Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 176-183
Mild Hemophilia A Caused by Increased Rate of Factor VIII A2 Subunit
Dissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIa
In Vivo
By
S.W. Pipe,
A.N. Eickhorst,
S.H. McKinley,
E.L. Saenko, and
R.J. Kaufman
From the Departments of Pediatrics and Biological Chemistry, Howard
Hughes Medical Institute, University of Michigan Medical Center, Ann
Arbor, MI; and the Holland Red Cross Laboratory, Rockville, MD.
 |
ABSTRACT |
Approximately 5% of hemophilia A patients have normal amounts of a
dysfunctional factor VIII (FVIII) protein and are termed cross-reacting
material (CRM)-positive. FVIII is a heterodimer (domain structure
A1-A2-B/A3-C1-C2) that requires thrombin cleavage to elicit
procoagulant activity. Thrombin-activated FVIII is a heterotrimer with
the A2 subunit (amino acid residues 373 to 740) in a weak ionic
interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2
subunit correlates with inactivation of FVIII. Recently, a phenotype of
CRM-positive hemophilia A patients has been characterized whose plasma
displays a discrepancy between their FVIII activities, where the
one-stage clotting assay displays greater activity than the two-stage
clotting assay. One example is a missense mutation where
ARG531 has been substituted by HIS531. An FVIII
cDNA construct was prepared containing the
ARG531HIS mutation and the protein was
expressed in COS-1 monkey cells by transient DNA transfection.
Metabolic labeling with [35S]-methionine demonstrated
that ARG531HIS was synthesized at an equal rate
compared with FVIII wild-type (WT) but had slightly reduced antigen in
the conditioned medium, suggesting a modest secretion defect. A time
course of structural cleavage of ARG531HIS
demonstrated identical thrombin cleavage sites and rates of proteolysis as FVIII WT. Similar to the patient phenotypes,
ARG531HIS had discrepant activity as measured
by a one-stage activated partial thromboplastin time (aPTT) clotting
assay (36% ± 9.6% of FVIII WT) and a variation of the two-stage
assay using a chromogenic substrate (COAMATIC; 19% ± 6.9% of FVIII
WT). Partially purified FVIII WT and ARG531HIS
proteins were subjected to functional activation by incubation with
thrombin. ARG531HIS demonstrated significantly
reduced peak activity and was completely inactivated after 30 seconds,
whereas FVIII WT retained activity until 2.5 minutes after activation.
Because the ARG531HIS missense mutation
predicts a charge change to the A2 subunit, we hypothesized that the
ARG531HIS A2 subunit could be subject to more
rapid dissociation from the heterotrimer. The rate of A2 dissociation,
using an optical biosensor, was determined to be fourfold faster for
ARG531HIS compared with FVIII WT. Because the
two-stage assay involves a preincubation phase before assay
measurement, an increased rate of A2 dissociation would result in an
increased rate of inactivation and reduced specific activity.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PLASMA COAGULATION factor VIII (FVIII)
functions within the blood coagulation cascade as a cofactor for factor
IXa in the proteolytic activation of factor X to factor Xa. A
quantitative or qualitative deficiency of FVIII leads to the phenotype
of the bleeding disorder hemophilia A. FVIII is synthesized as a
single-chain polypeptide of approximately 280 kD with the domain
structure A1-A2-B-A3-C1-C2.1-3 The A domains share 35% to
40% amino acid identity and are homologous to the A-domains of
ceruloplasmin. The C domains also display 35% to 40% amino acid
identity to each other and are homologous to phospholipid-binding
proteins, suggesting a role in phospholipid
interactions.1,2,4,5 The B domain shares no significant
homology with any known protein. Intracellular proteolytic processing
within the B domain after residues ARG1313 or
ARG1648 forms a heterogeneous FVIII heterodimer consisting
of approximately 90-to 220-kD heavy chain fragments (A1-A2-B)
associated with an 80-kD light chain fragment (A3-C1-C2) through a
monovalent copper ion-dependent linkage between the A1 and A3
domains.6
Thrombin activates FVIII through proteolytic cleavage after
ARG740, ARG1689, and ARG372,
generating an activated FVIII (FVIIIa) heterotrimer consisting of the
A1 subunit (50 kD) in a copper ion-dependent association with the
thrombin-cleaved light chain (73 kD) and a free A2 subunit associated
with the A1 domain through a weak ionic interaction.7-13 An
acidic amino acid-rich region at the carboxy-terminus of the A1 subunit
most likely interacts with positively charged residues within the A2
subunit to retain the heterotrimeric FVIIIa
configuration.14-16
FVIII activity is measured by either a one-stage or two-stage
procedure.17,18 The one-stage assay is based on the ability of FVIII-containing samples to correct the prolonged activated partial
thromboplastin time (aPTT) of FVIII-deficient plasma.19 The
two-stage assay uses the same principle as the one-stage method, yet is
split into two distinct phases.20 In the first phase, dilutions of FVIII are incubated with factor IXa, factor X,
Ca2+, and phospholipid. Activation of FVIII during the
first phase allows it to exhibit its procoagulant activity as a
cofactor for factor IXa, leading to the generation of factor Xa. The
incubation mixture is then subsampled into a second tube containing
Ca2+ and a source of prothrombin and fibrinogen, and the
time to fibrin formation is recorded. A modification of the two-stage
assay has also been used in which the second reaction is incubated with a factor Xa-sensitive chromogenic substrate where the rate or degree of
color change is proportional to the amount of FVIII added in the first
stage of the assay.21
Hemophilia A is a heterogeneous disorder, with severe phenotypes
associated with major disruptions of the FVIII gene. Patients with
severe phenotypes usually have FVIII antigen levels that are
undetectable and are termed cross-reacting material (CRM)-negative. Other genetic mutations are associated with inefficient secretion, with
patient plasmas having concomitantly reduced FVIII antigen and activity
levels, and are termed CRM-reduced. Approximately 5% of hemophilia A
patients have considerable FVIII antigen levels (at least 30% of
normal), but FVIII activity levels are significantly reduced,
suggesting a protein dysfunction.22 Interestingly, approximately 40% of the CRM-positive and CRM-reduced hemophilia A
patients contain missense mutations within the A2 domain.23 The A2 domain, representing only approximately 10% of the entire amino
acid sequence of FVIII, therefore contains a significant clustering of
missense mutations resulting in hemophilia A highlighting its
functional importance for FVIII procoagulant activity.
