased from General Diagnostics Organon Teknika Corporation (Durham, NC). COAMATIC chromogenic FVIII activity assay kit
was purchased from Pharmacia Hepar (Franklin, OH). Soybean trypsin
inhibitor, phenylmethylsulfonylfluoride (PMSF) and aprotinin were
purchased from Boehringer, Mannheim GmbH (Mannheim, Germany). Human
 -thrombin and O-Phenylenediamine Dihydrochloride (OPD) and
N--Leu-Leu-norleucinal (ALLN) were purchased from Sigma Chemical Co
(St Louis, MO). [35S]-methionine (>1,000 Ci/mmol) was
purchased from Amersham Life Science Inc (Arlington Heights, IL).
Dulbecco's Modified Eagle's Medium (DMEM), methionine-free DMEM,
Biotin N-Hydroxy Succinimide Ester, and Streptavidin-Horseradish
Peroxidase Conjugate were obtained from GIBCO BRL (Gaithersburg, MD).
Fetal bovine serum was purchased from PAA Laboratories Inc (Newport
Beach, CA). Recombinant N-glycanase was purchased from Genzyme
DIAGNOSTICS (Cambridge, MA). Patient's plasma containing FVIII variant
I566T (designated as ARC-22 or JH-11725 by the Holland
Laboratory, American Red Cross Blood Services, or the Center for
Medical Genetics, Johns Hopkins University School of Medicine,
respectively) was generously supplied by Carol Kasper (Orthopedic
Hospital, Los Angeles, CA) for research purposes.
Plasmid construction.
Mutagenesis was performed within the mammalian expression vector
pMT226 containing the wild-type (WT) full length FVIII
cDNA. Mutant plasmids were generated through oligonucleotide
site-directed mutagenesis using the gapped-heteroduplex procedure or
the polymerase chain reaction as described previously.27-29
Codon 527 was mutated from CGG to TGG predicting an amino acid change
from arginine to tryptophan, and the resultant mutant plasmid was
designated R527W. Codon 558 was mutated from TCT to either TTT or GCA
predicting an amino acid change from serine to either phenylalanine or
alanine, respectively, and the resultant mutant plasmids were
designated S558F and S558A. Codon 566 was mutated from ATA to ACA
predicting an amino acid change from isoleucine to threonine, and the
resultant mutant plasmid was designated I566T. Codon 634 was mutated
from GTG to either GCG or ATG predicting an amino acid change from valine to either alanine or methionine, respectively, and the resultant
mutant plasmids were designated V634A and V634M. Either codon 652 or
653, which are both TTC coding phenylalanine, was deleted and the
resultant 3-bp deletion mutant plasmid was designated DeltaF652/3. The
mutations were confirmed by DNA sequence analysis using the dideoxy
nucleotide sequencing method.30
DNA transfection and analysis.
Plasmid DNA was transfected into COS-1 cells by the diethyl
aminoethyl-dextran procedure as described.27 After 40 hours the cells were fed fresh medium containing 10% heat-inactivated fetal
bovine serum and samples of conditioned medium (CM) were harvested at
60 hours posttransfection for FVIII assay. Subsequently, protein
synthesis and secretion were analyzed by metabolically labeling cells
for 20 minutes with [35S]-methionine, followed by a chase
for 4 hours in medium containing a 100-fold excess of unlabeled
methionine and 0.02% aprotinin.27 Cell extracts (CE) were
prepared by lysis in Nonidet P-40 lysis buffer.27 For
analysis of the effect of cysteine protease inhibition, increasing
amounts of ALLN were included in the chase medium. CE and CM containing
labeled proteins were harvested as described previously31
and immunoprecipitated with F8 antibody coupled to CL-4B sepharose.
