Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DIAGNOSTIC TEST FOR THROMBOTIC
OR THROMBOEMBOLIC DISEASE
S
Background of the Invention
The invention is designed to improve the medical care of patients with
thrombotic or thromboembolic disease, such as deep venous thrombosis (DVT)
and pulmonary embolism (PE), by facilitating clinical diagnosis and by
providing a means by which the effectiveness of treatment can be measured.
The most challenging aspect of caring for patients with DVT and PE
concerns making the initial diagnoses. In fact, most PE fatalities occur
before
the disease has been detected. Clinical signs and symptoms are neither
sensitive
nor specific; and the subsequent evaluative process for diagnosing DVT and/or
PE is time consuming, expensive, and potentially invasive. The non-invasive
diagnostic techniques for these diseases focus on demonstrating defects in the
vascular anatomy, findings that are not specific for active thrombosis. For
example, compression ultrasonography (CUS) can be used to detect pathology in
the femoral vein. However, the distinction between a new thrombus and focal
wall thickening from a previous thrombus cannot be made reliably. In addition
CUS cannot be used to reliably detect asymptomatic DVT. As a result, attention
has been focused on the identification of a serologic marker that would
indicate
active thrombosis.
In the last decade, the utility of plasma D-Dimer levels for identifying
thromboembolic disease has been investigated extensively. D-Dimers are
formed as a result of the degradation of cross-linked fibrin, and plasma
levels
have been shown to be elevated in both DVT and PE. However, plasma D-
Dimer levels reflect the rate of fibrinolytic activity, but not necessarily
the rate of
fibrin formation. As a result, plasma D-Dimer levels are elevated in a variety
of
pathologic conditions involving previous fibrin formation, such as sepsis,
DIC,
pneumonia, and malignancy. In fact, only 22% of medical inpatients
(presumably without thomboembolic disease) do not have elevated D-Dimer
levels.
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Although anticoagulants have been used for decades to treat
thromboembolic disease, venous thromboembolism (VTE) in particular, the ideal
method of treating this disease is unresolved. It is generally accepted that
early
anticoagulation dramatically reduces short-term mortality (Douketis et al.
1998),
and it is becoming apparent that the incidence of long-term sequelae such as
recurrent DVT and PE are also dependant upon the intensity of treatment in the
first few days after diagnosis (Hull et al. 1997). The optimal method of early
anticoagulation for VTE is, however, a controversial issue. New anti-
thrombotic
strategies are constantly in development, including improved dosing regimens
for unfractionated heparin (Raschke et al. 1993, Lopaciuk et al. 1992, Hirsch
et
al. 1996) , low molecular weight heparins (Levine et a1.1996, Koopman et al.
1996m Meyer et al. 1995) and specific inhibitors of the coagulation enzymes
thrombin (Verstraete 1997) and factor Xa (Walenga et a1.1997). Each regimen
has a specific anti-thrombotic potency, defined as its ability to suppress in
situ
thrombus propagation. It is likely that the benefits of these newer
anticoagulant
strategies will depend on the relationship between early anti-thrombotic
effects
and long-term clinical outcomes.
Although anticoagulants may have different mechanisms of action, the
ultimate biochemical goal is the same, to prevent thrombin-mediated conversion
of fibrinogen to fibrin and thus stop thrombus propagation (anti-thrombosis).
Unfortunately, the anticoagulant potencies of these medications, measured by
in
vitro tests of activity such as the activated partial thromboplastin time
(aPTT)
and the plasma anti-Xa activity, do not reliably predict their anti-thrombotic
effects in animal models (Carrier et al. 1993), Carner et al. 1992, Morris et
al.
1998).
There is growing recognition that inadequate initial treatment of VTE
predisposes to fatal pulmonary emboli (Dalen 1986) and long-term recurrence
(Hull 1997). However, there are limitations to the data suggesting that low
antithrombotic activity itself in the early treatment of VTE leads to poor
clinical
outcomes. For example, anticoagulant activities of patients receiving
unfractionated heparin are generally measured using the plasma aPTT, which has
only a moderate correlation with actual plasma heparin levels (Gawoski et
a1.1987, Brandt et al. 1981, van den Besselaar et a1.1990)). Furthermore, even
the moderate correlation between anticoagulant activity and anti-thrombotic
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effect observed in animal models of thrombosis has not been validated in
humans. Finally, the assumption that the intensity of anti-thrombosis
correlates
with the clinical efficacy, though reasonable, has not been tested in humans.
For
example, there have been no clinical studies to correlate VTE recurrence with
the
anti-thrombotic effects of anticoagulation.
Measuring the anti-thrombotic effects of anticoagulants in humans with
VTE is difficult. The most commonly used non-invasive tests for diagnosis of
DVT (compression ultrasonography, impedance plethysmography and magnetic
resonance imaging) and PE (ventilation-perfusion scanning and helical CT
scanning) do not provide sufficient anatomical information to determine
reliably
whether thromboemboli have enlarged acutely. Invasive studies such as contrast
venography and angiography, while better at demonstrating gross changes in
thrombus size (Lopaciuk et a1.1992, National Heart 1970) can be painful and
are
often impractical for following treatment. Furthermore, they may not be able
to
detect subtle increases in clot dimensions due to ongoing thrombosis. Finally,
all of the anatomical tests described above share the limitation of being
unable to
differentiate the effects of anticoagulation (preventing clot enlargement)
from the
effects of the intrinsic fibrinolytic system (reducing clot size).
The most commonly used serological test for VTE, the D-dimer test, is
also unsuitable as a marker of acute thrombosis. Although increasingly
recognized as a sensitive indicator of VTE, the test measures thrombolytic
fragments from pre-existing clots, and would not correlate with thrombus
propagation. Likewise, serum markers of thrombin activation, such as
prothrombin F 1+2 fragments and thrombin-antithrombin III complexes, are not
direct indicators of fibrinogen) conversion and polymerization. Thus
anticoagulants with different spectra of activity against factor Xa and
thrombin
(for example heparin pentasaccharide and hirudin) would be expected to affect
these tests differently, even if their in vivo and anti-thrombotic effects
were the
same.
Therefore, an ongoing need exists for a reliable test for DVT and PE, and
also for a test to determine the effectiveness of different therapeutic
regimens.
Also the discovery of a marker with sufficient specificity and sensitivity in
detecting PE and/or DVT would aid in diagnostic accuracy, and facilitate cost-
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effective utilization of resources. Thus, only those patients with a positive
test
would require anticoagulation and further evaluation with the appropriate
tests.
Summar~of the Invention
The present invention provides a method for detecting thrombotic or
thromboembolic disease, such as PE and/or DVT, by measuring the levels of
fibrinopeptide B (FPB) in a physiological sample. The sample may be blood,
plasma or, preferably, urine. The present invention also provides methods for
monitoring the treatment of thrombotic or thromboembolic disease in a patient
by monitoring changes in the levels of FPB in blood, plasma or, preferably,
urine. The present invention also provides assay methods for conducting these
measurements. The invention also provides peptides that include sequences
from FPB; these peptides may be used as calibrators or controls in assays for
FPB, they may be linked to carrier proteins and used to generate antibodies
against FPB and/or they may be linked to labels or solid phases and used as
competitors in competitive assays for FPB. The invention also provides
reagents, compositions, and kits for carrying out immunoassays for FPB.
The present invention provides a fibronopeptide B (FPB) peptide defined
by an amino acid sequence indicated in SEQ ID NO:1, and an FPB peptide
defined by an amino acid sequence indicated in SEQ ID N0:2. These peptides
may be covalently linked to a carrier molecule, such as keyhole limpet
hemocyanin (KLH). Also these peptides and derivatives thereof may be
attached to a substrate, such as a gel, hydrogel, resin, bead, magnetic bead,
electrode, nitrocellulose, nylon filter, microtiter plate, culture flask, or
polymeric
material. The peptide may have a detectable moiety operably linked to it, and
the
detectable moiety may be a radionuclide, enzyme, specific binding pair
component, colloidal dye substance, fluorochrome, reducing substance, latex,
digoxigenin, metal, particulate, dansyl lysine, antibody, protein A, protein
G,
electron dense material, chemiluminescent substance, electrochemiluminescent
substance, electroactive compound or chromophore.
The present invention also provides an antibody or fragment thereof that
specifically recognizes an FPB peptide defined by an amino acid sequence
indicated in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ
ID NO:S or SEQ ID N0:6. Such a fragment may be an Fab, F(ab')Z, or Fv
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J
fragment. The antibody or fragment thereof may be attached to a substrate,
such
as a gel, hydrogel, resin, bead, magnetic bead, electrode, nitrocellulose,
nylon
filter, microtiter plate, culture flask, or polymeric material. The antibody
or
fragment thereof may have a detectable moiety operably linked to it, and the
detectable moiety may be a radionuclide, enzyme, specific binding pair
component, colloidal dye substance, fluorochrome, reducing substance, latex,
digoxigenin, metal, particulate, dansyl lysine, antibody, protein A, protein
G,
electron dense material, electrochemiluminescent substance, chemiluminescent
substance or chromophore.
