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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4188-4196
Inhibitory Anti-Factor V Antibodies Bind to the Factor V C2
Domain and Are Associated With Hemorrhagic Manifestations
By
Thomas L. Ortel,
Karen D. Moore,
Mary Ann Quinn-Allen,
Takashi Okamura,
Allen J. Sinclair,
John Lazarchick,
Ramaswamy Govindan,
Françoise Carmagnol, and
William H. Kane
From the Departments of Medicine and Pathology, Duke University
Medical Center, Durham, NC; the First Department of Internal Medicine,
Kyushu University, Fukuoka 812, Japan; Twin Falls Clinic and Hospital,
Twin Falls, ID; the Department of Laboratory Medicine, Medical
University of South Carolina, Charleston; the Department of Internal
Medicine, Cook County Hospital, Chicago, IL; and the Laboratoire de
biologie, Cannes, France.
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ABSTRACT |
Factor V inhibitors may develop as spontaneous autoantibodies, as
alloantibodies after exposure to bovine thrombin preparations, or in
factor V-deficient patients after plasma therapy. Clinical manifestations range from asymptomatic laboratory abnormalities to
life-threatening hemorrhage. We have characterized the anti-factor V
antibodies from 12 patients diagnosed with factor V inhibitors. In 8 patients, hemorrhagic complications (5 autoantibodies and 3 bovine
thrombin-induced alloantibodies) developed, and 4 were asymptomatic (2 autoantibodies and 2 alloantibodies). The IgG fractions from all 12 patients immunoprecipitated the factor Va light chain, but only the 8 IgG fractions associated with hemorrhage inhibited factor V activity in
a prothrombinase assay. Nine IgG fractions, including the 8 patients
with hemorrhage, immunoprecipitated the isolated second
C-type domain (C2). The 8 IgG fractions from the symptomatic
patients also immunoprecipitated recombinant chimeras containing only
the N-terminal third of the factor V C2 domain, and isolated
recombinant C2 domain abrogated the inhibitory effect of the
antibodies. Five of the inhibitory IgG fractions blocked binding of
factor V to phosphatidylserine. These results suggest that inhibitory
anti-factor V antibodies are associated with hemorrhagic manifestations and frequently bind to a common region within the C2
domain, whether originating spontaneously or after exposure to bovine
thrombin.
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INTRODUCTION |
COAGULATION FACTOR V is an essential
component of the "prothrombinase complex," accelerating
activation of the zymogen prothrombin to thrombin by the serine
proteinase factor Xa in the presence of calcium ions and a phospholipid
membrane surface.1 Factor V circulates in the plasma as a
single-chain protein composed of several domains defined by primary
amino acid sequence and arranged as A1-A2-B-A3-C1-C2.1
Structurally and functionally, factor V is similar to factor VIII, an
essential component of the "factor X-ase complex" that
accelerates the activation of factor X by factor IXa in the presence of
calcium ions and a phospholipid membrane
surface.2
Acquired inhibitors to factor V may develop spontaneously as
autoantibodies in previously normal patients, generally after surgical
procedures (without exposure to topical bovine thrombin), blood
transfusions, or antibiotic administration.3 These
autoantibodies are frequently associated with hemorrhagic symptoms,
usually mild but occasionally severe.4-6 Some patients are
asymptomatic, however, and the antibodies are frequently low titer and
transient.3 Factor V inhibitors may also develop in
patients with factor V deficiency after plasma infusions for treatment
of hemorrhagic complications, although this has been reported in only
two cases.7,8
Factor V inhibitors may also develop after exposure to bovine thrombin
preparations used as topical hemostatic agents.9-12 Bovine
thrombin preparations are frequently used in vascular, orthopedic, and
neurosurgical procedures, either applied directly to the bleeding site
or as a component of fibrin glue.13 However, these thrombin
preparations frequently contain additional bovine proteins, including
bovine factor V,10 and several investigators have
demonstrated that alloantibodies developing after exposure to bovine
thrombin may cross-react with the corresponding human proteins.9,10
Several recent studies suggest that the development of factor V
inhibitors after exposure to bovine thrombin may occur considerably more frequently than previously recognized. Bänninger et
al14 reported that 11 of 24 patients treated during
cardiovascular procedures with a fibrin glue preparation consisting of
human fibrinogen and bovine thrombin subsequently developed factor V inhibitors. Similarly, Carroll et al15 observed antibodies
to bovine factor V in nine patients after treatment with a bovine fibrin hemostatic agent. Six of the alloantibodies cross-reacted with
human factor V, although none of the patients showed the development of
hemorrhagic complications.15 In addition, we have observed
that more than 80% of patients exposed to topical thrombin
preparations during cardiovascular surgery develop a seropositive
response to the specific thrombin preparation applied.16 The clinical manifestations associated with these cross-reacting alloantibodies are extremely heterogeneous, however, similar to the
clinical manifestations observed with spontaneous
autoantibodies.11,12
We previously showed that the spontaneously arising factor V inhibitor
H1 bound to a discrete region within the second C-type domain, spanning
amino acids 2037 through 2087 in the N-terminal region of the
domain.17 This inhibitor, which was associated with fatal
hemorrhagic outcome,4 blocked binding of factor V to
phosphatidylserine.18 The purpose of this study was to determine whether differences in inhibitory activity or epitope specificity of the anti-factor V antibodies isolated from an
additional 11 patients diagnosed with factor V inhibitors correlated
with the observed clinical phenotypes. In addition, we wanted to
determine whether the anti-factor V antibodies that developed as
spontaneous autoantibodies differed in inhibitory activity or epitope
specificity from the bovine thrombin-induced cross-reacting
alloantibodies.