Immunoprecipitated proteins were washed with phosphate buffered saline
(PBS) containing Triton X-100 and resuspended in 50 mmol/L Tris-HCl pH
7.5, 150 mmol/L NaCl, 2.5 mmol/L CaCl2, and 5% glycerol
(buffer A). Immunoprecipitated proteins from conditioned medium were
resuspended in buffer A and treated with or without 2 U/mL of thrombin
at 37°C for 30 minutes. Samples were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under
reducing conditions and visualized by fluorography after treatment with
En3Hance (Dupont, Boston, MA).
FVIII assay.
FVIII activities were measured in a one stage clotting assay using
FVIII-deficient plasma as substrate or by the COAMATIC chromogenic assay according to the manufacturer. One unit of FVIII activity is that amount measured in 1 mL of normal human pooled plasma.
FVIII antigen was quantitated by a sandwich enzyme-linked immunosorbent
assay (ELISA)32 using two monoclonal antibodies to the
FVIII light chain. Purified recombinant FVIII protein was used as a
standard.
Protein purification.
Partially purified protein was obtained from 200 mL of conditioned
medium from COS-1 cells transfected with FVIII WT, S558F, S558A, I566T,
and V634A by immunoaffinity chromatography as described previously.31 The bound FVIII was eluted in buffer
containing 60% ethylene glycol and concentrated by dialysis against a
10% polyethylene glycol (MW 15K-20K) containing buffer33
and stored at  70°C.
N-glycanase digestion of WT and I566T mutant FVIII.
Immunoprecipitated FVIII WT and I566T mutant from radiolabeled CM was
resuspended with buffer A and divided into three aliquots for
incubation in the absence or presence of thrombin or thrombin and
subsequently N-Glycanase. Human thrombin was added to a final concentration of 5 U/mL. Recombinant N-Glycanase was added to a final
concentration of 10 U/mL in the presence of 4% Nonidet P-40 and 20 mmol/L PMSF. The resulting polypeptides were separated by SDS-PAGE and
visualized by autoradiography as described previously.
FVIII activity of samples from patient's plasma and conditioned medium
were measured after N-Glycanase digestion. Patient's plasma was
incubated with recombinant N-Glycanase (final concentration 10 U/mL) at
37°C. FVIII activity after increasing periods of time was measured
by the one stage clotting assay. Normal pooled plasma was used under
the same conditions as a control for this assay. For the evaluation of
recombinant FVIII protein, CM was mixed with an equal volume of
FVIII-deficient plasma to approximate physiological conditions. This
sample was treated with recombinant N-Glycanase and FVIII activity was
measured as described previously. WT FVIII conditioned medium was also
mixed with FVIII-deficient plasma as a control in this assay.
Peptide inhibition of intrinsic factor Xase activity.
The synthetic peptide SVDQRGNQ, which corresponds to FVIII residues 558 to 565, and a scrambled version of this peptide, NGSQDQRV, were
synthesized by the University of Michigan Protein and Carbohydrate Structure Facility (Ann Arbor, MI). Both peptides were greater than
90% pure as determined by high-liquid-performance-chromatography analysis and their identity was confirmed by mass spectrometry. The
effect of synthetic peptides on intrinsic factor Xase activity was
evaluated by the Factor Xa generation assay using COAMATIC chromogenic
factor VIII activity assay kit. Partially purified FVIII samples were
mixed with various concentrations of synthetic peptide, then factor
reagent containing factor IXa, factor X, thrombin, CaCl2,
and phospholipid was added. After 2 minutes incubation at 37°C,
S-2765 chromogenic substrate was reacted with thrombin inhibitor
I-2581, which prevents hydrolysis of S-2765 by thrombin. Factor Xa
activity was determined as initial rate values by measuring optical
density at 405 nm. Exponential best fit curves were determined from the
independent experiments presented.
Molecular modeling.
The effects of missense mutations within a structural model for the A
domains of FVIII34 were characterized by altering residue
side chains using the Biopolymer module of InsightII (Molecular Simulations Inc, Cambridge, UK) and the variant molecules were reminimized using Discover (Molecular Simulations Inc).