The present invention further provides a continuous cell line that produces
an antibody that specifically recognizes a target peptide, wherein the target
peptide is an FPB peptide defined by an amino acid sequence indicated in SEQ
ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S or SEQ
ID N0:6. The cell line may be a monoclonal antibody cell line.
The present invention further provides an animal that produces
polyclonal antibodies that specifically recognizes a target peptide, wherein
the
target peptide is an FPB peptide defined by an amino acid sequence indicated
in
SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S or
SEQ ID N0:6. The target peptide may be covalently linked to a Garner
molecule. It may be keyhole limpet hemocyanin (KLH).
The present invention provides diagnostic method for detecting
thrombotic or thromboembolic disease in a patient having the step of detecting
the presence or amount of FPB in a sample such as a physiological fluid taken
from the patient. to determine whether the patient has thrombotic or
thromboembolic disease. The thrombotic or thromboembolic disease to be
detected may be deep venous thrombosis (DVT) or pulmonary embolism (PE).
The physiological fluid to be tested may be a fluid, such as blood or urine.
Examples of techniques that can be used for the detection step include mass
spectrometry, peptide sequencing, chromatography (e.g., HPLC or TLC),
electrophoresis (e.g., capillary electrophoresis), enzyme-linked immunosorbent
assay, immunonephelometry, agglutination, precipitation, immunodiffusion,
immunoelectrophoresis, electrochemiluminescent immunoassay, electrochemical
immunoassay, chemiluminescent immunoassay, western blot,
immunofluorescence, radioimmunoassay, and immunohistochemistry. The
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amount of FPB present in the sample is considered "positive" for thrombotic or
thromboembolic disease if it is significantly above the normal range or if it
is in
a range that is indicative of thrombotic or thromboembolic disease. The exact
cutoff values used will vary depending on the desired assay sensitivity and
selectivity. In one embodiment, the amount of FPB present in a blood or plasma
sample is considered "positive" for thrombotic or thromboembolic disease if it
is
above 5 ng/ml, and in particular if it is above 10 ng/ml. In an alternative
embodiment, the amount of FPB present in a urine sample is considered
"positive" for thrombotic or thromboembolic disease if it is above 50 ng/ml,
and
in particular if it is above 100 ng/ml.
The present invention provides diagnostic method for detecting
thrombotic or thromboembolic disease in a patient having the steps of
contacting
a physiological sample suspected of containing fibrinopeptide B (FPB) and des-
arginine FPB with an amount of detection agent specific for FPB to form an
FPB:detection agent complex; wherein the detection agent is an antibody or
fragment thereof that specifically recognizes an FPB peptide defined by an
amino acid sequence indicated in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3,
SEQ ID N0:4, SEQ ID NO:S or SEQ ID N0:6; and detecting the presence or
amount of FPB:detection agent complex present in the sample to determine
whether the patient has thrombotic or thromboembolic disease. The method may
include a step of removing fibrinogen from the physiological sample. The
thrombotic or thromboembolic disease to be detected may be deep venous
thrombosis (DVT) or pulmonary embolism (PE). The physiological fluid to be
tested may be a fluid, such as blood, plasma or urine. The detection step may
be
by enzyme-linked immunosorbent assay, immunonephelometry, agglutination,
precipitation, immunodiffusion, immunoelectrophoresis,
electrochemiluminescent immunoassay, chemiluminescent immunoassay,
electrochemical immunoassay, western blot, immunofluorescence,
radioimmunoassay, or immunohistochemistry. The amount of FPB:detection
agent complex present in the plasma sample is considered "positive" for
thrombotic or thromboembolic disease if it is above 5 ng/ml, and in particular
if
it is above 10 ng/ml. The amount of FPB:detection agent complex present in the
urine sample is considered "positive" for thrombotic or thromboembolic disease
if it is above 50 ng/ml, and in particular if it is above 100 ng/ml.
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The present invention provides a method for monitoring the treatment of
thrombotic or thromboembolic disease in a patient by monitoring changes in the
levels of FPB in physiological samples such as blood, plasma, or ,preferably,
urine. The monitoring may comprise the steps of contacting a physiological
sample suspected of containing fibrinopeptide B (FPB) and des-arginine with an
amount of detection agent specific for FPB to form an FPB:detection agent
complex, wherein the detection agent is an antibody or fragment thereof that
specifically recognizes an FPB peptide defined by an amino acid sequence
indicated in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ
ID NO:S or SEQ ID N0:6; detecting the amount of FPB:detection agent
complex present in the sample; repeating the steps at a point later in time;
and
comparing the amounts determined at the two time points and correlating the
change in the amounts to determine whether the thrombosis or embolism is
diminishing in size. The method may include a step of removing fibrinogen
from the sample.
The present invention also provides a diagnostic method for detecting
thrombotic or thromboembolic disease in a patient involving contacting a urine
sample suspected of containing fibrinopeptide B (FPB) and des-arginine FPB
with an amount of detection agent specific for FPB to form an FPB:detection
agent complex, wherein the detection agent is an antibody or fragment thereof
that specifically recognizes an FPB peptide defined by an amino acid sequence
indicated in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ
ID NO:S or SEQ ID N0:6; detecting the presence or amount of FPB:detection
agent complex present in the sample to determine whether the patient has
thrombotic or thromboembolic disease.
Moreover, the present invention provides a diagnostic method for
monitoring the treatment of thrombotic or thromboembolic disease in a patient
by monitoring changes in the levels of FPB in the patients urine. Such
monitoring may involve contacting urine samples suspected of containing
fibrinopeptide B (FPB) and des-arginine FPB with an amount of detection agent
specific for FPB to form an FPB:detection agent complex; wherein the detection
agent is an antibody or fragment thereof that specifically recognizes an FPB
peptide defined by an amino acid sequence indicated in SEQ ID NO:l, SEQ ID
N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S or SEQ ID N0:6; and
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monitoring the changes in the urine concentration of FPB over time to
determine
if the thrombosis or embolism is diminishing in size.
The present invention also provides for kits that contain in one or more
containers one or more of the reagents or compositions used in carrying out
the
assays of the invention. These kits may also contain calibration samples or
standards.
Brief Description of Drawings
Figure 1. Specificity of FPB Antiserum by Competitive ELISA. Panel
A: Various concentrations of native human FPB (diamonds) or canine FPB
(sguares) were pre-incubated separately with FPB antiserum and then applied to
FPB-coated wells. After incubation, antibody binding to the wells was assessed
as described in Methods. FPB used in the pre-incubation mixtures was derived
from purified human or canine fibrinogen (2.5 mg/mL) that was clotted with
thrombin (2 units/mL) for one hour at room temperature. The clot liquor was
then subjected to centrifugal ultrafiltration, and the presence of FPB in the
ultrafiltrate was confirmed by HPLC. FPB concentrations are given in arbitrary
units (AU). Data are presented as the meantrange of duplicate determinations.
Panel B: Various concentrations of purified human fibrinogen (closed circles),
synthetic FPB (diamonds), des-arg FPB (triangles), or FPA (open circles) were
pre-incubated separately with FPB antiserum and then applied to FPB-coated
wells. After incubation, antibody binding to the wells was assessed as
described
in Methods. Data are presented as the mean of duplicate determinations and
expressed as a percentage of the maximal absorbance obtained when no
competitor was present in the pre-incubation mixture.
Figure 2. Plasma FPB Levels During Thromboembolism. Plasma FPB
levels were measured in all eight experimental animals (diamonds) before and
at
the indicated times after induction of bilateral femoral vein thrombosis. Four
hours after thrombosis, one of the femoral clots in each animal was embolized
to
the lungs. One hour before embolization, heparin therapy was initiated in
three
of the animals (triangles). The other five animals (squares) did not receive
heparin before embolization. Data are presented as the mean~SEM FPB level
for each group of animals. p<0.05, * p<0.01, ** p<0.005 compared to baseline
(time = 0) level.
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Figure 3. Total Urinary FPB During Thromboembolism. FPB levels
were measured in hourly urine collections before and after induction of
bilateral
femoral vein thrombosis. The baseline sample (BL) was taken from the hourly
collection just prior to insertion of the double balloon catheters used for
S induction of thrombosis. Total urinary excretion of FPB for each time
interval
was determined by multiplying the FPB concentration in the urine by the total
volume collected during the hourly interval. Four hours after thrombosis, one
of
the femoral clots in each animal was embolized to the lungs. One hour before
embolization, heparin therapy was initiated in three of the animals. Data are
presented as the mean+SEM urinary FPB for each group of animals. White bar:
all animals (n=8). Grey bar: animals not treated with heparin prior to
embolization (n=5). Black bar: animals treated with heparin prior to
embolization (n=3). Total urinary FPB was significantly elevated compared to
baseline (p<0.05) at all times after induction of thrombosis in all groups of
animals.
Figure 4 (example 4). Urine FPB levels were markedly higher in
patients with DVT (96+/- 41) ng/ml) than in patients without DVT (2.7+/- 1.9
ng/ml) and in healthy control subjects (2.15+/- 1.9 ng/ml). (All values
expressed
as mean +/- SEM.)