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MATERIALS AND METHODS |
Materials.
Human thrombin, factor Xa, prothrombin, and bovine prothrombin were
from Haemotologic Technologies (Essex Junction, VT).
L-( )-phosphatidylserine (bovine brain) was from Avanti
Polar Lipids (Birmingham, AL). The chromogenic substrate S2238
(D-Phe-L-pipecolyl-Arg-p-nitroanilide) was from
Chromogenix AB (Mölndal, Sweden). Immunoaffinity-purified polyclonal rabbit anti-human factor V antibodies and the murine monoclonal antibody (MoAb) 6A5 were prepared as described
previously.17,19 The murine MoAb HV-1, which binds to the
N-terminal region of the factor V C2 domain and blocks binding to
phosphatidylserine,17 and all other reagents were from
Sigma Chemical Co (St Louis, MO).
Patients.
Plasma or serum samples, or both, were obtained from patients diagnosed
with factor V inhibitors. The clinical laboratory criteria used for
diagnosis of a factor V inhibitor included (but were not limited to)
(1) a prolonged prothrombin time (PT) and activated partial
thromboplastin time (aPTT) that did not correct when patient plasma was
mixed 1:1 with pooled normal plasma; (2) a decreased or nonmeasurable
factor V level; and/or (3) demonstration of an inhibitor to
factor V by modification of the Bethesda method.20 For
determination of the factor V inhibitor titers, pooled normal plasma
was mixed 1:1 with patient plasma and incubated at 37°C for 2 hours. Factor V activity in the patient mixture was then determined and
divided by the factor V activity in a control plasma sample, also
incubated at 37°C for 2 hours. In this assay, one "inhibitor
unit" is defined as the amount inactivating one half of the factor V
activity in the patient mixture. Additional clinical laboratory testing
performed for certain patients included thrombin clotting times, factor
II levels, and fibrinogen levels.11 Several of these
patients were also evaluated for a lupus
anticoagulant.21,22
Recombinant factor V constructs.
Construction and expression of the recombinant human factor V (rHFV)
deletion mutants and chimeras used in this study have been described
elsewhere.17,19,23 Specific constructs used for epitope
mapping are described in the legends to Figs 1 and 3. Recombinant
chimeras substituted exon-size segments of the C2 domain of factor VIII
for the corresponding regions of the C2 domain of factor
V.17 Only one of these chimeras possessed significant
activity in the prothrombinase assay (chimera 5A), and none possessed
any activity in a factor VIII chromogenic assay.17
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| Fig 1.
Epitope mapping anti-factor V antibodies with
recombinant factor V (rHFV) deletion mutants. Recombinant mutants,
depicted schematically on the left side of the figure, included a
mutant lacking amino acids 811 through 1491 of the B domain (rHFV des B), and a mutant lacking amino acids 811 through 1491 and residues Gly-2037 through Tyr-2196 of the light chain (rHFV des B/C2). The
isolated heavy chain consists of amino acids Ala-1 through Arg-709
(rHFV HC); the isolated light chain consists of amino acids Ser-1546
through Tyr-2196 (rHFV LC); the isolated A3 domain consists of amino
acids Ser-1546 through Arg-1877 (rHFV A3); and the isolated C2 domain
consists of amino acids Gly-2037 through Tyr-2196 (rHFV C2). The IgG
fractions purified by protein A-Sepharose chromatography are listed on
the right side of the figure. Immunoprecipitation of
[35S]methionine metabolically labeled mutants and
analysis by SDS-PAGE was performed as previously
described.23 +, the IgG fraction immunoprecipitated the
specific factor V mutant;  , it did not. ND, not done.
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| Fig 3.
Epitope mapping anti-factor V antibodies with
recombinant factor V C2 domain chimeras. The complete domain structure
of rHFV des B is shown at the top, and the expanded domain structures of the light chain portions of the individual chimeras are shown below.