The study of missense mutations has contributed significantly to our
understanding of FVIII structure-function relationships and the
pathophysiology contributing to the hemophilia A disease phenotype.
Missense mutations have been identified at thrombin cleavage sites
critical for functional activation and at residues important in
interaction with von Willebrand factor (vWF).24-28 However,
to date, only two FVIII A2 domain missense mutations have been
characterized as to their mechanism leading to protein dysfunction.27,29 In both cases, the reduced specific
activity could be attributed to reduced interaction with factor IXa.
Recent reports have identified a CRM-positive hemophilia A phenotype in
which the patient plasmas exhibit a familial discrepancy in which the
FVIII procoagulant activity is higher when measured in a one-stage
assay compared with a two-stage assay.30-36 This discrepancy is unusual, because one-stage and two-stage assays have
been used interchangeably for some time as a standard determination of
FVIII activity and the majority of patients have similar results as
measured by either method.18 In this report, we have
studied the mechanistic basis for one of these patient phenotypes in
which an A2 domain missense mutation results in substitution of a
histidine for arginine at residue 531.31 Using
site-directed mutagenesis, the ARG531HIS
mutation was generated within the FVIII cDNA and the protein was
functionally characterized after expression in transiently transfected
COS-1 cells. ARG531HIS demonstrated only a
modest secretion defect, had reduced specific activity, and had
discrepant FVIII activity as measured by either the one-stage or
two-stage methods. Upon thrombin activation, the
ARG531HIS A2 subunit exhibited a fourfold
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 one-stage and
two-stage assays but would result in an increased disadvantage in the
two-step procedure 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.
| |
MATERIALS AND METHODS |
Materials.
Anti-heavy chain FVIII monoclonal antibody (MoAb; F-8) conjugated to
CL-4B Sepharose was a gift from Debra Pittman (Genetics Institute Inc,
Cambridge, MA). FVIII-deficient and normal pooled human plasma were
obtained from George King Biomedical, Inc (Overland Park, KS).
Activated partial thromboplastin (automated aPTT reagent) and
CaCl2 were purchased from General Diagnostics Organon
Teknika Corp (Durham, NC). Anti-light chain FVIII MoAbs, ESH-4 and
ESH-8, were purchased from American Diagnostica, Inc (Greenwich, CT). Human thrombin and aprotinin were purchased from Boehringer, Mannheim GmbH (Mannheim, Germany). [35S]-methionine (>1,000
µCi/mmol) was purchased from Amersham Corp (Arlington Heights, IL).
En3Hance was purchased from Dupont (Boston, MA).
Dulbecco's modified Eagle's medium (DMEM), methionine-free DMEM,
fetal bovine serum, biotin N-hydroxy succinimide ester, and
streptavidin-horseradish peroxidase conjugate were purchased from GIBCO
BRL (Gaithersburg, MD). COAMATIC was purchased from DiaPharma (West
Chester, OH).
Plasmid mutagenesis.
Mutagenesis was performed within the mammalian expression vector
pMT237 containing the FVIII cDNA
(pMT2VIII). Oligonucleotide-directed mutagenesis was used
to create a Spe I-Kpn I polymerase chain reaction
fragment in which codon 531 was mutated from CGC to CAC, predicting an
amino acid substitution of histidine for arginine, and was ligated into
Spe I-Kpn I-digested pMT2VIII. The
resulting mutant plasmid was designated
ARG531HIS. The plasmid containing the wild-type
FVIII cDNA sequence was designated FVIII WT. 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 diethylaminoethyl
(DEAE)-dextran method as previously
described.38 Conditioned medium was harvested at 64 hours
posttransfection in the presence of 10% fetal bovine serum. Protein
synthesis and secretion were analyzed by metabolically labeling cells
at 64 hours posttransfection for 30 minutes with
[35S]-methionine (300 µCi/mL in methionine-free
medium), followed by a chase for 4 hours in medium containing 100-fold
excess unlabeled methionine and 0.02% aprotinin. Cell extracts and
conditioned medium were harvested and immunoprecipitations were
performed and analyzed as described previously.38
Protein purification.
Partially purified ARG531HIS protein was
obtained from 200 mL of conditioned medium from transiently transfected
COS-1 cells by immunoaffinity chromatography,39 yielding
750 to 1,500 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 to 20,000) -containing
buffer14 and stored at 
70°C.
FVIII activity and antigen assay.
FVIII activity was measured by (1) one-stage aPTT clotting assay on an
MLA Electra 750 fibrinometer (Medical Laboratory
Automation, Inc, Pleasantville, NY) by reconstitution of human
FVIII-deficient plasma or (2) by modified two-stage assay using
the COAMATIC chromogenic assay according to the manufacturer's
instructions. For thrombin activation, protein samples were diluted
into 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 2.5 mmol/L
CaCl2, and 5% glycerol and incubated at room temperature
with 1 U/mL thrombin. After incubation for increasing periods of time,
aliquots were diluted and assayed for FVIII activity. One unit of FVIII
activity is the amount measured in 1 mL of normal human pooled
plasma. FVIII antigen was quantified using a sandwich
enzyme-linked immunosorbent assay (ELISA) method using anti-light chain
antibodies ESH-4 and ESH-8.40 Recombinant FVIII protein
purified in parallel was used as a standard.
Kinetic measurements using biosensor technology.