Figure 5 (example 5) The mean (+/- SD) levels of FBP in the urine of
209 patients without DVT, being admitted to the hospital for various reasons,
were 11.6 (+/- 25.4) ng/ml (figure 5). At the time of discharge (after
hospitalization for a least 48 hours), the urine FPB levels in those without
DVT
were 16.5 (+/- 34.3). These levels are markedly below those observed in
patients
with DVT or PE (see figure 4).
Detailed Description of the Invention
Fibrinopeptides are released at rapid rates during formation of fibrin clots
and blood/urine fibrinopeptide levels are, therefore, accurate measures of
thrombus propagation in thrombembolic disease. Fibrinopeptides A and B (FPA
and FPB) are short amino acid sequences situated at the amino termini of the
alpha and beta chains (respectively) of soluble fibrinogen. In one of the
final
steps in the coagulation pathway, thrombin converts soluble fibrinogen to
fibrin
by first cleaving off FPA to form fibrin I, which spontaneously polymerizes.
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l0
Thrombin then releases fibrinopeptide B (FPB) from the beta-chains of the
fibrin
I subunits to form fibrin II polymers, which laterally associate and cross-
link to
form a semi-solid network, while the fibrinopeptides remain soluble in plasma.
Plasma FPA levels are elevated during thrombosis and this measurement
could represent a valid marker for thrombosis and anticoagulation. FPA is, in
fact, detectable in plasma by immunoassays and by high-performance liquid
chromatography (HPLC). However, FPA is so easily cleaved from fibrinogen
that artifactually elevated plasma levels are a common confounding problem.
Even when collection tubes are supplemented with proteolytic inhibitors, the
sampling procedure itself can cause FPA release. Falsely elevated levels have
been associated with factors such as venipuncture techniques and phlebotomy
through indwelling catheters.
The inventors have discovered that FPB is better suited as a marker of
thrombotic activity than FPA. Without being bound by theory, it is believed
that
some of the advantages of FPB as a marker are associated with some of the
properties of FPB that are described below. The kinetics of thrombin-mediated
cleavage of FPB from fibrinogen are much slower than for FPA (Ng et al. 1993).
In fact, there is evidence that thrombin-mediated FPB release occurs only
after
FPA removal from fibrinogen is complete. Further, the release of FPB ex vivo
may be prevented by agents which inhibit fibrin I polymerization. Plasma
measurement of FPB is therefore less prone to artifactual error. FPB is
continuously cleaved from fibrinogen molecules during fibrin polymerization in
vivo. Once polymerization is halted by the administration of systemic
anticoagulation therapy, FPB cleavage ceases. Previous studies have shown that
plasma FPB levels rise sharply following intrauterine thrombosis, thereby
reflecting ongoing fibrin formation in vivo. Furthermore, FPB levels are not
significantly elevated in a variety of other diagnoses. Thus, the fibrin
formation
associated with active thrombosis leads to significantly higher plasma levels
of
FPB than are seen in other medical conditions. Thus, FPB is less susceptible
than FPA to artifactual elevation during sample collection. Measurement of
plasma FPB levels is therefore a more sensitive and specific serologic marker
for
acute thrombosis.
The present efforts to measure active in vivo thrombosis focused on
demonstrating that removal of FPB from fibrinogen on the surface of thrombi in
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situ correlated with local thrombotic activity and was extinguished during
anticoagulation. First, in vitro experiments were performed showing that
beta,s_
zz, the neo-epitope exposed on the beta-chain of fibrin after cleavage of FPB,
is
only transiently accessible to binding by a monoclonal antibody (anti-
beta,s_zz)
(Morns et al. 1997a). This phenomenon was explained by the fact that the
beta,s_z~ site is rapidly covered up by lateral associations between parallel
fibrin
monofilaments. Thus, fibrin subunits are only able to bind anti-beta,5_zz when
FPB has been cleaved off, but lateralization has not yet occurred.
Essentially,
the epitope is only accessible during active thrombosis.
Next, it was demonstrated that cleavage of FPB from fibrinogen also
correlated with in vivo thrombotic activity. In an in vivo experimental model
of
DVT and PE, actively propagating in situ thrombi bound radio-labeled anti-
beta,s_z, in sufficient quantities to be imaged with a gamma camera (Morris et
al.1993). Because the beta,s_,~ site is only transiently exposed (prior to
lateralization of the fibrin network), imaging of thrombi occurs only when
coagulation is ongoing. Systemic anticoagulation of subjects with pre-formed
thrombi, prior to radiolabeled anti- beta,s_z, administration, suppressed
antibody
binding. In fact, the degree of anticoagulation (as measured by blood heparin
levels) had a strong inverse correlation with localization of radiolabeled
anti-beta
,5_zz at the thrombi (Morns et al. 1997b).
A similar model of thrombosis was used to compare the anti-thrombotic
efficacies of four different anticoagulant regimens: intravenous
unfractionated
heparin, subcutaneous unfractioned heparin and two low molecular weight
heparin, both given subcutaneously (Morris et al. 1998). There were
significant
differences in anti-thrombotic effect among regimens, which did not correlate
with ex vivo anticoagulant tests (such as anti-Xa activities) measured during
the
experiments.
The measurement of the exposure of the beta,s_zz site as an indicator of
thrombosis is problematic due to the transient nature of the exposure and by
the
fact that the measurement requires the infusion of detecting agents
systematically. By contrast, we have now found that measurement of the
released FPB peptide is an excellent indicator of thrombotic activity and does
not
have the disadvantages described for betas-zz. Measurements of FPB in both in
vitro and in vivo models of thrombosis were taken using high performance
liquid
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chromatography (HPLC). Synthetic human FPB was easily distinguished from
synthetic FPA and other plasma proteins. Next, plasma samples from three
dogs, in which experimental thrombi had been induced, were pooled and
analyzed by HPLC. Fibrinopeptide concentrations were estimated by comparing
the areas under the peaks (A2o5 time) to those obtained by treating known
amounts of purified canine fibrinogen with thrombin. Peaks corresponding to
both FPA and FPB were low prior to thrombus induction, but steadily increased
as thrombi were induced and allowed to propagate in situ. These experiments
support the validity of using FPB levels as a measure of in vivo thrombosis.
The invention involves, in part, the creation of a non-invasive blood,
plasma, and/or urine test to detect active thrombosis, based on the
measurement
of FPB and/or des-arginine FPB (a degradation product that is formed rapidly
as
FPB is exposed to carboxypeptidases in normal plasma). Methods that can be
used to carry out this measurement include peptide sequencing, mass
1 S spectrometry, chromatography (e.g., HPLC or TLC), electrophoretic
separation
(e.g., capillary electrophoresis) and measurement through specific binding
interactions (e.g., immunoassays). Both FPB and des-arginine FPB may be
measured. The invention also includes an immunoassay for measuring FPB and
des-arginine FPB in these matrices..
The rationale for testing both peptides with the same assay is that, in
humans, carboxypeptidases present in vivo in normal plasma degrade circulating
FPB into des-arginine FPB. The relative proportions of FPB and des-arg FPB
present in the plasma depend, in part, on each individual's carboxypeptidase
activity. Elevated levels of both peptides in the plasma reflect the rate of
ongoing thrombosis. Therefore, in one embodiment of the invention, the
measurement of FPB is carried out using an immunoassay that is designed to
cross-react with both FPB and des-arginine FPB. Alternatively, the two
peptides
may be measured through the use of two independent assays each specific for
one of the peptides; the total amount of FPB derived peptides is then
determined
by summing the calculated concentrations of FPB and des-arginine FPB.
Thus, the present invention provides methods for measuring FPB in the
blood and in the urine as markers of ongoing thrombosis for use in the
diagnosis
of DVT, PE and other thrombotic disorders. In addition, the assays can be used
to determine the anti-thrombotic efficacy of different anticoagulation
regimens.
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The FPB test is more sensitive and specific for the presence of active DVT or
PE
than the D-dimer test. It can be performed using urine, so venipuncture is not
necessary.
An important aspect of the invention is the present discovery of the
utility of measurements of FPB in urine. Despite the fact that FPB is
generated
in blood, it has been found that the levels of FPB in urine are approximately
one
to two orders of magnitude greater than in blood or plasma and are thus
amenable to measurement with simpler, faster, and/or less sensitive assays.
Furthermore, in some cases the measurement of FPB in urine has, suprisingly,
been found to correlate better with thrombotic activity or disease (relative
to
measurements in blood or plasma). An additional benefit of urine measurements
is the fact that the time constant for changes in FPB concentration associated
with thrombotic activity tends to be longer in urine than in blood; urine
measurements are therefore not only indicative of current thrombotic activity
but
will also indicate thrombotic activity in the recent past.
An immunoassay test for the total concentration of FPB and des-arg FPB
has been developed and the reagents have been or can be produced in mass
quantities easily. The assay has been tested in in vitro models of thrombosis;
in
animal models using experimentally induced deep venous thrombi (DVT) and
pulmonary emboli (PE) and in a human clinical study, where it has reliably
detected the presence of DVT and PE in hospitalized patients. The invention
can
be used as a point-of care diagnostic tool to detect and assist in the
management
of DVT and PE. The immunoassay can be performed easily and inexpensively
at the patient's bedside. In addition, since the assay detects ongoing
thrombus
propagation, it is also useful for guiding therapy.