The white boxes indicate factor V-derived sequences, and the shaded
boxes indicate factor VIII-derived sequences. Chimera 1A substituted
amino acids 2282 through 2332 of factor VIII for amino acids 2149 through 2196 of factor V. Chimera 2A substituted amino acids 2223 through 2332 of factor VIII for amino acids 2088 through 2196 of factor
V. Chimera 3A substituted amino acids 2173 through 2332 of factor VIII
for amino acids 2037 through 2196 of factor V. Chimera 5A substituted
amino acids 2173 through 2222 of factor VIII for amino acids 2037 through 2087 of factor V. Chimera 7A substituted amino acids 2223 through 2281 of factor VIII for amino acids 2088 through 2148 of factor
V. The antibodies that immunoprecipitated the factor V C2 domain are
listed on the right side. Immunoprecipitation of
[35S]methionine metabolically labeled mutants and
analysis by SDS-PAGE was performed as described in Materials and
Methods. +, the antibody immunoprecipitated the specific factor V
mutant; , it did not.
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Recombinant C2 domain was obtained for protein purification by
subcloning the cDNA encoding the factor V C2 domain into the baculovirus transfer vector pBluebac2. Sf9 cells were cotransfected with rHFV C2 and WT Autographa californica nuclear
polyhedrosis virus, and recombinant baculovirus was obtained by plaque
purification. Insect cells were infected in spinner flasks, and
recombinant C2 domain was purified to homogeneity from conditioned
media using cation-exchange chromatography on Mono S (Amersham
Pharmacia Biotech, Piscataway, NJ).24
Purification of IgG fractions.
Plasma or serum samples, or both, were fractionated by affinity
chromatography on Protein A Sepharose (Amersham Pharmacia Biotech), as previously described.18 The IgG
fractions were concentrated using a microconcentrator (Centricon 30;
Amicon, Beverly, MA), and both binding and nonbinding fractions were
assessed for inhibitory activity.
Purification of anti-factor V C2 domain antibodies.
Two plasma samples (RS and H5) were also fractionated by affinity
chromatography with Sepharose to which purified rHFV C2 domain had been
coupled. For patient RS, the Protein A Sepharose binding fraction from
0.5 mL of plasma was dialyzed into 10 mmol/L Tris-HCl, pH 7.5/0.15
mol/L NaCl, concentrated, and then applied to a C2 domain affinity
column ( 0.9 mL; 8.62 mg/mL). The resin was then serially developed
with 100 mmol/L glycine-HCl, pH 2.5; 100 mmol/L triethylamine, pH 11.5;
and 50 mmol/L Tris-HCl, pH 7.4/50% ethylene glycol. After each buffer
change, 10 mL of elution buffer flow-through was pooled, dialyzed
into phosphate-buffered saline, pH 7.4, and concentrated to 0.5 mL
final volume on a CM30 microconcentrator (Amicon). Protein
concentration was determined by absorbance at 280 nm. For patient H5,
initial fractionation on Protein A Sepharose was not performed because
this fractionation step did not isolate significant inhibitory activity
from H5 plasma (see Results). Instead, 10 mL of H5 plasma was diluted
1:10 into 10 mmol/L Tris-HCl, pH 7.5, and applied directly to the C2
domain affinity column. The resin was then serially developed as
described for patient RS. For a negative control, 10 mL of citrated
plasma from a single normal donor was fractionated on the C2 domain
affinity column, as described for patient H5.
Immunoprecipitation of factor V deletion mutants and chimeras.
Immunoprecipitation analyses of [35S]methionine
metabolically labeled factor V mutants and sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed as
previously described.23 Immunoprecipitation of
metabolically labeled factor V mutants with (1) an
immunoaffinity-purified polyclonal rabbit anti-human factor V antibody,
and (2) a Protein A Sepharose IgG preparation from pooled normal
plasma, were used as positive and negative controls, respectively.
Prothrombinase assays.
Factor Va activity in the presence of IgG from inhibitor plasmas was
determined using a chromogenic prothrombinase assay, as previously
described.18 Briefly, the factor V sample (rHFV des B or
Russell's viper venom-activated rHFV) was incubated with patient IgG
and then added to 50 mmol/L Tris HCl, pH 7.9, 175 mmol/L NaCl, 5 mg/mL
bovine serum albumin (BSA), containing either rabbit brain cephalin
(8.0% vol/vol) or 25% phosphatidylserine/75% phosphatidylcholine
vesicles (0.8 mmol/L), 0.20 µmol/L prothrombin, and 8.0 mmol/L CaCl2. Preliminary experiments confirmed that
inhibition occurred rapidly and was complete within 15 minutes for
those IgG fractions containing inhibitory activity. The prothrombinase reaction was subsequently initiated by the addition of 0.2 nmol/L factor Xa and then stopped after 2 minutes by the addition of 20 mmol/L
EDTA. Aliquots of the reaction mixtures were transferred to microtiter
plate wells, and the amount of thrombin generated during the reactions
was determined by adding 0.2 mmol/L S2238 and measuring the change in
absorbance at 405 nm using a Vmax microtiter plate reader
(Molecular Devices, Menlo Park, CA).18
For inhibition of chimera 5A, conditioned media containing the
recombinant chimera (13 mU/mL) was preincubated with each of the
factor V inhibitors (50 µg/mL) for 15 minutes at 37°C. Residual
activity was then determined in the prothrombinase assay using rabbit
brain cephalin as the phospholipid membrane source, as described above.