The kinetics of the A2 subunit dissociation from thrombin-activated
FVIII WT and ARG531HIS was studied by surface
plasmon resonance using the IASys biosensor (Fisons, Cambridge, UK),
which measures protein binding and subsequent dissociation in real
time.41 Binding of 1 ng of protein per square millimeter of
the biosensor chip produces a resonance signal of 200 Arc seconds. MoAb
ESH8 (50 µg/mL) in 10 mmol/L sodium acetate, pH 5.0, was covalently
coupled to the activated carboxymethyldextran-coated biosensor cuvette
via amino groups using succinimide ester chemistry.41 The
carboxymethyldextran chip and reagents for its activation, N-ethyl-N
-(dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and deactivation, ethanolamine, were purchased from Fisons. FVIII WT and ARG531HIS binding to
ESH8, their dissociation from the antibody, and activation by thrombin
were measured in 200 µL of 20 mmol/L HEPES, 0.15 mol/L NaCl, 5 mmol/L CaCl2, 0.01% Tween 20, pH 7.4. The chip was
regenerated by the addition of 0.1 mol/L 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 ARG531HIS from immobilized ESH8 and
those for dissociation of the A2 subunits upon thrombin activation of
immobilized FVIII proteins were determined by fitting the dissociation
kinetics data to the following equation describing a single-phase
dissociation process: dR/dt = koffR,42 where R is observed surface plasmon resonance signal. The fitting procedure was performed using Sigmaplot 1.02 software (Jandel Scientific, San Raphael, CA).
| |
RESULTS |
Synthesis and secretion of ARG531HIS.
The synthesis and secretion of FVIII WT and
ARG531HIS was compared by transient DNA
transfection into COS-1 monkey cells. At 64 hours posttransfection, the
rates of synthesis were analyzed by immunoprecipitation of cell
extracts from [35S]-methionine pulse-labeled cells.
Intracellular FVIII WT and ARG531HIS were
detected in their single chain forms and migrated at approximately 250 kD (Fig 1, lanes 3 and 5).
ARG531HIS exhibited similar band intensity to
the FVIII WT, suggesting that the missense mutation did not interfere
with efficient protein synthesis. After a 4-hour chase period, FVIII WT
was lost from the cell extract (Fig 1, lane 4) and was recovered from
chase conditioned medium as a 280-kD single chain, a 200-kD heavy
chain, and an 80-kD light chain (Fig 1, lane 8).
ARG531HIS was also lost from the cell extract
over the 4-hour chase period (Fig 1, lane 6), similar to
FVIII WT, but had a reduced recovery from the chase-conditioned medium
(Fig 1, lane 9), suggesting a modest secretion defect.
ARG531HIS was also detected within the
chase-conditioned medium in single-chain, heavy-chain, and light-chain
forms of identical molecular mobility as FVIII WT, suggesting similar
posttranslational processing and proper heavy and light chain
association. From the relative band intensities within the
chase-conditioned medium, ARG531HIS-secreted
protein was determined to be 56% of FVIII WT. FVIII antigen
determinations by ELISA were performed on unlabeled conditioned media
collected from 24 to 64 hours posttransfection.
ARG531HIS protein was detected at 46% and 76%
of FVIII WT in two independent transfection experiments, consistent
with the modest secretion defect indicated by the pulse-chase analysis.
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| Fig 1.
Synthesis and secretion of FVIII WT and
ARG531HIS expressed in COS-1 cells. FVIII WT
and ARG531HIS plasmids were transfected into
COS-1 monkey cells. At 64 hours posttransfection, cells were
pulse-labeled with [35S]-methionine for 30 minutes and
cell extracts were harvested. Duplicate labeled cells were chased for 4 hours in medium containing excess unlabeled methionine and then cell
extracts and conditioned medium were harvested. Equal proportionate
volumes of cell extract (lanes 1 through 6) and conditioned medium
(lanes 7 through 9) were immunoprecipitated with anti-FVIII-specific
antibody and equal aliquots were analyzed by SDS-PAGE. Mock indicates
cells that did not receive plasmid DNA. Cell extract pulse (P) and
chase (C). The migration of FVIII from the cell extracts is indicated
at the right by an arrow. FVIII from the conditioned medium is
indicated at the far right as single-chain (SC), heavy-chain (HC), and
light-chain (LC) forms. Molecular weight markers are shown on the
left.
|
|
Recombinant-derived ARG531HIS protein
demonstrates a similar functional phenotype to patient plasmas.
Conditioned medium was collected from COS-1 cells transiently
expressing FVIII WT and ARG531HIS from 24 to 64 hours posttransfection. FVIII activity (Fig
2) was measured by a one-stage aPTT clotting assay or by a modified two-stage method using the COAMATIC chromogenic assay. Similar to the
patient phenotypes reported previously,
ARG531HIS had discrepant activity as measured
by the one-stage aPTT clotting assay (36% ± 9.6% of FVIII WT) as
compared with the two-stage chromogenic assay (COAMATIC; 19% ± 6.9% of FVIII WT). The activity measurements for the patient plasmas
represent all of the reported data obtained from the HAMSTeRS
hemophilia A mutation database.43 The slightly higher
two-stage activity results obtained with the recombinant-derived
ARG531HIS are consistent with those obtained
from the few reported patient plasmas in which a chromogenic assay
rather than the classical two-stage assay was used.31 After
immunoaffinity purification of FVIII WT and
ARG531HIS from the conditioned medium, similar
results were obtained. Immunoaffinity purified
ARG531HIS-specific activity was 62% of FVIII
WT by one-stage aPTT clotting assay as compared with 32% of FVIII WT
as determined by the two-stage chromogenic assay.
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| Fig 2.
Recombinant-derived ARG531HIS
demonstrates a similiar phenotype to described
ARG531HIS patient plasmas. Plasma activity data
were derived from the HAMSTeRS hemophilia A mutation database for all
reported patients identified with ARG531HIS for
which both one-stage ( ) and two-stage ( ) results were available.