For purposes of more clearly and accurately describing the invention
herein, certain terminological conventions have been adopted in the following
discussion. These conventions are intended to provide a practical means for
enhancing description of the invention, but are not intended to be limiting,
and
the skilled artisan will appreciate that other and additional, albeit not
inconsistent, interpretations can be implied.
An "analog" or "variant" of FPB is a polypeptide that is not completely
identical to native FPB. Such an analog of FPB can be obtained by altering the
amino acid sequence by insertion, deletion or substitution of one or more
amino
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acids. The amino acid sequence of the protein is modified, for example by
substitution, to create a polypeptide having substantially the same or
improved
qualities as compared to the native polypeptide. The substitution may be a
conserved substitution. A "conserved substitution" is a substitution of an
amino
acid with another amino acid having a similar side chain. A conserved
substitution would be a substitution with an amino acid that makes the
smallest
change possible in the charge of the amino acid or size of the side chain of
the
amino acid (alternatively, in the size, charge or kind of chemical group
within
the side chain) such that the overall peptide retains its spatial conformation
but
has altered biological activity. For example, common conserved changes might
be Asp to Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and
Ser to Cys, Thr or Gly. Alanine is commonly used to substitute for other amino
acids. The 20 essential amino acids can be grouped as follows: alanine,
valine,
leucine, isoleucine, proline, phenylalanine, tryptophan and methionine having
nonpolar side chains; glycine, serine, threonine, cystine, tyrosine,
asparagine and
glutamine having uncharged polar side chains; aspartate and glutamate having
acidic side chains; and lysine, arginine, and histidine having basic side
chains.
L. Stryer, Biochemistry (2d ed.) p. 14-15; Lehninger, Biochemistry, p. 73-75.
It is known that analogs of polypeptides can be obtained based on
substituting certain amino acids for other amino acids in the polypeptide
structure in order to modify or improve biologic activity, such as antigenic
or
immunogenic activity. For example, through substitution of alternative amino
acids, small conformational changes may be conferred upon a polypeptide which
result in increased activity. Alternatively, amino acid substitutions in
certain
polypeptides may be used to provide residues which may then be linked to other
molecules to provide peptide-molecule conjugates which retain sufficient
biologic properties of FPB. Certain analogs that are linked to labels or solid
phases but retain the ability to bind to anti-FPB antibodies, may be used as
competitors in competitive immunoassays for FPB.
The degree of homology (percent identity) between a native and a variant
sequence may be determined, for example, by comparing the two sequences
using computer programs commonly employed for this purpose. One suitable
program is the GAP computer program described by Devereux et al. (Nucl. Acids
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Res. 12:387, 1984), which is available from the University of Wisconsin
Genetics Computer Group.
The amino acid sequence of the analog of FPB corresponds essentially to
the native FPB amino acid sequence. As used herein "corresponds essentially
S to" refers to a polypeptide sequence that will elicit a biological response
substantially the same as the response generated by native FPB. Such a
response
may be at least 60% of the level generated by native FPB, and may even be at
least 80% of the level generated by native FPB. A variant of the invention may
include amino acid residues not present in the corresponding native FPB or
deletions relative to the corresponding native FPB.
An amino acid is "operably linked" when it is placed into a functional
relationship with another amino acid sequence. Generally, "operably linked"
means that the amino acid sequences being linked are contiguous
An "antibody" in accordance with the present specification is defined
broadly as a protein that binds specifically to an epitope. Monoclonal
antibodies
may be produced by methods known in the art. These methods include the
immunological method described by Kohler and Milstein ( 1975) and by
Campbell (1985); as well as the recombinant DNA method described by Huse et
al. ( 1989).
As used herein, the term "monoclonal antibody" (or "mAb") refers to any
homogeneous antibody or antigen-binding region thereof that is reactive with,
preferably specifically reactive with, a single epitope or antigenic
determinant.
The term "monoclonal antibody" as used herein may, however, refer to
homogeneous antibodies that are native, modified, or synthetic, and can
include
hybrid or chimeric antibodies. The term does not include "polyclonal
antibodies," as that term is commonly understood. A "polyclonal antibody" is a
group of heterogeneous antibodies that all recognize a single epitope or
antigenic
determinant.
The term "antigen-binding region" refers to a naturally occurring,
modified, or synthetic fragment of an antibody of the invention that is
reactive
with an epitope. Such antigen-binding regions include, but are not limited to,
Fab, F(ab')2, and Fv fragments.
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/ ~o
Functional equivalents of the antibody of the invention further include
fragments of antibodies that have the same binding characteristics as, or that
have binding characteristics comparable to, those of the whole antibody. Such
fragments may contain one or both Fab fragments or the F(ab')z fragment.
Preferably, the antibody fragments contain all six complement determining
regions ("CDRs") of the whole antibody, although fragments containing fewer
than all of such regions, such as three, four or five CDRs, may also be
functional. Fragments may be prepared by methods described by Lamoyi et al.
(1983) and by Parham (1983). Other functional equivalents of the antibody of
the invention include other molecules that specifically bind FPB, for example,
receptors that bind FPB and peptides or nucleic acids that have been selected
for
their ability to bind FPB (e.g., by phage display or SELEX methods).
The antibodies of the present invention have been found to be
specifically reactive with an epitope FPB, which is found in a plurality of
related
protein moieties, including intact fibrinogen as well as fragments thereof,
including fibrinopeptide B, des-Arg fibrinopeptide B, the N-DSK fragment of
fibrinogen resulting from cleavage with cyanogen bromide, and the peptides
defined by SEQ. ID. NOs:l and 2. The term "anti-FPB" refers to the ability of
the antibody of the present invention to react specifically with this epitope,
which is characteristic of fibrinogen, fibrinopeptide B, des-Arg
fibrinopeptide B,
N-DSK, and related peptides.
Accordingly, the antibodies of the invention is specifically reactive with
an epitope defined by an amino acid sequence characteristic of SEQ ID NOs:l
and 2, and other functionally equivalent sequences, i.e., those amino acid
sequences that exhibit similar binding capacities. The antibody is not
significantly cross-reactive with moieties lacking the defining epitope.
Among other properties of the antibody of the invention, it is
demonstrated herein that the antibody is reactive with peptides defined by SEQ
ID NOs:l and 2, which differ at their N-termini from native FPB, as well as
with
SEQ ID NOs 3-6, which are native forms of FPB found in physiological
samples. Accordingly, the antibody of the invention is understood to react
specifically with an epitope defined by the amino acid sequence
CQGVNDNEEGFFSAR (SEQ ID NO:1) and CQGVNDNEEGFFSA (SEQ ID
N0:2). Proteins containing SEQ ID NO:1 or SEQ ID N0:2, or similar
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/7
sequences, (for example: QGVNDNEEGFFSAR (SEQ ID N0:3);
QGVNDNEEGFFSA (SEQ ID N0:4); cyclised pyroglutamic acid-
GVNDNEEGFFSAR (SEQ ID NO:S); and cyclised pyroglutamic acid-
GVNDNEEGFFSA (SEQ ID N0:6)) within their primary structures, and lacking
significant steric interference from higher order structures will, therefore,
bind
with one or more of the detecting antibodies or polyclonal antisera of the
invention. Such proteins may be naturally occurring, such as Fibrin I, or
synthetically made, e.g., produced by conventional synthetic or recombinant
methods such as are known in the art. Homologs of the amino acid sequence
characterized by SEQ ID NOs:I and 2, and proteins containing the epitope-
defining sequence, are also expected to be reactive with one or more of the
detecting antibodies or polyclonal antisera of the invention. However, the
antibodies and antisera exhibit no substantial cross-reaction with moieties
lacking this epitope. Peptides or proteins containing this epitope can be
detected
using the immunoassay of the invention, can be used as calibrators or
standards,
or can be labeled or immobilized and used as competitors in immunoassays for
FPB.
The term "fibrinogen" without more is intended to include any type of
fibrinogen. Fibrinogen, therefore, refers to monomeric and dimeric fibrinogen
molecules having the monomer structure (AaB~3~y), as well as molecules having
the monomer structure (Aa EB(3'y), and other hybrid molecules, whether
naturally occurring, modified, or synthetic. The term "fibrinogen" refers
generally to fibrinogen from humans, but may include fibrinogen of any
species,
especially mammalian species. In addition, the term may be specifically
limited
to a particular species in particular contexts, such as "human fibrinogen."
Generally, to be useful as an immunogen, a peptide fragment must
contain sufficient amino acid residues to define the epitope of the molecule
being
detected. If the fragment is too short to be immunogenic, it may be conjugated
to a carrier molecule. Some suitable Garner molecules include keyhole limpet
hemocyanin and bovine serum albumin. Conjugation may be carried out by
methods known in the art. One such method is to combine a cysteine residue of
the fragment with a thiol-reactive moiety on the carrier molecule such as a
cysteine residue or a maleimide group. In the present invention, a cysteine
residue has specifically been covalent attached to the amino-terminus of the
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molecule so as to provide the necessary cysteine. Further, by attaching the
cysteine to the amino-terminus, the carrier molecule will preferentially
attach to
this particular end, allowing the antigenic carboxy-end to be exposed.