For the chromogenic assay, 1 unit is defined as the amount of factor V
activity present in 1 mL of activated pooled human
plasma.19
Phospholipid binding enzyme-linked immunosorbent assay (ELISA).
The ability of anti-factor V antibodies to interfere with the binding
of recombinant factor V to phosphatidylserine (PS) was investigated
using a solid-phase ELISA, as previously described.23 Briefly, microtiter plate wells were coated overnight with 3 µg/mL PS
in methanol and then blocked with 0.5% gelatin.23 Aliquots of conditioned media containing recombinant factor V des B (0.7 nmol/L) were first incubated for 15 minutes at 37°C with IgG
fractions from the individual patients, an IgG fraction from pooled
normal plasma, or the MoAb HV-1, followed by 1-hour incubation of the conditioned media-antibody mixture in the PS-coated microtiter plate
wells. The 15-minute preincubation step with the antibodies was
included for consistency with the prothrombinase assays. Binding of
factor V to PS was determined with either biotin-labeled MoAb 6A5, or
biotinylated immunoaffinity-purified rabbit polyclonal anti-human
factor V, as previously described.17
Anti-bovine prothrombin ELISA.
Bovine prothrombin (5 µg/mL in PBS, pH 7.4) was coated onto 96-well
microtiter plates (Costar EIA, Cambridge, MA; 50 µL/well) and
incubated overnight at 4°C. Unbound protein was removed by washing
the wells three times with 0.1% Tween 20/phosphate-buffered saline, pH
7.4 (PBS). Wells were then blocked by the addition of 200 µL/well of
1% BSA/0.1% Tween 20/PBS (block buffer) for 2 hours at 22°C.
Patient plasma or purified IgG samples were diluted in the block buffer
and 100-µL aliquots were incubated in each well for 2 hours at
22°C. Pooled normal plasma was used to determine nonspecific
antibody binding. Bound IgG was detected by incubation with a goat
-human IgG peroxidase conjugate (1/500 dilution into block buffer;
100 µL/well; 1 hour at 22°C) followed by peroxidase substrate.
Absorbance was measured at 405 nm, as described above.
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RESULTS |
Patients.
Clinical laboratory results obtained on the 12 patients investigated in
this study are summarized in Table 1. Eight
patients presented with hemorrhagic manifestations, ranging from oozing around a vascular graft site (EM) to fatal exsanguination (H1). Six of
these patients had been treated with antibiotics, five with
cephalosporins (patients H3, RB, AR, EM, and RH) and one with an
aminoglycoside antibiotic (H1).4 Patient RB initially developed hematuria and gastrointestinal bleeding after a course of
therapy with cephradine. The symptoms resolved with discontinuation of
the antibiotic, only to recur 2 years later after exposure to
cephalexin. Two of the patients had been treated with bovine thrombin,
either as a topical hemostatic agent (RS) or as a component of fibrin
glue (EM). In a third patient, hemorrhagic symptoms developed after
coronary artery bypass grafting (WH), during which bovine thrombin is
frequently used.
Four patients presented with abnormal coagulation studies in the
absence of any hemorrhagic manifestations (inhibitors H4, H5, MJ, and
BH). Two patients (H5 and MJ) had been recently treated with bovine
thrombin preparations,11,25 and one (H5) had also been
treated with an antibiotic (Table 1). Patient H4 presented with a
pleural effusion related to congestive heart failure and underwent
thoracentesis without bleeding. Patient BH presented with chest pain
and was found to have abnormal coagulation studies during a
precatheterization evaluation. Neither patient H4 nor BH had been
previously exposed to bovine thrombin or antibiotics. These four
patients presented with prolonged PT and aPTT results that did not
correct when mixed 1:1 with normal plasma, markedly decreased factor V
levels, and low-titer factor V inhibitors by Bethesda analysis in two
of the patients (Table 1).
IgG isolated from patients diagnosed with factor V inhibitors contain
antibodies that bind to the factor Va light chain.
Protein A Sepharose was used to purify the IgG fractions from these 12 patients. Anti-factor V antibodies were shown in the IgG fraction from
each patient by ELISA (data not shown) and by immunoprecipitation
analysis (Fig 1). None of the IgG fractions contained antibodies that bound to the heavy chain of factor V (rHFV
HC), but all 12 IgG fractions immunoprecipitated recombinant constructs
containing an intact light chain (rHFV; rHFV des B; and rHFV LC; Fig
1).
Recombinant factor V light chain deletion mutants were used to further
delineate the epitopes recognized by these anti-factor V antibodies.
All eight IgG fractions from the symptomatic patients (H1, H3, RS, RB,
WH, AR, EM, and RH), as well as the IgG fraction from one of the
patients without bleeding (BH) immunoprecipitated the isolated C2
domain (rHFV C2). The IgG fraction from one of the patients without
hemorrhagic symptoms (MJ) immunoprecipitated the light chain deletion
mutant lacking the C2 domain (thrombin-activated rHFV des B/C2) but not
the isolated A3 domain (rHFV A3), suggesting a possible epitope in the
C1 domain (Fig 1). The other two IgG fractions (H4 and H5) did not
immunoprecipitate any construct smaller than the entire light chain
(Fig 1).