The data from recombinant-derived ARG531HIS
were obtained from assaying the activity in the conditioned medium from
four independent transfection experiments. Activity for
ARG531HIS patient plasmas and
recombinant-derived ARG531HIS is presented as
the percentage of wild-type (normal plasma or recombinant FVIII WT,
respectively).
|
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ARG531HIS demonstrates similar thrombin
proteolysis compared with FVIII WT.
[35S]-methionine-labeled FVIII WT and
ARG531HIS proteins were immunoprecipitated from
chase conditioned medium of transiently expressing COS-1 cells labeled
at 60 hours posttransfection. After Triton X-100 washes as described,
the immunoprecipitated complexes were incubated with thrombin (0.1 U/mL) for increasing periods of time at 37°C before sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Both
FVIII WT and ARG531HIS were initially detected
in their 280-kD single-chain forms, and dimers of a 200-kD heavy chain
in association with an 80-kD light chain
(Fig 3, lanes 2 and 8). Both FVIII WT and
ARG531HIS were sequentially cleaved into a
heterotrimer of fragments consistent with a 50-kD A1 subunit, 43-kD A2
subunit, and 73-kD thrombin-cleaved light chain, A3-C1-C2 (Fig 3, lanes
3 through 7 and 9 through 13). A 90-kD fragment appeared immediately
after incubation with thrombin consistent with an A1-A2 heavy chain
fragment due to cleavage after arginine 740. With higher concentrations
of thrombin, this fragment is further cleaved into 50-kD A1 and 43-kD
A2 subunits (data not shown). The similar pattern of electrophoretic
mobility and rate of appearance of proteolytic fragments over time
suggests that ARG531HIS, compared with FVIII
WT, has identical sites of thrombin cleavage and sensitivity to
proteolysis.
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| Fig 3.
Thrombin proteolysis of FVIII WT compared with
ARG531HIS. [35S]-methionine
labeled FVIII WT and ARG531HIS proteins
immunoprecipitated from the chase conditioned medium of transiently
expressing COS-1 cells were divided into equal aliquots and incubated
with thrombin (0.1 U/mL) for increasing periods of time at 37°C.
Reactions were terminated with SDS-PAGE sample buffer and protein
fragments were separated by 10% SDS-PAGE. Time is in minutes, with 0 representing the absence of thrombin. Mock indicates medium from cells
that did not receive plasmid DNA. FVIII protein forms are indicated at
the right as follows: SC, single chain; HC, heavy chain; LC, light
chain; A1+A2, A1, and A2, thrombin-cleaved heavy chain fragments;
LC+IIa, thrombin-cleaved light chain; FVIIIa, predicted
thrombin-activated FVIII heterotrimer. Molecular weight markers (m) are
indicated on the left.
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ARG531HIS exhibits reduced peak activity and
increased rate of inactivation after functional activation by thrombin.
Having demonstrated a similar pattern and sensitivity of
ARG531HIS to thrombin cleavage, the functional
consequence of the ARG531HIS missense mutation
on activation and inactivation was examined in an in vitro functional
assay. Equal concentrations of immunoaffinity purified FVIII WT and
ARG531HIS were incubated with thrombin and
assayed for FVIII activity by a one-stage aPTT clotting assay
(Fig 4). Upon treatment with thrombin, FVIII WT was maximally activated within 10 seconds and then inactivated over the next 5 minutes. ARG531HIS also reached
peak activity after 10 seconds of incubation with thrombin, but at
approximately fivefold lower activity compared with FVIII WT. In
addition, ARG531HIS was completely inactivated
after 30 seconds incubation with thrombin, suggesting some increased
instability of the thrombin-activated heterotrimer.
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| Fig 4.
Activation and inactivation of FVIII WT and
ARG531HIS by thrombin. Immunoaffinity purified
FVIII WT () and ARG531HIS ( ) proteins
(0.5 nmol/L) were incubated with thrombin (1 U/mL) at room temperature
and assayed over time for FVIII activity by aPTT. The results are from
a single thrombin activation experiment and are typical of multiple
independent experiments.
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ARG531HIS exhibits increased rate of A2
subunit dissociation from the thrombin-activated heterotrimer.
Because the A2 subunit is retained within the thrombin-activated FVIIIa
heterotrimer through a weak electrostatic interaction with the acidic
amino acid rich region at the carboxy terminus of the A1 subunit, we
hypothesized that the charge change resulting from the
ARG531HIS mutation would make its A2 subunit
susceptible to more rapid dissociation. The relative rates of A2
dissociation for FVIII WT and ARG531HIS were
determined using an optical biosensor (Fig
5). An anti-light chain antibody, ESH8, was covalently immobilized on a
carboxymethyldextran-coated biosensor chip. Similar amounts (1.12 ng/mm2) of immunoaffinity-purified FVIII WT or
ARG531HIS protein (2.5 nmol/L) 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 s1, were
similar for FVIII WT and ARG531HIS.
Subsequently, thrombin was added to a final concentration of 1 U/mL.
Because the FVIII preparations are bound to an ESH8-coated chip via
their light chains, this interaction will not be disturbed by thrombin
activation and the A1 subunit will remain associated with the light
chain 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. The koff values for the FVIII WT and
ARG531HIS A2-subunits were (6.7 ± 0.6) × 103 s1 and (2.9 ± 0.22) × 102 s1,
respectively. The half lives of A2-dissociation for FVIII WT and
ARG531HIS calculated as ln2/koff
are 103 and 24 seconds, respectively. Because A2-dissociation
correlates with inactivation, the ARG531HIS
thrombin-activated heterotrimer would be expected to inactivate fourfold faster compared with FVIII WT. Similar results were obtained via the same method, substituting an anti-A2 domain antibody (MoAb 413;
Holland Red Cross Laboratory) for immobilization of the FVIII proteins
(data not shown). The single exponential dissociation of the A2 subunit
observed for FVIII WT in this study is comparable to the decay of
FVIIIa previously reported.14,44
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| Fig 5.