The present invention provides animals that produce polyclonal
antibodies reactive with an epitope of fibrinopeptide B and fibrinogen and
fragments thereof containing the epitope. The invention also provides
hybridoma cell lines that produce monoclonal antibodies reactive with an
epitope
of fibrinopeptide B and fibrinogen and fragments thereof containing the
epitope.
The antibodies produced by these animals and hybridomas are also important
aspects of the invention.
The hybridoma technology originally described by Kohler and Milstein
(1975) can be used to prepare hybridoma cell lines whose secretory product,
monoclonal antibodies, are reactive with an epitope or antigenic determinant
of
fibrinopeptide B. A general method of preparing hybridoma cell lines of the
invention is described below. Those skilled in the art will recognize that the
present invention, including the monoclonal antibodies and hybridoma cell
lines
described in detail herein, provide a variety of ways to make the hybridomas,
and
thus the antibodies of the invention. Hybridoma cell lines of the invention
can
be prepared using the fibrinopeptide B peptide defined by SEQ ID NO:1 or a
desarginine-FPB peptide defined by by SEQ ID N0:2 for activation of
immunologically relevant spleen cells. Generally, a host mammal is inoculated
with a peptide or peptide fragment as described above, and then boosted.
Spleens are collected from inoculated mammals a few days after the final
boost.
Antibody-producing spleen cells are then harvested and immortalized by fusion
with mouse myeloma cells. The hybrid cells, called hybridomas, are continuous
cell lines resulting from the fuision, which are then selected and screened
for
reactivity with the peptide. The artisan is referred to Kohler and Milstein
(1975);
Kennett et al. (1980); and Goding (1986) for further details on hybridoma
technology. See also Campbell (1985).
The specific anti-FPB antibodies described herein are merely illustrative
of the invention, and all antibodies that are specifically reactive with the
fibrinopeptide B peptide defined by SEQ ID NO: l or the desarginine-FPB
peptide defined by by SEQ ID N0:2, regardless of species of origin or
immunoglobulin class or subclass designation, including IgG, IgA, IgM, IgE,
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I
and IgD, are included in the scope of this invention. The present invention
also
provides antigen-binding fragments of the anti-FPB antibodies. The ability to
bind to fibrinopeptide B as opposed to non-FPB-containing substances is a
general characteristic of the specific antibodies according to the present
invention.
As discussed above, antibodies of the invention can be constructed and
isolated by immunization of animals, preparation of hybridomas, and
identification of antibodies with a reactivity to fibrinopeptide B and
fibrinogen
similar to that of the anti-FPB antibodies described. However, the present
invention also provides means for identifying monospecific antibodies of the
invention that does not require determination of antibody reactivity with a
broad
number of B~3-related fragments. Antibodies of the invention can be identified
also by immunoprecipitation and competitive binding studies using the antibody
produced by the cell lines described herein.
Immunoprecipitations using the anti-FPB monospecific antibody can be
used to determine antigenic identity. Confirmation of identity can be obtained
by depleting the antigen from testable samples such as plasma samples, using
excess amounts of one anti-FPB antibody and observing the inability of another
antibody to immunoprecipitate a Bpi-chain fragment from the treated sample.
Also, in instances in which the antibodies bind with the same epitope or
closely
associated epitopes, each antibody will compete with the others) for binding
to
fibrinopeptide B. Competitive binding studies are generally known in the art,
and one conventional type is presented in the examples below.
Treatment of antibody preparations with proteolytic enzymes such as
papain and pepsin generates antibody fragments, including the Fab and F(ab')z
species, that retain antigen-binding activity. Treatment of the antibodies of
the
invention with such enzymes can therefore be used to generate fibrinopeptide B
antigen-binding fragments of the invention. The preparation of antigen-binding
fragments of the antibodies of the invention and their diagnostic and
therapeutic
usefulness, as well as other applications, suggest themselves to the skilled
artisan. Antigen-binding fragments of the anti-FPB antibody are especially
useful in therapeutic embodiments of the present invention.
Those skilled in the art will recognize that the antigen-binding region of
the antibodies and antibody fragments of the invention is a key feature of the
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ao
present invention. The anti-FPB hybridoma cells of the invention serve as a
preferred source of DNA that encodes such antigen-binding regions of the
invention. This DNA, through recombinant DNA technology, can be attached to
DNA that encodes any desired amino acid residue sequence to yield a novel
"hybrid," or "chimeric," DNA sequence that encodes a hybrid, or chimeric,
protein. In such a fashion, chimeric antibodies of the invention, in which one
portion of the antibody is ultimately derived from one species and another
portion of the antibody is derived from another species, can be obtained.
However, the present invention also comprises any chimeric molecule that
contains an FPB antigen-binding region.
Antibodies of the present invention can also be labeled by conjugation to
any detectable group, such as fluorescent labels, enzyme labels, and
radionuclides to identify expression of fibrinogen, or cleavage products
including fibrinopeptide B or parts thereof. Suitable detectable labels may be
selected from among those known in the art, including, but not limited to,
radionuclides, enzymes, specific binding pair components, colloidal dye
substances, fluorochromes, reducing substances, latexes, digoxigenin, metals,
particulates, dansyl lysine, antibodies, protein A, protein G, electron dense
materials, chromophores, electrochemiluminescent substances,
chemiluminescent substances, electroactive substances and the like.
Effectively,
any suitable label, whether directly or indirectly detectable, may be
employed.
One skilled in the art will clearly recognize that these labels set forth
above are
merely illustrative of the different labels that could be utilized in this
invention.
Methods for labeling antibodies have been described, for example, by
Hunter et al. (1962) and by David et al. (1974). Additional methods for
labeling
antibodies have been described in U.S. Pat. Nos. 3,940,475 and 3,645,090.
The label may be radioactive, i.e., contain a radionuclide. Some
examples of useful radionuclides include 32P,'zsI,'3'I,"'In, and 3H. Use of
radionuclides have been described in U.K. patent document No. 2,034,323, U.S.
Pat. Nos. 4,358,535, and 4,302,204.
Some examples of non-radioactive labels include enzymes,
chromophores, atoms and molecules detectable by electron microscopy, and
metal ions detectable by their magnetic properties.
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~l
Some useful enzymatic labels include enzymes that cause a detectable
change in a substrate. Some useful enzymes and their substrates include, for
example, horseradish peroxidase (pyrogallol and o-phenylenediamine), beta-
galactosidase (fluorescein beta-D-galactopyranoside), and alkaline phosphatase
(5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The use of
enzymatic labels have been described in U.K. 2,019,404, EP 63,879, and by
Rotman (1961).
Useful chromophores include, for example, fluorescent,
chemiluminescent, and bioluminescent molecules, as well as dyes. Some
specific chromophores useful in the present invention include, for example,
fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol and
luminescent bipyridyl or phenanthrolyl containing complexes of ruthenium or
osmium..
The labels may be conjugated to the antibody probe by methods that are
well known in the art. The labels may be directly attached through a
functional
group on the probe. The probe either contains or can be caused to contain such
a
functional group. Some examples of suitable functional groups include, for
example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.
Alternatively, labels such as enzymes and chromophoric molecules may be
conjugated to the antibodies by means of coupling agents, such as dialdehydes,
carbodiimides, dimaleimides, and the like.
The label may also be conjugated to the antibody probe by means of a
ligand attached to the probe by a method described above and a receptor for
that
ligand attached to the label. Any of the known ligand-receptor combinations is
suitable. Some suitable ligand-receptor pairs include, for example, biotin-
avidin
or -streptavidin, and antibody-antigen. The biotin-avidin combination is
preferred. Thus, the anti-FPB antibodies of the invention can be derivatized
by
conjugation to biotin, and used, upon addition of species of avidins that have
been rendered detectable by conjugation to fluorescent labels, enzyme labels,
radionuclides, electron dense labels, substrates, etc., in a multiplicity of
immunochemical and immunohistological applications.
The antibodies of the invention may also be attached or bound to
substrate materials according to methods known to those skilled in the art.
Such
materials are generally substantially solid and relatively insoluble,
imparting
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as
stability to physical and chemical disruption of the antibodies, and
permitting the
antibodies to be arranged in specific spatial distributions. Among substrate
materials, materials may be chosen according to the artisan's desired ends,
and
include materials such as gels, hydrogels, resins, beads, magnetic particles
or
S beads, nitrocellulose, nylon filters, microtiter plates, culture flasks,
polymeric
materials, and the like, without limitation.
The antibodies of the present invention, whether labeled or unlabeled,
can be used in immunological assays to determine the presence of fibrinogen or
FPB-associated peptides in tissue samples from human or animal subjects. Fluid
samples of subjects, such as plasma or urine, as well as samples from blood
banks, can be evaluated for the presence of fibrinogen and FPB using an anti-
FPB antibody of this invention. Moreover, suitable pharmaceutical preparations
according to the invention may be employed for in vivo use, such as for the
visualization of fibrinogen or FPB-containing substances and structures in a
living subject.