Factor V activity is inhibited by the IgG fractions from patients
with hemorrhagic symptoms.
The IgG fractions purified from the eight patients with hemorrhagic
manifestations all inhibited the activity of rHFV des B in a
prothrombinase assay (Fig 2). In general,
the severity of the hemorrhagic complications appeared to correlate
with the amount of inhibitory activity in the patient plasma. Three of the IgG fractions did not completely inhibit rHFV des B activity, however, with 25% to 50% residual activity at IgG concentrations in excess of 200 µg/mL (patients H3, RH, and EM; Fig 2).
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| Fig 2.
Inhibition of activity of recombinant human factor V des
B (rHFV des B) by IgG fractions. The individual IgG preparations were
incubated with 3 nmol/L rHFV des B in conditioned media at the
concentrations shown for 15 minutes at 37°C. The sample was then
diluted 1:10 into 50 mmol/L Tris-HCl, pH 7.9, 175 mmol/L NaCl, 5 mg/mL
BSA, and residual activity was determined by the chromogenic
prothrombinase assay as described in Materials and Methods. The IgG
fractions were H1 ( ); H3 ( ); RS ( ); RB ( ); WH ( ); AR
( ); EM ( ); RH ( ); H4 ( ); H5 ( ); MJ ( ); and BH ( ).
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By contrast, the IgG fractions purified from the four patients with no
hemorrhagic manifestations had essentially no effect on the activity of
rHFV des B (Fig 2; patients H4, H5, MJ, and BH), although antibody
binding to factor V at concentrations used in the functional assay
could be documented by ELISA (data not shown). The Protein A Sepharose
nonbinding fractions from these patient plasmas also had no inhibitory
effect in this assay, indicating that these patients did not have an
IgM (or other non-protein A Sepharose binding isotype) inhibitory
antibody (data not shown). Similar results were obtained with these IgG
fractions and thrombin-activated factor Va, indicating that the absence
of inhibitory activity was not due to a unique property of the B domain
deletion mutant (data not shown).
Inhibitory IgG fractions contain anti-factor V antibodies that bind
to the N-terminal region of the C2 domain.
We further characterized the nine anti-factor V antibodies that bound
to the C2 domain (H1, H3, RS, RB, WH, AR, EM, RH, and BH) by using
recombinant factor V/factor VIII C2 domain chimeras (Fig 3). The immunoprecipitation results
for patient RS are shown in Fig 4. The IgG
fraction from patient BH immunoprecipitated only the WT C2 domain and
none of the chimeras (Fig 3). By contrast, the eight IgG fractions
associated with hemorrhagic manifestations (H1, H3, RS, RB, WH, AR, EM,
and RH) immunoprecipitated those chimeras containing factor VIII
sequences substituted for the central and C-terminal thirds of the
domain (eg, chimeras 1A and 2A), but not those chimeras that
substituted the N-terminal portion of the factor VIII C2 domain for the
corresponding segment of the factor V C2 domain (eg, chimeras 3A and
5A; Fig 3). The anti-factor V antibody RH differed slightly from the
other inhibitory antibodies in that it did not immunoprecipitate
chimera 7A, which substitutes only the central 59 amino acids of the
factor VIII C2 domain for the corresponding region of the factor V C2
domain (Fig 3).
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| Fig 4.
Immunoprecipitation of thrombin-activated C2 domain
chimeras by IgG fraction from patient RS. Recombinant factor V
constructs metabolically labeled with [35S]methionine
included rHFV des B, chimera 1A, chimera 2A, chimera 3A, chimera 7A,
and chimera 5A. Samples were first incubated with 2 nmol/L thrombin for
1 minute at 37°C and then immunoprecipitated with either the
polyclonal rabbit anti-human factor V antibody or the IgG fraction from
patient RS, as shown. The immunoprecipitates were analyzed by 6%
SDS-PAGE and visualized by autoradiography. A nonspecific,
high-molecular-weight band (>200 kD) is seen in all the
immunoprecipitations of conditioned media, including the control IgG
fraction from pooled normal plasma. Molecular-weight standards are
shown on the left, and the positions of the factor Va heavy chain and
light chain on the right.
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Recombinant factor V C2 domain neutralizes inhibitory anti-factor V
antibodies.
To determine whether antibody binding to the C2 domain in the intact
protein resulted in the inhibition of activity, we investigated whether
preincubation of the IgG fractions with purified recombinant factor V
C2 domain could neutralize the inhibitory activity. As shown in
Fig 5, preincubation of the IgG fraction
from patient RB with the isolated C2 domain completely abrogated the
inhibitory effect of the IgG fraction on the activity of factor V. Similar results were obtained with all of the inhibitory anti-factor V antibodies (data not shown).