Determination of the kinetic parameters for
nonproteolytic and thrombin-mediated dissociation of FVIII WT and
ARG531HIS from MoAb ESH8. MoAb ESH8 was
covalently immobilized to a biosensor chip at 20 ng/mm2.
FVIII WT or ARG531HIS (2.5 nmol/L) were bound
to ESH8 at 1.12 ng/mm2. A resonance response of 200 Arc seconds corresponds to 1 ng of protein bound per square millimeter
of the biosensor chip surface. The kinetics of FVIII WT or
ARG531HIS 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
thrombin-mediated 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 as described under Materials and Methods.
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| |
DISCUSSION |
The one-stage and two-stage methods for assaying FVIII activity have
been used clinically for more than 40 years. Several studies have
demonstrated little differences in precision between the two
methods.18 The one-stage assay, being technically simpler and easier to automate, has typically been the method of choice in
recent years. Discrepancy in FVIII activity results have only been
described under a few unique conditions. These have included the
measurement of FVIII concentrates against plasma standards and the
measurement of concentrates against concentrate standards and in the
assessment of in vivo recovery of FVIII concentrate infusions.18 The discrepancies have included higher
two-stage versus one-stage activities or the converse as investigated
in this report. An explanation for these discrepancies has been
elusive, although the Al(OH)3 adsorption step, which is
used in the preparation of samples in the two-stage assay, has been
suggested as a major cause of the discrepancy in assays of concentrates
versus plasma. However, this discrepancy was an average of only 26%
higher activity in the two-stage assay compared with the one-stage
assay.45 Other suggested causes have included the choice of
predilution buffer, thrombin activation of the samples, nonspecific
contaminants such as lipids, and the influence of the presence of
vWF.18 In the latter case, a patient with von Willebrand's
disease demonstrated a discrepancy in FVIII activity in which, even
though the plasma FVIII activity measured by a one-stage assay was
consistent with the antigen level, there was a 75% decrease in the
plasma FVIII activity when a two-stage assay was used.35
The patient was later characterized as having a von Willebrand disease
Normandy defect in which there was a weaker interaction of FVIII with
the mutant vWF leading to increased susceptibility of the FVIII to adsorption by Al(OH)3.36 The apparent
discrepancy was corrected by adding hemophilic plasma or purified
hemophilic vWF.
Several recent reports have now observed these discrepancies as part of
a hemophilia A phenotype. The patients have typically been mild in
phenotype and the discrepancy, a more than twofold higher activity
result by the one-stage assay versus the two-stage, was observed in all
affected family members.31 This phenotypic subgroup was
subjected to DNA analysis to determine a responsible FVIII gene
mutation.31 Several single missense mutations were identified either within the FVIII A1, A2, or A3 domains. The best
characterized missense mutation was the
ARG531HIS mutation chosen for this study. Three
independent reports collected by the hemophilia A mutation database
have shown that the patients with the ARG531HIS
mutation have FVIII antigen levels from approximately 30% to 100% of
normal, suggesting at worst a modest protein secretion defect.43 All the patients analyzed by both one-stage and
two-stage assays demonstrated at least twofold higher results in the
one-stage assay. Where a chromogenic based assay was also used, there
was still an approximate twofold difference in the activities, although the chromogenic two-stage results were somewhat higher than those for
the classical two-stage assay.31
The ARG531HIS mutation does not lie within
major identified functional FVIII epitopes, such as the terminal
portion of the C2 domain containing the binding site for
phospholipids,46,47 the vWF binding sites (residues
1648-1689 of the A3 domain and epitopes within the terminal C2
domain),48,49 or the proposed factor IXa interaction site
within the A2 domain (residues 558-565).50,51 Therefore,
this particular missense mutation may require an alternative mechanism
for dysfunction not yet characterized. Insights into the differences
between the one-stage and two-stage assays allow a hypothesis as to a
possible mechanism. The two-stage assay, divided into two separate
steps, requires a prolonged phase in the first step under conditions in
which FVIII becomes activated and generates the predicted FVIIIa
heterotrimeric structure to exert its cofactor function with factor
IXa. Previous studies have highlighted the instability of the FVIIIa
heterotrimer. 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.14 The data
supporting the idea that the amino acid region 558 to 565 within the A2 subunit represents a factor IXa interaction site are
consistent with this observation. Porcine FVIIIa exhibits an increased
affinity for its A2 subunit compared with human FVIIIa and,
accordingly, demonstrates an increased specific
activity.44,52,53 A genetically engineered recombinant FVIII molecule in which the A2 subunit remains covalently linked to the
heterodimer after activation by thrombin exhibited a fivefold increase
in specific activity.54 These observations highlight the
role of A2 dissociation in limiting FVIIIa activity. The observations are consistent with positively charged residues within the A2 subunit
interacting with acidic amino acid residues at the carboxy-terminus of
the A1 subunit maintaining a weak electrostatic interaction to preserve
the FVIIIa heterotrimer. Loss of residues 337-372 of the A1 subunit
after cleavage by activated protein C or further proteolysis by
thrombin leads to rapid inactivation of FVIIIa via A2 subunit
dissociation.14,15,55,56 A genetically engineered mutant of
recombinant FVIII, containing ARG336ILE, was
resistant to proteolytic cleavage by thrombin and activated protein C
and had an increased specific activity, as determined by a chromogenic
assay, attributable to increased stability of the
heterotrimer.12 Many of the amino acid substitutions in the
porcine compared with the human A2 subunit lead to a charge alteration
and may be responsible for the increased stability of the porcine
FVIIIa heterotrimer. Because the two-stage assay involves the
preincubation phase before subsampling, an FVIII protein with increased
heterotrimeric stability would be predicted to have an increased
activity in the two-stage assay. Consistent with this
finding, when porcine FVIII concentrates were assayed against human concentrates by the classical two-stage method, the
activity results were two to three times higher than by one-stage assays.18
However, the phenotype characterized in relation to the
ARG531HIS missense mutation is one in which not
only the one-stage activity is reduced from that expected by the
antigen levels present, but also the two-stage assay activity results
are at least twofold lower than by the one-stage assay. The
ARG531HIS mutation predicts a loss of charge
within the A2 subunit predicting a weakened electrostatic interaction
after thrombin activation. Thus, FVIIIa dissociation of a
ARG531HIS A2 subunit, compared with FVIII WT,
would be hypothesized to be more rapid. Because the recombinant-derived
ARG531HIS protein exhibited a similar phenotype
to the patient plasmas, we were able to analyze the purified protein
for its rate of A2 dissociation. The data using the optical biosensor
confirmed the fourfold increased rate of A2 subunit loss after thrombin
activation of ARG531HIS. The reduced peak
activity observed for the ARG531HIS protein
compared with FVIII WT can also be attributed to the increased rate of
A2 subunit dissociation. Under the conditions of the aPTT clotting
assay, thrombin was added to the FVIII samples in buffer and the
thrombin-activated samples were then prediluted before incubation with
the clotting assay reagents. The mutant would undergo spontaneous decay
more rapidly than FVIII WT before assay determination. Accordingly,
extrapolating from the initial slope of the inactivation phase, if
FVIII WT was incubated four times longer with thrombin before assay
determination, the remaining apparent peak activity would be similar to
the apparent peak activity observed for the thrombin-activated
ARG531HIS protein. Therefore, there is no need
to invoke an additional functional defect for this mutation.