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RESULTS |
CRM-Positive mutants are synthesized and secreted similarly to WT
FVIII.
The profile of the mutations analyzed in this study is summarized in
Table 1.12,25,35 We constructed
six mutations within the A2-domain that correlate with mutations
observed in five CRM-positive and one CRM-reduced hemophilia A. FVIII
WT and the A2-domain mutants were compared by transient DNA
transfection of the cDNA expression vectors into COS-1 monkey cells. At
60 hours after transfection, the rates of synthesis were analyzed by
immunoprecipitation of CE from [35S]-methionine
pulse-labeled cells. Intracellular FVIII WT was detected in its single
chain form and migrated at approximately 280 kD
(Fig 1, lane 1). The primary translation
products for each of the A2-domain mutants were detected at 280 kD at
similar levels to WT (Fig 1, lanes 2 to 7). Thus, there was no
significant effect of these mutations on the rate of FVIII protein
translation. Analysis of the CE after a 4-hour chase indicated that
FVIII WT and all mutants disappeared from the CE at approximately
similar rates (Fig 1, lanes 10 to 16).
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Table 1.
Comparison of Phenotype of Hemophilia A Patients and
Expression of Their Respective Recombinant Proteins
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| Fig 1.
Synthesis of FVIII WT and mutants in COS-1 cells. WT and
mutant expression plasmids were transfected into COS-1 monkey cells. At
60 hours posttransfection, cells were pulse-labeled with
[35S]-methionine for 20 minutes and cell extracts (CE)
were harvested. Duplicate plates were chased for 4 hours in medium
containing excess unlabeled methionine and then CE were harvested.
Equal volumes of CE were immunoprecipitated with anti-FVIII antibody and equal aliquots were analyzed by SDS-PAGE. Mock indicates cells that
did not receive plasmid DNA. The migration of FVIII in the CE is
indicated at the right as FVIII. Molecular weight size markers are
shown on the left.
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The secretion into the CM was analyzed by immunoprecipitation of CM
from [35S]-methionine pulse-labeled transfected cells
chased for 4 hours in medium containing excess unlabeled methionine. On
SDS-PAGE analysis, FVIII WT was detected as a 300-kD single chain, a
200-kD heavy chain, and an 80-kD light chain
(Fig 2, lane 1). The amount of secreted
polypeptides from DeltaF652/3 mutant transfected cells was greatly
reduced in the CM (Fig 2, lane 11), whereas the five CRM-positive
mutants were secreted at levels similar to FVIII WT (Fig 2, lanes 3, 5, 7, 9, and 13).
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| Fig 2.
Secretion and thrombin cleavage of WT and mutant FVIII.
The secretion of each mutant was analyzed by immunoprecipitation
of conditioned medium from [35S]-methionine
pulse-labeled transfected cells chased for 4 hours in
medium containing excess unlabeled methionine. Immunoprecipitated FVIII
molecules were analyzed by SDS-PAGE before () and after (+)
thrombin (IIa) digestion. Mock indicates cells that did not receive
plasmid DNA. Molecular weight size markers are shown on the left.
Single chain (Single), heavy chain (Heavy), and light chain (Light) are
indicated for undigested samples. A3-C1-C2, A1, and A2 fragments are
indicated for digested samples. The symbol * represents the A2 fragment
with reduced mobility.
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The thrombin cleavage fragments for all mutants, except I566T, were
indistinguishable from WT FVIII.
Immunoprecipitated FVIII molecules were treated with thrombin before
analysis by SDS-PAGE. Thrombin cleavage of all mutants, except I566T,
generated the light chain migrating at 73 kD and the heavy chain
derived fragments corresponding to the 50-kD A1-domain and 43-kD
A2-domain (Fig 2, lanes 4, 8, 10, 12, and 14) that were indistinguishable from the FVIII WT (Fig 2, lane 2). However, the
A2-domain fragment from the I566T mutant showed reduced mobility at 46 kD compared with FVIII WT (Fig 2, lane 6).