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| Fig 5.
Neutralization of the inhibitory anti-factor V
antibodies from patient RB by recombinant human factor V C2 domain.
Purified IgG from patient RB (5 µg/mL) was incubated with purified
recombinant factor V C2 domain at the concentrations shown for 15 minutes at 37°C. Conditioned media containing rHFV des B (0.5
nmol/L) was then incubated with either the inhibitor IgG/C2 domain
mixture () or the same concentration of the C2 domain alone ()
for 15 minutes at 37°C. Residual activity was then determined by
the chromogenic prothrombinase assay, as described in Materials and Methods.
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Inhibitory anti-factor V antibodies do not inhibit a recombinant
chimera that substitutes the N-terminal portion of the factor VIII C2
domain for the homologous region of the factor V C2 domain.
The recombinant chimera 5A possesses 20% of the activity of factor
V des B and is resistant to the inhibitory effect of the IgG fraction
from patient H1.17 Similarly, this chimera was also
resistant to the IgG fractions from all of the patients with hemorrhagic complications (data not shown), consistent with the immunoprecipitation data obtained with the
[35S]methionine metabolically labeled factor V mutants
(Fig 3). This chimera was also not inhibited by any of the IgG
fractions from the nonhemorrhagic patients (H4, H5, MJ, and BH; data
not shown).
Inhibition of factor V binding to phosphatidylserine by IgG
fractions.
The IgG fractions from patients H1, RS, and RB consistently inhibited
binding of rHFV des B to immobilized PS (<25% residual binding at
IgG concentrations 50 mg/mL; Fig 6 and data
not shown). The IgG fractions from patients WH and AR partially
inhibited binding of rHFV des B to immobilized PS (25% to 50%
residual binding at IgG concentrations  50 µg/mL; Fig 6). By
contrast, the IgG fractions from the patients H3, RH, and EM had
minimal or no inhibitory effect on binding to PS (>50% residual
binding at IgG concentrations 50 µg/mL; Fig 6 and data not shown).
These three IgG fractions also did not completely neutralize the
activity of rHFV des B in the prothrombinase assay (Fig 2). Of the IgG
fractions from the patients without hemorrhagic symptoms, H4 partially
blocked binding to PS (25% to 50% residual binding at IgG
concentrations 500 µg/mL; Fig 6). The other three IgG fractions
(H5, MJ, and BH), however, had no effect on the binding of factor V to
PS (>75% residual binding at IgG concentrations 500 µg/mL; data
not shown).
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| Fig 6.
Inhibition of rHFV des B binding to phosphatidylserine by
IgG fractions. Recombinant factor V des B was incubated with the IgG
fractions at the concentrations shown for 15 minutes at 37°C, and
then incubated in the PS-coated wells as described in Materials and
Methods. Binding to PS was determined by ELISA, as previously described.18 The IgG fractions shown are: H1 (); H3
(); RS (); RB (); WH (); AR (); EM (); RH (); and
H4 (). Controls include the IgG fraction from pooled normal plasma
( ) and the murine MoAb HV-1 ( ).
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Plasma from patient H5 does not contain inhibitory anti-C2 domain
antibodies in low titer.
One possible explanation for the differences in the inhibitory activity
of the IgG fractions from the hemorrhagic and nonhemorrhagic patients
is that the latter simply contain lower titers of inhibitory anti-C2
domain antibodies. To address this possibility, plasma samples from
patients RS and H5 were fractionated by C2 domain affinity
chromatography, as described in Materials and Methods. For patient RS,
all inhibitory activity (from an initial 0.5 mL of patient plasma)
bound to the C2 domain affinity column and was eluted with 100 mmol/L
glycine-HCl, pH 2.5 (Fig 7). The total amount of bound IgG that was eluted from the C2 column was 45 µg,
or 0.6% of the total IgG loaded on the column.
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| Fig 7.
Inhibition of factor V des B activity by factor V C2
domain-affinity purified RS and H5 fractions. The Protein A Sepharose binding fraction from 0.5 mL RS plasma and 10 mL of unfractionated H5
plasma were applied to 1 mL of factor V C2 domain-Sepharose and
serially eluted with glycine-HCl (elution step 1), triethylamine (elution step 2), and ethylene glycol (elution step 3), as described in
Materials and Methods. A total of 10 1-mL fractions after each
buffer change were pooled, concentrated to a final volume of 0.5 mL,
and then evaluated for inhibitory activity in the prothrombinase assay.
Residual factor V activity is plotted against the individual elution
steps for the RS ( ) and H5 ( ) fractions.