Several other genetic mutations have been described with discrepant
one-stage and two-stage activities. All reported patients have mild to
moderate hemophilia A phenotype and all of the mutations occur within
either the A1, A2, or A3 domains.31 Typically, they involve
amino acid substitutions that alter charge (eg,
ARG698TRP) or hydrophobicity (eg,
ALA284GLU and
SER289LEU). It can be postulated that these
missense mutations, although not confined to either the acidic region
of the A1 domain or to the A2 subunit itself, may still interfere with
the weak electrostatic interaction at this critical interaction site,
thereby leading to similar instability of the FVIIIa heterotrimer.
Because the patients with the ARG531HIS
mutation have a mild hemophilia A phenotype, the observed FVIII
dysfunction observed in vitro in these assays is apparently also
important in vivo. This is an important observation for several
reasons. Firstly, it is not known in vivo whether FVIIIa procoagulant
activity is limited by spontaneous A2 subunit dissociation or further
proteolysis. The observations from this CRM-positive mutant and porcine
FVIII would suggest that the inherent instability of FVIIIa also limits its activity in vivo and is either further compromised by mechanisms that lead to increased A2 dissociation or partially abrogated by
mechanisms that lead to reduced A2 dissociation. Secondly, an increased
plasma level of FVIII has now been identified as a risk factor for
thrombosis.57,58 If proteolytic inactivation by activated
protein C was of primary importance in regulating FVIIIa, then one
could predict that a mutation leading to resistance to activated
protein C would also lead to an increased risk of thrombosis.
Comprehensive analysis of patients with thrombophilia has failed to
identify any mutations at activated protein C cleavage sites within
FVIII despite the prominent association of factor V Leiden (resistant
to activated protein C) with this cohort of patients.59
Finally, this study predicts that even minor modifications of the A2
subunit can have major functional impacts both in vitro and in vivo.
This provides further insight into research efforts directed at
producing a new generation of recombinant FVIII molecules with
increased specific activity. Based on the conclusions from this study
and the others summarized here, we propose that the most significant
mechanism of FVIIIa inactivation in vivo is dissociation of the A2
subunit.
| |
FOOTNOTES |
Submitted May 11, 1998;
accepted August 24, 1998.
Supported by National Institutes of Health (NIH) Grant No. HL52173 and
by National Institute of Child Health and Human Development (NICHD) HD28820.
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.
Address reprint requests to R.J. Kaufman, PhD, Howard Hughes Medical
Institute, University of Michigan Medical Center, 4570 MSRB II, 1150 W
Medical Center Dr, Ann Arbor, MI 48109-0650; e-mail:
kaufmanr{at}umich.edu.
| |
REFERENCES |
1.
Vehar GA, Keyt B, Eaton D, Rodriguez H, O'Brien DP, Rotblat F, Oppermann H, Keck R, Wood WI, Harkins RN:
Structure of human factor VIII.
Nature
312:337, 1984[Medline]
[Order article via Infotrieve]
2.
Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Buecker JL, Pittman DD, Kaufman RJ, Brown E, Shoemaker C, Orr EC:
Molecular cloning of a cDNA encoding human antihaemophilic factor.
Nature
312:342, 1984[Medline]
[Order article via Infotrieve]
3.
Kaufman RJ, Wasley LC, Dorner AJ:
Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells.
J Biol Chem
263:6352, 1988[Abstract/Free Full Text]
4.
Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick RM, Kaufman RJ, Mann KG:
Complete cDNA and derived amino acid sequence of human factor V.
Proc Natl Acad Sci USA
84:4846, 1987[Abstract/Free Full Text]
5.
Koschinsky ML, Funk WD, van Oost BA, MacGillivray RT:
Complete cDNA sequence of human preceruloplasmin.
Proc Natl Acad Sci USA
83:5086, 1986[Abstract/Free Full Text]
6.
Tagliavacca L, Moon N, Dunham WR, Kaufman RJ:
Identification and functional requirement of Cu(I) and its ligands within coagulation factor VIII.
J Biol Chem
272:27428, 1997[Abstract/Free Full Text]
7.
Eaton D, Rodriguez H, Vehar GA:
Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity.
Biochemistry
25:505, 1986[Medline]
[Order article via Infotrieve]
8.
Fulcher CA, Roberts JR, Zimmerman TS:
Thrombin proteolysis of purified factor VIII procoagulant protein: Correlation of activation with generation of a specific polypeptide.
Blood
61:807, 1983[Abstract/Free Full Text]
9.