Thus, the invention provides a method for binding fibrinopeptide B,
fibrinogen or a fragment thereof comprising the amino acid sequence defined by
SEQ ID NOs: l and 2 by means of the anti-FPB monospecific antibody.
Accordingly, fibrinogen and fibrinopeptide B, natural, modified, and synthetic
variants thereof, as well as fragments thereof, may be detected and measured
by
means of the antibodies of the invention.
In the FPB binding method of the invention, the method includes
contacting a testable system, in which the presence or absence of FPB is to be
determined, with a composition comprising an anti-FPB antibody or antigen-
binding region thereof. The method then involves measuring an amount of
specific association or binding between an analyte of the testable system and
the
antibody. In this method, specific binding of the antibody in the system
indicates the presence of the analyte, i. e., fibrinogen or FPB-containing
fragments thereof in the system
The present invention further provides a method of detecting the presence
of fibrinopeptide B in a sample. The method involves use of a labeled probe
that
recognizes protein/peptide present in a biological sample such as a blood or
urine
sample. The probe may be an antibody according to the invention that
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a .~
recognizes FPB-containing analytes present in the sample. Other testing
methods known in the art can be adapted to use the antibody of the invention.
A typical method involves the differential separation of degradation
products, such as separation of the products by ultracentrifugation. The
products
are then measured by contacting the products with antibodies that are
specifically
reactive with or specifically associate with one or more domains of
fibrinogen.
Preferably, such antibodies are specifically reactive with a single
degradation
product, thereby permitting characterization of the product in relation to
other
products.
In one embodiment, the detection method employs a anti-FPB antibody
that has been detectably labeled with a marker moiety. In other embodiments,
the method may employ an antibody of the invention that has been bound to a
substrate material. In the method, the composition may also include other
reagents such as other antibodies that differentially detect other fibrinogen
subunits or subtypes. This method can be further adapted for use with at least
one other antibody having specificity for alternative fragments, permitting
differential analysis or characterization of free FPB or of FPB-containing
fragments and other fragments in the same sample. For example, two or more
antibodies conjugated to distinct fluorescent labels can be employed as probes
in
protein separations or other immunometric techniques.
The FPB binding method of the invention includes methods known in the
art that employ antibodies to bind target substances specifically. Preferred
methods include immunochemical methods, such as enzyme-linked
immunosorbent assay (ELISA) methods, western blot, immunonephelometry
methods, agglutination methods, precipitation methods, immunodiffusion
methods, immunoelectrophoresis methods, immunofluorescence methods,
radioimmunoassay methods, surface plamon resonance, and immunoassay
methods based on the detection of chemiluminescent , fluorescent,
phosphorescent, electrochemiluminescent , bioluminescent or electroactive
compounds.
Assays for detecting the presence of proteins with antibodies have been
previously described, and follow known formats, such as standard blot and
ELISA formats. These formats are normally based on incubating an antibody
with a sample suspected of containing the protein and detecting the presence
of a
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a~
complex between the antibody and the protein. The antibody is labeled either
before, during, or after the incubation step. The protein is preferably
immobilized prior to detection. Immobilization may be accomplished by
directly binding the protein to a solid surface, such as a microtiter well or
bead,
S or by binding the protein to immobilized antibodies.
Methods for conducting immunoassays are well known. Techniques that
can be used include direct binding formats and competitive binding formats.
One common example of the direct binding format is the sandwich binding
assay. In a typical example of a competitive assay for FPB, FPB in a sample
competes with labeled FPB (or an analog thereof) for binding to a labeled anti-
FPB antibody. In some common "solid phase" binding assay formats, the label
one of the two labeled species is a solid phase support or a capture moiety
that is
used to bring the reagent onto a solid phase support. Examples of appropriate
immunoassay techniques may be found in the Immunassay Handbook, Wild D.,
Editor, Stockton Press: New York, 1994, hereby incorporated by reference.
The invention further includes a method for determining or diagnosing
the existence of thrombotic or thromboembolic disease, such as deep thrombosis
or pulmonary embolism in a subject. In this method, FPB or a fragment thereof
is measured by means of a composition including an anti-FPB antibody of the
invention. The measured amount of the FPB analyte is compared with an
amount of FPB that is recognized or known to be associated with thrombotic or
thromboembolic disease. The method then involves the determination from the
measured and standard values) of FPB the presence or likelihood of thrombotic
or thromboembolic disease in the subject. The method can include measuring or
detecting FPB peptides in vivo, such as by imaging or visualizing the location
and/or distribution of fibrinopeptide B, in the body. Alternatively, the
method
includes obtaining a medical sample from the subject and measuring FPB ex vivo
or in vitro..
Polyclonal antisera:
Peptide Synthesis - An analogue of human fibrinopeptide B (FPB)
containing a cysteine residue at the amino terminal end of the peptide was
prepared by Peninsula Laboratories (San Carlos, CA). The peptide was designed
to permit directional coupling to maleimide-activated carrier protein for
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o~J
immunization of rabbits. Peptide purity was >90% as judged by mass spectral
analysis and by HPLC using two different solvent systems. The amino acid
sequence of the peptide is as follows: CQGVNDNEEGFFSAR (SEQ ID NO:1).
An analogue of des-arginine FPB (amino acid sequence: CQGVNDNEEGFFSA,
(SEQ ID N0:2)) was prepared in a similar fashion.
Preparation of FPB Antisera - An immunoconjugate of human FPB was
prepared by coupling 5 mg of N-cysteinyl-FPB to 10 mg of maleimide-activated
keyhole limpet hemocyanin (Pierce Chemical, Rockford, IL) according to a
protocol provided by Pierce (Protocol #0135). Polyclonal antiserum to FPB was
raised in New Zealand White rabbits. Three rabbits each received a primary
subcutaneous injection of immunoconjugate (one mg) in complete Freund's
adjuvant followed by two subcutaneous booster injections (one mg each) in
incomplete Freund's adjuvant given at four week intervals. Rabbits were bled
at
two-week intervals starting two weeks after the primary injection. After
allowing
the blood to clot overnight at 4 C, antiserum was collected by centrifugation
(2000xg, 10 min) and stored at -20 C until analyzed. The FPB antibody titer of
each antiserum was determined in a direct-binding ELISA. Briefly, microplate
wells were coated with synthetic human FPB (Sigma Chemical, St. Louis, MO)
and blocked with BSA. Wells were then incubated with serial dilutions of FPB
antiserum followed by peroxidase-conjugated goat anti-rabbit IgG detecting
antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Wells were
developed with o-phenylenediamine (OPD) substrate solution, and the
absorbance (490 nm) of each well was measured in a microplate reader
(Molecular Devices, Sunnyvale, CA). The antibody titer was arbitrarily defined
as the minimal antiserum dilution resulting in an absorbance of >1Ø The
process was repeated on separate rabbits using an iminunoconjugate of des-
arginine FPB and keyhole limpet hemocyanin.
Specificity of the Antiserum - A competitive ELISA was developed to
evaluate the specificity of the FPB antiserum. Unless otherwise noted, all
steps
were performed at room temperature, and the microplates were washed three
times in between each step with 0.02 M NaHZP04, 0.15 M NaCI, 1 mM EDTA,
pH 7.4 (PNE) containing 0.1 % tween-20. Microplate wells were first coated
with
synthetic human FPB (2 mg/mL in 0.2 M NaHC03, 100 mL/well) overnight at 4
C, and then "blocked" with 1% BSA in PNE (PNEA) for one hour. Meanwhile,
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~? 6
serial dilutions of each competitor were prepared. The following competitors,
all
of human origin except where noted, were tested: native FPB (human and
canine), synthetic FPB, synthetic des-arg FPB, synthetic fibrinopeptide A, and
purified fibrinogen. Twenty mL of each competitor dilution were pre-incubated
separately with 200 mL of FPB antiserum (1:2500) in polypropylene microfuge
tubes for 90 min. Dilutions of the competitors and of antiserum were prepared
in
dilution buffer (PNEA containing 0.1 % tween-20). At the end of the pre-
incubation period, 100 mL of each mixture were applied to duplicate FPB-coated
wells and incubated for 90 min. After washing, 100 mL of peroxidase-
conjugated goat anti-rabbit IgG detecting antibody (1:5000 in dilution buffer)
were added to each well and incubated for one hour. After a final wash, each
well was developed with 100 ml of OPD substrate solution for exactly five
minutes. The reaction was stopped by addition of an equal volume of 3 N HZS04,
and the absorbance (490 nm) of each well was measured in the microplate
reader.
Although high titers were demonstrated for several antisera, and the
present invention includes any antisera produced in this fashion, the best
reactivity profile obtained by the inventors to-date is was disclosed for
84097,
bleed I3. Competition experiments using this antiserum indicated that native
human FPB in solution was an effective competitive inhibitor of antibody
binding to surface-bound synthetic human FPB. In contrast, native canine FPB
in
solution did not compete for antibody binding to surface-bound synthetic human
FPB suggesting that canine FPB does not cross-react with the antiserum. The
cross-reactivity of des-arg FPB, defined as ICSO of FPB (6.7 nM) divided by
IC50 of des-arg FPB (8.9 nM) times 100, was 75%. Thus, the FPB assay is
sensitive to both FPB and its primary metabolite, des-arg FPB. FPA exhibited
essentially no cross-reactivity (<0.1 %).