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For patient H5, 10 mL of plasma was diluted into the starting buffer
and applied to the C2-domain column. A total of 140 µg of protein
was eluted with 100 mmol/L glycine-HCl, pH 2.5, but no inhibitory
activity was present in this fraction (Fig 7). SDS-PAGE showed
predominantly two bands consistent with Ig heavy and light chains,
respectively (data not shown). As with patient RS, essentially no
protein was eluted with either the triethylamine or ethylene glycol
buffers. Fractionation of a second 10-mL plasma sample from patient H5
confirmed the presence of a noninhibitory fraction that could be eluted
with the glycine-HCl buffer. By contrast, fractionation of 10 mL of
plasma from a normal donor on the C2 domain affinity column resulted in
all of the protein remaining in the nonbinding fraction, with no
protein being obtained from any of the elution steps (data not shown).
Insufficient plasma samples from patients MJ, BH, and H4 were available
for similar analyses.
Binding to bovine prothrombin distinguishes spontaneous
autoantibodies from topical thrombin-associated alloantibodies.
None of the above data would distinguish patients in whom anti-factor
V antibodies developed after exposure to bovine thrombin preparations
from patients with spontaneous inhibitors. To determine this, we tested
for the presence of antibodies to bovine prothrombin in the patient
plasma samples. All four patients in whom anti-factor V antibodies
developed after exposure to bovine thrombin also manifested antibodies
to bovine prothrombin (Fig 8). In addition, patient WH, who had previously undergone coronary artery bypass grafting (CABG), had antibodies to bovine prothrombin, suggesting that
topical bovine thrombin was likely used during this patient's surgical
procedure (Fig 8). By contrast, none of the spontaneous anti-factor V
antibody plasmas contained antibodies to bovine prothrombin (Fig
8).
View larger version (22K):
[in this window]
[in a new window]
| Fig 8.
Anti-bovine prothrombin antibodies in patients with
antibodies to factor V. Plasma or sera samples from the patients were diluted into block buffer and incubated in microtiter plate wells coated with bovine prothrombin (for patient WH, a 1:100 dilution of the
IgG fraction was used instead of plasma). Antibody binding to bovine
prothrombin was detected with a peroxidase-conjugated goat anti-human
IgG antibody. The IgG fractions were: H1 (); H3 (); RS (); RB
(); WH (); AR (); EM (); RH (); H4 (); H5 (); MJ
(); and BH ().
|
|
| |
DISCUSSION |
The clinical heterogeneity associated with factor V inhibitors was
exemplified by the 12 patients included in this study (Table 1). Eight
patients manifested hemorrhagic symptoms during their clinical course
(H1, H3, RS, RB, WH, AR, EM, and RH), whereas four patients had no
bleeding complications (H4, H5, MJ, and BH). Seven patients presented
with spontaneous autoantibodies (H1, H3, RB, AR, RH, H4, and BH),
whereas in five patients cross-reacting alloantibodies developed after
exposure to topical bovine thrombin (RS, WH, EM, H5, and MJ). Seven
patients developed antibodies to factor V in the setting of antibiotic
therapy (H1, H3, RB, AR, EM, RH, and H5), with one patient (RB)
developing a factor V inhibitor on two separate occasions after
treatment with different cephalosporin antibiotics (both
first-generation cephalosporins that are similar in structure).
All 12 patients clearly had anti-factor V antibodies (Fig 1). However,
even though anti-factor V antibodies were present, only the IgG
fractions prepared from the patients with hemorrhagic symptoms
inhibited the activity of factor V in the prothrombinase assay. By
contrast, the IgG fractions (as well as the Protein A Sepharose
nonbinding fractions) from the asymptomatic patients were noninhibitory
in the prothrombinase assay (Fig 2). IgG binding to factor V at the
concentrations used in the prothrombinase assays was confirmed by ELISA
for the noninhibitory fractions, but this binding may not have
interfered with prothrombinase function or have been weaker than the
interactions of factor V with the other components of the
prothrombinase complex. Two of the patients with noninhibitory
anti-factor V antibodies did have a measurable inhibitor titer,
however, as determined by a modified Bethesda assay (patients MJ and
BH; Table 1). This may reflect differences in assay methodology
(plasma-based v purified proteins) or the fact that plasma
samples for this study were not necessarily collected on the same day
as determination of the inhibitor titer.
The anti-factor V antibodies from all 12 patients bound to the light
chain of factor V (Fig 1). Factor V inhibitors binding to the light
chain have also been described in two other patients, both associated
with hemorrhagic manifestations.6,9 A spontaneously occurring factor V inhibitor that bound to the heavy chain of factor Va
has also been described.26 This spontaneous autoantibody was identified in a patient presenting with extensive ecchymoses, hematuria, and melena.27 None of the patients we studied
had anti-factor V antibodies that bound to the heavy chain or to the large connecting region (B domain).