Fay PJ, Anderson MT, Chavin SI, Marder VJ:
The size of human factor VIII heterodimers and the effects produced by thrombin.
Biochim Biophys Acta
871:268, 1986[Medline]
[Order article via Infotrieve]
10.
Fay PJ:
Subunit structure of thrombin-activated human factor VIIIa.
Biochim Biophys Acta
952:181, 1988[Medline]
[Order article via Infotrieve]
11.
Fay PJ, Haidaris PJ, Smudzin TM:
Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit.
J Biol Chem
266:8957, 1991[Abstract/Free Full Text]
12.
Pittman DD, Kaufman RJ:
Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII).
Proc Natl Acad Sci USA
85:2429, 1988[Abstract/Free Full Text]
13.
Lollar P, Parker CG:
Subunit structure of thrombin-activated porcine factor VIII.
Biochemistry
28:666, 1989[Medline]
[Order article via Infotrieve]
14.
Fay PJ, Beattie TL, Regan LM, O'Brien LM, Kaufman RJ:
Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa-catalyzed proteolysis.
J Biol Chem
271:6027, 1996[Abstract/Free Full Text]
15.
Fay PJ, Haidaris PJ, Huggins CF:
Role of the COOH-terminal acidic region of A1 subunit in A2 subunit retention in human factor VIIIa.
J Biol Chem
268:17861, 1993[Abstract/Free Full Text]
16.
O'Brien LM, Huggins CF, Fay PJ:
Interacting regions in the A1 and A2 subunits of factor VIIIa identified by zero-length cross-linking.
Blood
90:3943, 1997[Abstract/Free Full Text]
17.
Denson KWE, Biggs R:
Laboratory diagnosis, tests of clotting function and their standardization, in
Biggs R
(ed):
Human Blood Coagulation, Haemostasis and Thrombosis. Oxford, UK, Blackwell Scientific, 1998, p 310.
18.
Barrowcliffe TW:
Comparisons of one-stage and two-stage assays of factor VIII:C.
Scand J Haematol Suppl
41:39, 1984[Medline]
[Order article via Infotrieve]
19.
Over J:
Methodology of the one-stage assay of factor VIII (VIII:C).
Scand J Haematol Suppl
41:13, 1984[Medline]
[Order article via Infotrieve]
20.
Barrowcliffe TW:
Methodology of the two-stage assay of factor VIII (VIII:C).
Scand J Haematol Suppl
41:25, 1984[Medline]
[Order article via Infotrieve]
21.
Rosen S:
Assay of factor VIII:C with a chromogenic substrate.
Scand J Haematol Suppl
40:139, 1984[Medline]
[Order article via Infotrieve]
22.
Lazarchick J, Hoyer LW:
Immunoradiometric measurement of the factor VIII procoagulant antigen.
J Clin Invest
62:1048, 1978
23.
McGinniss MJ, Kazazian HH Jr, Hoyer LW, Bi L, Inaba H, Antonarakis SE:
Spectrum of mutations in CRM-positive and CRM-reduced hemophilia A.
Genomics
15:392, 1993[Medline]
[Order article via Infotrieve]
24.
Arai M, Inaba H, Higuchi M, Antonarakis SE, Kazazian HH Jr, Fujimaki M, Hoyer LW:
Direct characterization of factor VIII in plasma: Detection of a mutation altering a thrombin cleavage site (arginine-372
histidine).
Proc Natl Acad Sci USA
86:4277, 1989[Abstract/Free Full Text]
25.
O'Brien DP, Pattinson JK, Tuddenham EG:
Purification and characterization of factor VIII 372-Cys: A hypofunctional cofactor from a patient with moderately severe hemophilia A.
Blood
75:1664, 1990[Abstract/Free Full Text]
26.
Johnson DJ, Pemberton S, Acquila M, Mori PG, Tuddenham EG, O'Brien DP:
Factor VIII S373L: Mutation at P1 site confers thrombin cleavage resistance, causing mild haemophilia A.
Thromb Haemost
71:428, 1994[Medline]
[Order article via Infotrieve]
27.
Aly AM, Higuchi M, Kasper CK, Kazazian HH Jr, Antonarakis SE, Hoyer LW:
Hemophilia A due to mutations that create new N-glycosylation sites.
Proc Natl Acad Sci USA
89:4933, 1992[Abstract/Free Full Text]
28.
Aly AM, Hoyer LW:
Factor VIII-East Hartford (arginine 1689 to cysteine) has procoagulant activity when separated from von Willebrand factor.
J Clin Invest
89:1382, 1992
29.
Amano K, Sarkar R, Pemberton S, Kemball-Cook G, Kazazian HH Jr, Kaufman RJ:
The molecular basis for cross-reacting material-positive hemophilia A due to missense mutations within the A2-domain of factor VIII.
Blood
91:538, 1998[Abstract/Free Full Text]
30.
Duncan EM, Duncan BM, Tunbridge LJ, Lloyd JV:
Familial discrepancy between the one-stage and two-stage factor VIII methods in a subgroup of patients with haemophilia A.
Br J Haematol
87:846, 1994[Medline]
[Order article via Infotrieve]
31.
Rudzki Z, Duncan EM, Casey GJ, Neumann M, Favaloro EJ, Lloyd JV:
Mutations in a subgroup of patients with mild haemophilia A and a familial discrepancy between the one-stage and two-stage factor VIII:C methods.
Br J Haematol
94:400, 1996[Medline]
[Order article via Infotrieve]
32.
Hathaway WE, Christian MJ, Jacobson LJ:
Variant mild hemophilia. Discrepancy in one stage and two stage factor VIII assays.
Thromb Haemost
50:357a, 1993 (abstr)
33.
Parquet-Gernez A, Mazurier C, Goudemand M:
Functional and immunological assays of FVIII in 133 haemophiliacsCharacterization of a subgroup of patients with mild haemophilia A and discrepancy in 1- and 2-stage assays.
Thromb Haemost
59:202, 1988[Medline]
[Order article via Infotrieve]
34.