The antiserum showed significant cross-reactivity to parent (human)
fibrinogen. The IC50 of fibrinogen (2.3 nM) was about one-half the IC50 of
FPB, not unexpected since each fibrinogen molecule harbors two potentially
cross-reacting FPB sequences. It is, therefore, preferable to remove cross-
reacting fibrinogen molecules (340,000 kD) from plasma and urine samples by
centrifugal ultrafiltration. Fibrinogen can be separated from fibrinopeptides
in
solution using a variety of filtration membranes, with molecular weight
cutoffs
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a~
ranging from 50,000-100,000kD. However, the best results were obtained using
centrifugal ultrafiltration with Biomax-100 membranes (100,000 kD cut-off).
It is important to note that other polyclonal antisera produced using this
method
in additional animals did not cross-react with fibrinogen. The invention also
includes the use of this antisera in immunoassays, in which case fibrinogen
would not need to be removed from the patient samples.
Monoclonal antibodies:
Conjugation of Peptide: N-cysteinyl FPB (or des-arg FPB) is conjugated
to maleimide-activated keyhole limpet hemocyanin as previously described.
Five Balb/c mice are each immunized with a SO ug priming dose followed by
several boosting doses of 50 ug each. Test bleeds are screened for antibody
production and titer using the direct binding ELISA previously described.
A splenocyte fusion is performed on the best responder as determined by
the direct binding and competitive ELISA. The lymphocytes are fused to an
Sp2/0-Agl4 hybridoma cell line using an optimized polyethylene glycol-
mediated fusion protocol. Expansion and Screening: Fused cells are plated into
96-well plates and monitored weekly for cell growth. Wells exhibiting cell
growth are screened by ELISA at four weeks and then again at six weeks.
Cells from positive wells are harvested and subcloned by limiting
dilution. Subclones are screened by ELISA. Positive subclones are expanded and
screened again. The most promising clones, as determined by direct binding and
competitive ELISA, are stored frozen and used for ascites production.
Ascites fluid is produced in Balb/c mice following intra-peritoneal
injection of the selected hybridoma cell line derived from subcloning. The
fluid
is screened (by ELISA) and subjected to antibody isotyping.
FPB/des-arg FPB Assays:
The following procedure was found to be the most optimal one for
determination of the fibrinopeptide B (FPB) concentration in ultrafiltered
plasma
and urine samples. In general, because of the high levels of FPB in urine, it
is
customary to run urine ultrafiltrates in the "standard assay" (see below) and
plasma ultrafiltrates in the "sensitive assay". Note that the plasma
ultrafiltrate
starts out at a 1:2 dilution.
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Materials used: anti-FPB antiserum (rabbit 4097, bleed I3); synthetic
FPB control solution; FPB stock solution; IgG-HRP stock solution; Coating
buffer (0.2 M sodium bicarbonate); wash buffer (0.02 M NaH2P04, 0.15 M
NaCI, 1 mM EDTA, pH 7.4, containing 0.1% tween-20); dilution buffer (0.02 M
NaH2P04, 0.15 M NaCI, 1 mM EDTA, pH 7.4, containing 1% BSA and 0.1%
tween-20); Hydrogen peroxide, 30% (H2O2, Sigma #H1009); o-phenylene
diamine tablets, 10 mg (OPD, Sigma #P8287); OPD solution A (0.5 M citric
acid); OPD solution B (0.11 M sodium phosphate (dibasic)); OPD stop solution
(3 N sulfuric acid ); PNE l OX stock solution (0.2 M sodium phosphate
(monobasic), 1.5 M sodium chloride, lOmM EDTA (disodium salt), pH 7.4);
PNEA ( PNE (1X) containing 1% (w/v) bovine serum albumin); Tween-20
(Sigma #P6585); Tween-20, 10% (Sigma #P8942).
Methods: Microwell plates are prepared as follows: Dilute FPB stock
solution to 2 mg/mL with coating buffer. Add 100u1 FPB solution (2 mg/mL) to
each microplate well. Seal wells with a plate sealer and place in the
refrigerator
overnight.
The "standard assay" is used to detect FBP/des-arginine FPB
concentrations of 1.56 - 100 ng/mL. The samples are prepared as follows:
Dilute FPB stock solution to 2 mg/mL with CB. Add 100u1 FPB solution (2
mg/mL) to each microplate well. Seal wells with a plate sealer and place in
the
refrigerator overnight. Thaw ultrafiltered samples in a room temperature bath.
Dilute anti-FPB antiserum 1:2500 with DB. Dilute FPB stock solution to 100
ng/mL with DB and then make two-fold serial dilutions (501 + 501 DB) using the
P200. Use the following FPB concentrations for the standard curve: 100, 50,
25,
12.5, 6.25, 3.13, 1.56, and 0 ng/mL. Dilute FPB control 1:2,000 with dilution
buffer. Combine 20u1 standard/control/sample with 200u1 anti-FPB (1:2500) in
separate 1.7-mL microfuge tubes, vortex to mix. (These are referred to as pre-
incubation mixtures.) Incubate 1 hr 45 min.
The "sensitive assay" is used to detect FBP/des-arginine FPB
concentrations of 0.313 - 20 ng/mL. The samples are prepared as follows:
Dilute FPB stock solution to 2 mg/mL with CB. Add 100u1 FPB solution (2
mg/mL) to each microplate well. Seal wells with a plate sealer and place in
the
refrigerator overnight. Thaw ultrafiltered samples in a room temperature bath.
Dilute anti-FPB antiserum 1:250 with DB. Dilute FPB stock solution to 20
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ng/mL with dilution buffer and then make two-fold serial dilutions (250u1 +
250u1 dilution buffer). Use the following FPB concentrations for the standard
curve: 20, 10, 5, 2.5, 1.25, 0.625, 0.313, and 0 ng/mL. Dilute FPB control
1:10,000 with dilution buffer. Combine 20u1 anti-FPB (1:250) with 2001
standard/control/sample in separate 1.7-mL microfuge tubes, vortex to mix.
(These are referred to as pre-incubation mixtures.) Incubate 1 hr 45 min.
During the antibody pre-incubation period, remove the FPB-coated plate
from the refrigerator and rinse each well twice with 250u1 washing buffer. Add
1001 PNEA to each well to "block" residual protein binding sites on the wells.
Cover the plate and allow 1 hr to "block". At the end of the antibody pre
incubation period, rinse each well twice with 2501 washing buffer.
The assays are performed as follows: Add 1001 of each pre-incubation
mixture to duplicate wells. Cover the plate and incubate 1 hr 15 min and then
rinse each well three times with 2501 washing buffer. Add 1001 IgG-HRP
( 1:4000 in dilution buffer) to each well. Cover the plate and incubate 1 hr
15
min. At the end of the IgG-HRP incubation rinse each well three times with
2501 washing buffer.
Complete the substrate solution by first dissolving a 10-mg OPD tablet in
the OPD A/OPD B mixture and then adding 101 H2O2. Invert several times to
mix. Add 1001 substrate solution to each well. Incubate exactly 5 min and then
add 1001 OPD stop solution to each well.
Read the absorbance of each well in the Vmax microplate reader using
the following instrument setup: dual wavelength end-point mode (L1 = 490 nm,
L2 = 650 nm), automix on. Read the plate 14 min after initiating the OPD
substrate reaction.
Generate a standard curve from a four-parameter fit of the FPB standards. Use
Softmax to interpolate the FPB concentrations of the samples and the FPB
control from the standard curve.
The invention will be further described by reference to the following
detailed examples.
Example 1 ~ Measurement of FPB and des-arginine FPB in plasma samples
The objective of this experiment was to determine if the immunoassay
could detect synthetic FPB and synthetic des-arginine FPB in the presence of
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normal human plasma. The assay was also used to measure FPB levels in serum
(clotted plasma), serially diluted in anticoagulated plasma, in order to
simulate
the concentrations expected when a small thrombus releases FPB into the
circulating blood volume
5 Methods: Known concentrations of synthetic FPB or des-arg FPB (0 to
100 ng/ml) diluted in buffer or anticoagulated, ultrafiltrated plasma were pre-
incubated with a fixed dilution of high-titer anti-FPB antiserum for 90
minutes at
room temperature in polypropylene tubes. These pre-incubation mixtures were
then added to FPB-coated microplate wells and incubated for an additional hour
10 at room temperature. The plates were then further developed with anti-
rabbit
IgG-HRP and OPD as described above. After measuring the absorbance at 490
nm, a standard curve is generated. Parallelism was evaluated by comparing
standards diluted in buffer to standards diluted in plasma.
Results: The assay did not detect FPB in normal human anticoagulated
15 plasma. When plasma was "spiked" with purified FPB (final concentration 10
ng/ml), the assay measured 7.72 ~ 0.89 ng/ml. Similar results were obtained
using purified des-arginine FPB (results not shown). In addition, serum
(obtained by clotting plasma with thrombin) was diluted (1:800) with plasma
pretreated with an excess of heparin. The FPB concentration was 8.03 ~ 0.51
20 ng/ml, which is within the range one would expect if 1 % of the plasma
fibrinogen had been converted to fibrin. Anti-FPB did not cross-react with
canine fibrinopeptides, or with canine intact fibrinogen (results not shown).