Specific activity for each mutant correlated with published data for
the patients' plasma-derived FVIII.
CM from cells transfected with FVIII WT or the A2-domain mutants were
harvested at 60 hours posttransfection for FVIII assay. The activity
and antigen in the CM of FVIII WT showed 195 mU/mL and 37 ng/mL
(average of 3 different transfections), respectively. The specific
activity for the WT FVIII was 5,270 U/mg, consistent with the value
obtained for plasma-derived FVIII. The activities determined by the
COAMATIC assay and antigen levels (represented as percent of FVIII WT)
in the CM from cells transfected with the A2-domain mutants are
compared with the patients' phenotype (represented as percent of
normal) in Table 1. Compared with FVIII WT, the activity values
obtained by the COAMATIC assay were similar to those obtained by
clotting assay (data not shown). The S558F mutant CM showed a lower
amount of activity than the patient's plasma-derived FVIII and the
V634M mutant CM showed a slightly higher amount of activity than the
patient. The other A2-domain mutants exhibited activities similar to
the patients' values. Compared with antigen levels of FVIII WT in the
CM or FVIII in normal plasma, the levels of FVIII antigen in the CM from most of the FVIII mutant transfected cells were lower than those
obtained in the patients' plasma. Quantitation of the specific activity showed that all A2-domain mutants, except for R527W, had
values less than 10% of FVIII WT (Fig 3).
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| Fig 3.
The specific activity for WT and mutant FVIII. CM of WT
and mutant FVIII were harvested at 60 hours posttransfection for FVIII assay. The activity and antigen in the CM were measured by COAMATIC chromogenic assay and ELISA using an anti-FVIII light chain antibody, respectively. Specific activity is expressed as percent of WT. Bars are
expressed as a mean ± standard deviation (SD) of three independent
transfection experiments.
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Mutant S558A has increased specific activity over S558F.
As mentioned previously, the amino acid region 558 to 565 within the A2
subunit is proposed to be a factor IXa interaction site.16
Because of the significant difference in the amino acid structure
between serine and phenylalanine, the S558F mutant may have a reduced
interaction with factor IXa. To test this possibility, a more
conservative mutation of serine to alanine (S558A) was made. Analysis
of pulse-chase [35S]-methionine labeled CE and CM before
and after thrombin cleavage by SDS-PAGE showed that the S558A mutant
was synthesized, secreted, and cleaved by thrombin similar to FVIII WT
and S558F (Fig 1, lanes 8 and 17; Fig 2, lanes 15 and 16). However, its
specific activity was increased sixfold over the S558F mutant (Table 1 and Fig 3). The results suggest that eliminating the large
side-chain of phenylalanine increased procoagulant activity.
I566T creates a new N-linked glycosylation site that is used.
Substitution of isoleucine at residue 566 for threonine creates a
potential new N-linked glycosylation site at asparagine 564. Analysis
of plasma-derived FVIII from a patient harboring the I566T mutation
suggested that this glycosylation site is used resulting in reduced
FVIII activity.25 The thrombin-derived A2-domain fragment
of the recombinant-derived I566T mutant had a reduced mobility (Fig 2,
lane 6 and Fig 4A, lanes 2 and 5) consistent with
addition of oligosaccharides to asparagine 564. To show that the
reduced mobility results from additional glycosylation,
immunoprecipitated WT and I566T mutant FVIII from radiolabeled CM were
treated with thrombin and N-glycanase before SDS-PAGE
analysis. After N-glycanase digestion the A2 fragment from I566T
comigrated with the WT A2 fragment (Fig 4A, lanes 3 and 6). This result
is consistent with the previous data characterizing FVIII in patient's
plasma.25 To test whether N-glycanase digestion would
increase FVIII activity for the I566T mutant, we analyzed the FVIII
clotting activity after increasing periods of incubation with
N-glycanase. N-glycanase digestion increased the activity for
the I566T recombinant protein in CM to 30%, and this increase was
similar to that observed for the I566T patient's plasma-derived FVIII
(Fig 4B).