Gaucher C, Jorieux S, Parquet-Gernez A, Mazurier C:
Two mutations of the factor VIII (FVIII) gene, R527W and R531H, are identified in 4 unrelated haemophilia A patients with discrepancy in 1- and 2-stage assays of FVIII coagulant activity.
Haemophilia
2:223a, 1996 (abstr, suppl 1)
35.
Montgomery RR, Hathaway WE, Johnson J, Jacobson L, Muntean W:
A variant of von Willebrand's disease with abnormal expression of factor VIII procoagulant activity.
Blood
60:201, 1982[Abstract/Free Full Text]
36.
Kroner PA, Foster PA, Fahs SA, Montgomery RR:
The defective interaction between von Willebrand factor and factor VIII in a patient with type 1 von Willebrand disease is caused by substitution of Arg19 and His54 in mature von Willebrand factor.
Blood
87:1013, 1996[Abstract/Free Full Text]
37.
Kaufman RJ:
Vectors used for expression in mammalian cells.
Methods Enzymol
185:487, 1990[Medline]
[Order article via Infotrieve]
38.
Pittman DD, Kaufman RJ:
Site-directed mutagenesis and expression of coagulation factors VIII and V in mammalian cells.
Methods Enzymol
222:236, 1993[Medline]
[Order article via Infotrieve]
39.
Michnick DA, Pittman DD, Wise RJ, Kaufman RJ:
Identification of individual tyrosine sulfation sites within factor VIII required for optimal activity and efficient thrombin cleavage.
J Biol Chem
269:20095, 1994[Abstract/Free Full Text]
40.
Zatloukal K, Cotten M, Berger M, Schmidt W, Wagner E, Birnstiel ML:
In vivo production of human factor VII in mice after intrasplenic implantation of primary fibroblasts transfected by receptor-mediated, adenovirus-augmented gene delivery.
Proc Natl Acad Sci USA
91:5148, 1994[Abstract/Free Full Text]
41.
Johnsson B, Lofas S, Lindquist G:
Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors.
Anal Biochem
198:268, 1991[Medline]
[Order article via Infotrieve]
42.
O'Shannessy DJ, Brigham-Burke M, Soneson KK, Hensley P, Brooks I:
Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: Use of nonlinear least squares analysis methods.
Anal Biochem
212:457, 1993[Medline]
[Order article via Infotrieve]
43.
Kemball-Cook G, Tuddenham EG:
The Factor VIII Mutation Database on the World Wide Web: The haemophilia A mutation, search, test and resource site. HAMSTeRS update (version 3.0).
Nucleic Acids Res
25:128, 1997[Abstract/Free Full Text]
44.
Lollar P, Parker ET, Fay PJ:
Coagulant properties of hybrid human/porcine factor VIII molecules.
J Biol Chem
267:23652, 1992[Abstract/Free Full Text]
45.
Barrowcliffe TW, Kirkwood TB:
An international collaborative assay of factor VIII clotting activity.
Thromb Haemost
40:260, 1978[Medline]
[Order article via Infotrieve]
46.
Arai M, Scandella D, Hoyer LW:
Molecular basis of factor VIII inhibition by human antibodies. Antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid.
J Clin Invest
83:1978, 1989
47.
Foster PA, Fulcher CA, Houghten RA, Zimmerman TS:
Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine.
Blood
75:1999, 1990[Abstract/Free Full Text]
48.
Saenko EL, Shima M, Rajalakshmi KJ, Scandella D:
A role for the C2 domain of factor VIII in binding to von Willebrand factor.
J Biol Chem
269:11601, 1994[Abstract/Free Full Text]
49.
Saenko EL, Scandella D:
The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von Willebrand factor.
J Biol Chem
272:18007, 1997[Abstract/Free Full Text]
50.
Fay PJ, Beattie T, Huggins CF, Regan LM:
Factor VIIIa A2 subunit residues 558-565 represent a factor IXa interactive site.
J Biol Chem
269:20522, 1994[Abstract/Free Full Text]
51.
O'Brien LM, Medved LV, Fay PJ:
Localization of factor IXa and factor VIIIa interactive sites.
J Biol Chem
270:27087, 1995[Abstract/Free Full Text]
52.
Lollar P, Parker ET:
Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog.
J Biol Chem
266:12481, 1991[Abstract/Free Full Text]
53.
Lollar P, Parker CG:
pH-dependent denaturation of thrombin-activated porcine factor VIII.
J Biol Chem
265:1688, 1990[Abstract/Free Full Text]
54.
Pipe SW, Kaufman RJ:
Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa.
Proc Natl Acad Sci USA
94:11851, 1997[Abstract/Free Full Text]
55.
Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS:
Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V.
Blood
63:486, 1984[Abstract/Free Full Text]
56.
Amano K, Michnick DA, Moussalli M, Kaufman RJ:
Mutation at either Arg336 or Arg562 in factor VIII is insufficient for complete resistance to activated protein C (APC)-mediated inactivation: Implications for the APC resistance test.
Thromb Haemost
79:557, 1998[Medline]
[Order article via Infotrieve]
57.
Koster T, Blann AD, Briet E, Vandenbroucke JP, Rosendaal FR:
Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis.
Lancet
345:152, 1995[Medline]
[Order article via Infotrieve]
58.
O'Donnell J, Tuddenham EG, Manning R, Kemball-Cook G, Johnson D, Laffan M:
High prevalence of elevated factor VIII levels in patients referred for thrombophilia screening: Role of increased synthesis and relationship to the acute phase reaction.
Thromb Haemost
77:825, 1997[Medline]
[Order article via Infotrieve]
59.
Roelse JC, Koopman MM, Buller HR, ten Cate JW, Montaruli B, van Mourik JA, Voorbert J:
Absence of mutations at the activated protein C cleavage sites of factor VIII in 125 patients with venous thrombosis.
Br J Haematol
92:740, 1996[Medline]
[Order article via Infotrieve]
Top
Abstract
Introduction