The IgG fractions from the eight patients with hemorrhagic
manifestations could be further shown to bind to the
N-terminal region of the C2 domain of the light chain,
encoded by exon 23 of the factor V gene.28 By contrast, two
of the IgG fractions from the four asymptomatic patients
immunoprecipitated only the recombinant factor V constructs containing
the entire light chain (H4 and H5; Fig 1), and one of the IgG fractions
appeared to bind to the C1 domain of the light chain (MJ). The IgG
fraction from the fourth patient (BH) immunoprecipitated the isolated
C2 domain but did not immunoprecipitate any of the recombinant C2
domain chimeras (Fig 3). Although the IgG fraction from patient H5 did not immunoprecipitate the C2 domain, a C2 domain binding fraction could
be obtained from 10 mL of patient plasma by immunoaffinity chromatography on a C2 domain column (Fig 7). Nevertheless, neither the
IgG fraction from patient BH nor the C2 domain binding fraction from
patient H5 inhibited the activity of factor V in the prothrombinase assay. These results suggest that noninhibitory as well as inhibitory antibodies bind to the C2 domain of factor V.
We have previously shown that binding of factor V to immobilized PS
requires the presence of the C2 domain, and that the N-terminal portion
of the domain is involved in this binding.17,23 The IgG
fractions from five of the patients with hemorrhagic symptoms bound to
this region of the C2 domain and at least partially blocked binding of
rHFV des B to PS (H1, RS, RB, WH, and AR; Fig 6). The IgG fractions
from the other three patients with hemorrhagic symptoms bound to the
same region of the C2 domain but did not block rHFV des B binding to PS
(H3, EM, RH). These three IgG fractions also did not completely inhibit
factor V activity in the prothrombinase assay (Fig 2), which may have
been due to lower antibody titers or affinity.
By contrast, although the IgG fraction from patient BH
immunoprecipitated the C2 domain, it did not inhibit binding of rHFV des B to PS. The MoAb 6A5 also binds to the C2 domain and does not
inhibit binding of rHFV des B to PS, although it is inhibitory in the
prothrombinase assay.17 We have previously shown that the
IgG fraction from patient H1 and the MoAb 6A5 bind to nonoverlapping epitopes in the C2 domain,17 and it is possible that the
IgG fraction from patient BH recognizes a separate region in this domain. Interestingly, although the IgG fraction from patient H4
neither immunoprecipitated the C2 domain nor inhibited factor V in the
prothrombinase assay, it did partially inhibit binding of factor V to
PS. A second PS-binding site has been identified in the A3 domain of
the factor Va light chain,29 but the IgG fraction from this
patient did not immunoprecipitate the A3 domain either. Therefore, the
mechanism for this inhibition is unknown.
Factor VIII binding to PS also involves the second C-type domain of the
light chain, but, in contrast to factor V, the C-terminal portion of
the factor VIII C2 domain appears to be involved in binding.30 Factor VIII inhibitors that bind to the C2
domain of factor VIII disrupt binding of factor VIII to anionic
phospholipids31 and/or von Willebrand
factor.32 Certain factor VIII inhibitors that bind to the
C-terminal region of the factor VIII C2 domain recognize an epitope
that overlaps with the putative phospholipid-binding site.33 However, we have recently identified several factor VIII inhibitors that bind to the N-terminal region of the C2 domain and
interfere with the binding of factor VIII to PS,34 similar to the corresponding region in the factor V C2 domain recognized by
factor V inhibitors. Clarification of the mechanism(s) whereby antibodies that bind to the C2 domains of factor V or factor VIII result in inhibition of procoagulant activity and PS binding of the
respective cofactors will require fractionation of antibody subsets and
detailed functional characterization.
Finally, this study showed that inhibitory anti-factor V antibodies
associated with hemorrhagic symptoms bound to a common epitope within
the C2 domain of the factor Va light chain, regardless of whether the
antibody developed spontaneously or after exposure to bovine thrombin.
Nevertheless, bovine thrombin-associated factor V inhibitors could be
clearly distinguished from spontaneous autoantibodies by the presence
of antibodies to bovine prothrombin (Fig 7). It is possible that the
hemorrhagic symptoms manifested by patients exposed to bovine thrombin
may require additional cross-reacting alloantibodies to human
coagulation proteins, although this has not been previously observed.
| |
FOOTNOTES |
Submitted July 30, 1997;
accepted January 26, 1998.
Supported in part by Grant-In-Aid No. NC-93-GS-17 from the American
Heart Association North Carolina Affiliate (T.L.O.), a Basil O'Connor
Starter Scholar Research Award (No. FY96-0556) from the March of Dimes
Birth Defects Foundation (T.L.O.), and by National Institutes of Health
Grant No. HL43106 (W.H.K.). T.L.O. is a Pew Scholar in the Biomedical
Sciences.
Presented in part at Biomedicine '96, American Federation for Clinical
Research, Washington, DC, May 3-6, 1996.
Address reprint requests to Thomas L. Ortel, MD, PhD, Divisions of
Hematology and Oncology, Department of Medicine, Duke University Medical Center, Box 3422, Durham, NC 27710.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank the Duke Clinical Coagulation Laboratory for performing the
factor V assays and inhibitor titers on plasma samples from these
patients. We also thank John Brandt, David McGlasson, W. Muntean, and
W.A. Dittman for providing us with plasma samples from patients AR, RH,
H5, and BH, respectively.
| |
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Abstract
Introduction
Abstract