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| Fig 4.
Effect of N-glycanase for I566T mutant FVIII. (A)
Immunoprecipitated WT and I566T mutant from radiolabeled CM were
treated with nothing (IIa ,
N-Gly) or thrombin (IIa+,
N-Gly) or N-glycanase after thrombin
(IIa+, N-Gly+) before SDS-PAGE as described
in the Materials and Methods. Molecular weight size markers are shown
on the left. A3-C1-C2, A1, A2, and mutant A2 fragments are indicated at
the right. (B) FVIII in conditioned medium as well as in patient's
plasma were incubated with 10 U/mL of N-glycanase at 37°C. FVIII
activity after increasing times was determined by one stage clotting
assay. Data are plotted as the percent activity of the mutant compared
with the WT-recombinant ( ) or plasma-derived ( ) FVIII,
respectively. Data represent the average of two independent
experiments.
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I566T and S558F FVIII are more sensitive to inhibition by a synthetic
peptide corresponding to a factor IXa interaction site.
The results above show that FVIII activity increased after removal of
either the large side-chain of phenylalanine for the S558F mutant or of
the carbohydrate at N564 for the I566T mutant. We propose that these
moieties may sterically prevent FVIII interaction with factor IXa.
Previously, a synthetic peptide corresponding to FVIII residues 558 to
565 prevented Xa generation by factor IXa, presumably because of
preventing factor IXa interaction.16 To measure the factor
IXa interaction with the mutants, we tested the ability of the
synthetic peptide 558 to 565 to inhibit FVIII activity.
Equal amounts (4 nmol/L) of partially purified FVIII samples were mixed
with increasing concentrations of synthetic peptide and assayed for
FVIII activity. An exponential best fit curve was determined from three
independent experiments to extrapolate the concentration of peptide
required for 50% inhibition of activity (IC50) for WT FVIII and for
V634A mutant FVIII to be 3,340 (±80) µmol/L and 2,240 (±330)
µmol/L, respectively (Fig
5A). In contrast, the IC50 for the I566T and S558F mutant FVIII
molecules were measured to be 800 (±130) µmol/L and 960 (±60)
µmol/L, respectively. These results support that the I566T and S558F
mutants are more sensitive than WT to inhibition by the peptide. The
inhibition curve for S558A was shifted right and the IC50 for S558A
increased to 1,690 (±300) µmol/L (Fig 5A). N-glycanase digestion
did not change the inhibition profile of WT FVIII, but increased the
IC50 for I566T to 1,400 (±170) µmol/L (Fig 5B). Thus I566T and
S558F were inhibited at lower concentrations of peptide than WT FVIII.
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| Fig 5.
Inhibition of intrinsic factor Xase activity by
synthetic peptide 558 to 565. (A) Equal amounts (4 nmol/L) of partially
purified FVIII samples were mixed with increasing concentrations of
synthetic peptide and assayed for FVIII activity as described in the
Materials and Methods. The data represent the average of three
independent experiments. FVIII activity for each molecule is expressed
as a percent of that obtained in the absence of the peptide. The symbols represent WT FVIII (), S558F ( ), S558A ( ), I566T ( ) and V634A ( ). (B) WT and I566T mutant FVIII were treated with N-glycanase for 3 hours at 37°C, then samples were tested in the peptide inhibition assay. The results represent the average of three
(filled symbols) and two (open symbols) independent experiments. The
symbols represent WT FVIII (), WT FVIII with N-glycanase (),
I566T () and I566T with N-glycanase ( ). (C) Equal amounts (4 nmol/L) of partially purified FVIII samples were mixed with increasing
concentrations of scrambled synthetic peptide and assayed for FVIII
activity as described in the Materials and Methods. Two independent
experiments are shown for WT FVIII (), S558F (), I566T () and
V634A (). (D) Four different concentrations of WT FVIII (4 nmol/L
[], 0.8 nmol/L [], 0.4 nmol/L [], 0.08 nmol/L [])
were tested in the peptide inhibition assay. The results are the
average of two independent experiments.
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To confirm that the effect of this peptide was specific for interaction
between factor IXa and FVIII, a scrambled synthetic peptide having the
same composition was used in the same assay. The scrambled peptide did
not inhibit either WT or any mutant FVIII (Fig 5C). Equal antigenic
amounts of each FVIII protein were used in the peptide inhibition
assays. Therefore, the initial activity for the WT and each mutant was
different because of differences in specific activity. Therefore, we
tested if the amount of initial activity affected the peptide
inhibition. The effects on increasing peptide 558 to 565 on four
different concentrations of WT FVIII were measured. The inhibition
profiles for all samples were indistinguishable (Fig 5D), suggesting
that the results did not depend on initial activity of sample.
Intracellular accumulation of DeltaF652/3 mutant in the presence of
inhibitors of intracellular degradation.
Although the DeltaF652/3 mutant was synthesized and chased from the CE
similar to WT FVIII (Fig 1), it displayed significantly reduced
appearance in the conditioned medium (Fig 2). Thus, this protein was
either degraded within the cell or was secreted and rapidly degraded in
the conditioned medium. The cysteine-protease inhibitor, ALLN, inhibits
intracellular degradation and previous studies showed increased
intracellular accumulation of a mutant FVIII (R2307Q) that was
inefficiently secreted.29 These results support that this
mutant did not accumulate in the CM because of a block in secretion
with subsequent degradation within the secretory pathway and not as a
consequence of instability in the CM. The effect of ALLN on the
secretion of DeltaF652/3 mutant was studied by analyzing
[35S]-methionine labeled FVIII protein. Addition of ALLN
to the CM resulted in a greater accumulation of the DeltaF652/3 in the
CE (Fig 6, lanes 6-8) compared with WT (Fig
6, lanes 2-4). However, ALLN treatment did not increase the secretion
of either WT or DeltaF652/3 into the CM (Fig 6, lanes 13-18). These
results suggest that defective secretion with subsequent intracellular
degradation is responsible for loss of the mutant FVIII from the CE,
leading to the CRM-reduced phenotype.
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| Fig 6.
Inhibition of intracellular degradation causes
intracellular accumulation of DeltaF652/3. Parallel plates of
transfected COS-1 monkey cells were labeled at 60 hours
posttransfection with [35S]methionine for 30 minutes,
chased for 4 hours in the absence (lanes 2, 6, 10, 13, 16, and 19) or
presence of increasing amounts of ALLN (lanes 3, 4, 7, 8, 11, 12, 14, 15, 17, 18, 20, and 21). CE and conditioned medium (CM) were harvested
and equal proportionate volumes of CE and CM were immunoprecipitated
with anti-FVIII specific antibody for analysis by SDS-PAGE. Mock
indicates cells that did not receive plasmid DNA. Molecular weight size
markers are shown on the left of each CE and CM.
|
|
| |
DISCUSSION |
The study of missense mutations in the FVIII gene that result in
hemophilia A has elucidated the functional requirements for FVIII
activity. Previous studies showed that the A2 subunit is essential for
FVIIIa activity.8,9,13-15 In this study, we have characterized six different mutants within the A2-domain using site-directed mutagenesis. The goal of these studies was to confirm the
genetic basis for the phenotype of hemophilia A and to elucidate the
mechanism by which these missense mutations within the A2-domain result
in hemophilia A.
We compared the phenotype of recombinant mutant FVIII proteins with
their respective mutants analyzed in patients' plasma. SDS-PAGE
analysis showed that there was no significant effect of these mutations
on the rate of FVIII synthesis and all the CRM-positive mutants were
secreted into the CM similar to WT. However, the activity in the CM for
all the CRM-positive mutants was reduced and correlated with the
reduced activity reported in patients' plasma. Quantitation of the
antigen levels in the CM by ELISA showed that most mutants also
displayed lower antigen levels in the CM compared with WT and these
were lower than the values obtained from respective patients' plasma.
We speculate that the synthesis and/or secretion of the FVIII
mutants in vivo might be stimulated to compensate for the low plasma
antigen levels. Although there was variation in the antigen levels, the
specific activity of all mutants, except for the R527W mutant, were
very low as predicted by the hemophilia A phenotype. Interestingly, the
antigen level for the R527W was low as measured by ELISA, although
SDS-PAGE analysis showed similar amounts of FVIII secretion compared
with WT. It is likely that the ELISA quantitation for this mutant was
reduced as a consequence of poor reactivity with the antibodies used in
the ELISA. In this context, it is essential that quantitation of
antigen in patients' plasma also consider the particular antibodies
used in the assay. The reduced quantity of FVIII antigen measured by
ELISA for the R527W mutant resulted in an apparently higher specific
activity. If we assume the antigen level was similar to WT as indicated
by the characterization of the secretion of the R527W mutant, then the
specific activity for the R527W mutant was also significantly reduced.
Therefore, the analysis of all the recombinant proteins supports that
each mutation is responsible for the respective patients' phenotype, confirming the basis of the genetic defect.
How do the mutations within the A2-domain of FVIII reduce procoagulant
activity? SDS-PAGE analysis showed that all mutants were susceptible to
thrombin cleavage, eliminating thrombin resistance as a cause for the
defect in activity. However, after thrombin cleavage, all mutants
studied displayed no significant increase in activity (data not shown),
indicating a functional defect in factor VIIIa. Previous studies have
identified two regions within FVIII that are likely responsible for
factor IXa interaction. First, peptide inhibition studies suggest that
residues 558 to 565 comprise a factor IXa interaction
site.16 In addition, residues 1,778 to 1,840 within the
FVIII light chain were proposed to be involved in factor IXa binding
because an antibody directed against this region inhibited the binding
of factor IXa.36 This second factor IXa binding site has
been more localized to the residues 1,811 to 1,818 by using synthetic
peptides.37 Thus, present data support two interaction
sites for factor IXa. It has been suggested that the binding site on
the FVIII light chain is responsible for complex assembly via the first
EGF-like domain of factor IXa,38 whereas the factor IXa
interaction with the A2-domain might induce changes within the factor
IXa active site.17 In this study we have shown that mutants
I566T and S558F are defective in factor IXa interaction by an assay
that measured the functional consequence of the FVIII-factor IXa
interaction. We confirmed that I566T creates a new N-linked
glycosylation site that is used and this prevents appearance of FVIII
activity. In addition, removal of the large side-chain of phenylalanine
for the S558F mutant, by mutagenesis of S558 to alanine, improved the
FVIII activity. The activities of the I566T and S558F mutants were
inhibited at lower concentrations of peptide 558 to 565 than WT FVIII
or V634A. In our experiments, inhibition of WT FVIII required
approximately 2 mmol/L peptide, compared with the value of 0.1 mmol/L
previously reported.16 The greater amount of peptide
required to inhibit WT FVIII in our assays may be because of the higher
concentration of factor IXa (4 nmol/L) present in our assays compared
with those of Fay et al16 (1 nmol/L). Either removal of the
bulky phenylalanine side chain at residue 558 in mutant S558F by
mutation of S558A or removal of N-linked oligosaccharides in the I566T
mutant by N-glycanase increased both the specific activity of the FVIII and the IC50 of the peptide. These results support the hypothesis that
the defects in the I566T and S558F FVIII molecules are caused by a
reduced specific activity resulting from steric hindrance for
interaction with factor IXa.
A structural model of the FVIII A domains based on the 3-angstrom
structure of ceruloplasmin showed that both sequences 558 to 565 and
1,811 to 1,818 are surface exposed and oriented toward the same
direction.34 Structural modeling of I566T indicates that
side-chain of the new N-glycosylation site (N564) is surface exposed
and lies within the proposed factor IXa binding site
(Fig 7A). Modeling of S558F shows that
residue 558 is surface exposed and the substitution of serine to
phenylalanine introduces a bulky hydrophobic side chain, altering the
conformation of the loop in that region and leading to steric strain
around the proposed factor IXa binding site (Fig 7B). Thus, addition of
a bulky side-chain or N-linked oligosaccharide would be expected to
prevent, or severely reduce, the interaction with factor IXa.
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| Fig 7.
Structural model of FVIII mutations I566T and S558F. (A)
Homology model of the triplicated A domains of FVIII is shown as viewed
perpendicular to threefold axis with "top" of molecule to top of
Fig Red, blue, and green represent A1, A2, and A3 subunit, respectively. Binding loop of factor IXa is shown as magenta CPK spheres. N564 (new N-glycosylation site) and T566 are colored by atom
(carbon, green; hydrogen, white; oxygen, red; nitrogen, blue). (B)
Factor IXa binding loop 558 to 565 shown in CPK spheres: residue 558 colored by atom, 559 to 565 in gray. Blue ribbons represent the
alpha-carbon trace of neighboring residues in the A2 domain. Left, WT
S558; Right, variant F558 (reminimized).
|
|
In contrast to the missense mutations S558F and I566T, the mechanism of
the molecular defect of the CRM-positive mutants R527W, V634A, and
V634M remains unknown. Because these mutants were efficiently secreted
as heterodimers, they are likely not significantly defective in protein
folding or chain assembly. V634A was inhibited by peptide 558 to 565 similar to WT FVIII, suggesting no defect in its factor IXa
interaction. Additional studies are required to elucidate the mechanism
responsible for the absence of procoagulant activity for these mutants.
In contrast to the CRM-positive mutants studied, the secretion of the
one CRM-reduced mutant DeltaF652/3 was reduced. According to the
structural model, residues 652 and 653 are close to the A3-domain and
at the interface of the A2 and A3 subunit.39 Therefore, deletion of this residue might prevent formation of the tightly packed
structure of FVIII, resulting in aberrant folding and a block to
transport through the secretory pathway. Previously, R2307Q mutant
FVIII protein showed reduced secretion with increased intracellular
degradation, although the low level of secreted protein displayed WT
specific activity.29 DeltaF652/3 also displayed defective
secretion; however, the low level of secreted mutant protein had, in
addition, a low specific activity. Inhibition of intracellular
degradation resulted in intracellular accumulation of the DeltaF652/3
mutant; however, it was not secreted, presumably as a result of failure
to bypass the "quality control" machinery of the secretory
pathway. Thus, even if it were possible to rescue secretion of this
mutant, it would not likely improve the severity of hemophilia A caused
by the DeltaF652/3 mutation because of its reduced specific activity.
| |
FOOTNOTES |
Submitted March 17, 1997;
accepted September 15, 1997.
Supported by National Institutes of Health Grants No. HL53777 and
HL52173 (R.J.K.) and the Tokyo Medical College (K.A.).
Address reprint requests to Randal J. Kaufman, PhD, Department of
Biological Chemistry and the Howard Hughes Medical Institute, University of Michigan Medical Center, 4554 Medical Science Research Building II, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0650.
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.
| |
ACKNOWLEDGMENT |
We thank Peter Lenting for helpful comments regarding the peptide
inhibition experiments, Debra Pittman for assistance in the early phase
of this study, and Steven Pipe for valuable discussion throughout the
course of these studies.
| |
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Abstract
Introduction
Abstract