Discussion: These experiments validate the accuracy of the
immunoassay for measuring FPB and des-arginine FPB. Furthermore, the FPB
25 levelsmeasured would be able to detect a thrombosis of 0.1 % of the total
in vivo
blood volume, which in a 70 kg person, would constitute a 4 ml thrombus.
Conclusions: The plasma FPB immunoassay is capable of detecting
active thrombosis in vivo. The assay is useful for diagnosing DVT and PE, as
well as for comparing the ability of anticoagulants to suppress clot
propagation
30 in vivo.
Example 2: FPB Levels In An Animal Model Of Thrombosis
Objectives: In a well-established model used in previous experiments,
plasma and urine samples from dogs during experimental thrombosis were
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31
analyzed to measure FPB levels. Prior to the induction of thrombosis, the
animals were depleted of canine fibrinogen and transfused with purified human
fibrinogen. Elevated levels of human FPB in the plasma and urine reflect
thrombotic activity.
Methods: Adult mongrel dogs were anesthetized and their native
fibrinogen depleted. After the ancrod has been eliminated from the
circulation,
during a time when the native fibrinogen activity is minimal, experimental
thrombi are induced as previously described (Morris et al. 1997b). The dogs
are
anesthetized with halothane, intubated and mechanically ventilated to maintain
arterial blood gases within normal limits. The animals received a bolus of
tranexamic acid at this time and every 6 hours thereafter to completely
inhibit
fibrinolysis (Marsh et al. 1994). After this, they were transfused with 6
grams of
purified human fibrinogen in normal saline to achieve a fibrinogen level of at
least 300 mg/dl. Under these conditions, fibrinogen levels were maintained at
>200 mg/dl throughout the study period.
Double balloon catheters were advanced via hind-leg saphenous veins to
the femoral veins on each side. The balloons were then inflated, creating a 5
ml
sealed chamber within the veins. Through a port between the double balloons,
200 units of thrombin were infused into each venous lumen to induce
thrombosis. After one hour, all the balloons were deflated and the induced
thrombi are aged in situ for an additional three hours.
For each animal, one balloon catheter was left in the femoral vein to
prevent embolization of the thrombus. The balloon catheter in the
contralateral
femoral vein was removed and the thrombus was embolized by passive leg
motion.
After embolization, anticoagulation was achieved with a heparin bolus
(300 units/kg) followed by continuous infusion (90 units/kg/hr), adjusted to
keep
the plasma heparin level greater than 1.0 units/ml. Anticoagulation at this
dose
was associated with complete suppression of thrombus propagation (Morns et al.
1997b). In separate experiments, lower doses of heparin were used (80 units/kg
loading dose and 18 units/kg/hr infusion rates) and the dose was adjusted to
maintain a "therapeutic heparin level" (0.2-0.4 units/ml).
Blood and urine samples were collected into titrated tubes (containing
protease inhibitors) at baseline, after defibrinogenation, at regular
intervals after
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thrombus induction, and after anticoagulation. Plasma was obtained by
centrifugation (2500 X g for 10 minutes) and then subjected to ultrafiltration
(Millipore) to remove fibrinogen and other plasma proteins. All samples were
stored at 70 C until analyzed. FPB,o~ levels in urine and ultrafiltrated
plasma are
determined by immunoassay as described above.
The means and standard deviations of the FPB,o~ levels, as determined by
immunoassay, associated with each time point are calculated. Baseline values
are compared to values during thrombus induction, propagation and
anticoagulation. A difference between means of at least two standard
deviations
is considered statistically significant.
Results: Preliminary experiments were performed on three dogs, all of
whom received anticoagulation with excess doses of heparin after embolization
of the experimentally induced thrombi (see figure below). The measurements in
the first few hours of the experiment demonstrate that transfusion of large
amounts of human fibrinogen do not significantly elevate plasma or urine
FPB,o,
levels, prior to thrombus induction.
However, after thrombi are formed in the deep veins, the plasma FPB~ot
levels immediately rise. Plasma FPB~o, remains elevated above baseline during
clot propagation. Another peak in the plasma FPB,o~ levels occurs immediately
after embolization. Once excess heparin is begun, thrombosis is halted. FPB is
no longer produced and the FPB~o~ remaining in the plasma clears rapidly (t",
of
approximately 45 minutes).
The total amount of FPB~o~ in the urine (measured at 30-minute intervals)
was also low after human fibrinogen transfusion, but steadily rose after
thrombi
were formed. Predictably, the amount of FPB~o, recovered in the urine
paralleled
the rise in plasma levels, albeit several hours later. The actual
concentration of
FPB~p~ in the urine was, in general, two orders of magnitude higher than the
levels measured in the plasma, presumably reflecting preferential clearance of
the peptide by the kidney.
Interestingly, FPB levels in both the plasma and urine increased after the
thrombi were embolized, despite the fact that no further thrombin was infused.
As before, the plasma levels rose immediately, whereas the amounts cleared in
the urine rose several hours after the event.
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Discussion: These experiments indicate that plasma and urine levels of
FPB are potential markers of ongoing thrombosis in VTE. Preliminary data
suggest that one or both of these levels will increase significantly during
active
thrombosis, but revert towards normal during increasingly intense
anticoagulation regimens. These findings demonstratethe validity of FPB as a
marker of thrombosis.
Example 3' Fibrinopeptide B in a Model of Acute Pulmonary Embolism
The objectives of this experiment were to determine if the thrombotic
activity of pulmonary embolism can be measured using a novel assay for plasma
levels of fibrinopeptide B and to test the hypothesis that embolization itself
causes pre-existing thrombi to thrombose further, possibly leading to clot
enlargement and downstream micro-embolization.
The cardiopulmonary effects of major pulmonary embolism are
incompletely understood. In particular the role of active thrombosis in the
detrimental effects on the pulmonary circulation is not known. In fact, it has
yet
to be determined whether emboli in the high-flow circulation of the pulmonary
arteries increase their thrombotic activity relative to the activity occurring
prior
to embolisation. Using a recently developed plasma marker for active
thrombosis, fibrinopeptide B (FPB) the following experiments were performed
to determine if the thrombotic activity of venous thrombi increases following
embolisation.
Most patients with pulmonary emboli survive their illness when treated
with "standard doses" of anticoagulants. However, a sub-population of patients
with massive PE have a high mortality even when treated with standard
anticoagulation and thrombolytics. The pathophysiology of their deaths is not
clear, since a large proportion of patients die several days after the initial
event
and the size of the embolization does not directly predict mortality. It is
hypothesized that embolization itself promotes further thrombotic activity,
which
may either propagate the existing clot or lead micro-embolization into small
pulmonary arteries, worsening right ventricular failure.
Clinicians treating patients with PE do not currently have a practical
method of determining the degree to which anticoagulation has arrested the
process of clotting. The doses of anticoagulants necessary to treat PE are
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determined in lar a art b outcome measures in atients who are clinicall
g p Y p Y
stable. An assay is described below for fibrinopeptide B (FPB) and its
metabolites, which are released into the circulation only during active
thrombosis. This assay was used in an animal model to determine if pulmonary
embolization itself accelerates the thrombotic activity of pre-existing deep
vein
thrombi.
Since the immunoassay for human FPB does not recognize canine FPB,
the canine thrombosis model is modified so that both the pre-existing thrombi
and the circulating fibrinogen are predominantly of human origin. Therefore,
the
dogs have their native fibrinogen inactivated to a substantial degree.
Materials: Maxisorp ELISA plates were obtained from Nunc
(Naperville, IL). Synthetic human fibrinopeptide B and fibrinopeptide A (both
99% pure), canine fibrinogen (>60% clottable), bovine serum albumin (>98%
pure), ancrod, and tranexamic acid were purchased from Sigma (St. Louis, MO).
N-cysteinyl fibrinopeptide B (>90% pure) and des-arg fibrinopeptide B (>98%
pure) were custom synthesized by Peninsula Laboratories (San Carlos, CA) and
Immuno-Dynamics (La Jolla, CA) respectively. Human fibrinogen (>95%
clottable) and aprotinin were purchased from Calbiochem (La Jolla, CA).
Affinity purified goat anti-rabbit IgG (Fc fragment specific, peroxidase-
conjugated) was purchased from Jackson ImmunoResearch Laboratories (West
Grove, PA). Maleimide-activated keyhole limpet hemocyanin and Freund's
adjuvant (complete and incomplete) were purchased from Pierce (Rockford, IL).
Topical thrombin (bovine) and unfractionated heparin (porcine) were obtained
from Jones Pharma (St. Louis, MO) and Elkins-Sinn (Cherry Hill, NJ),
respectively. Propofol and halothane were obtained from Zeneca
Pharmaceuticals (Wilmington, DE) and Halocarbon Laboratories (River Edge,
NJ), respectively. All other chemicals were reagent grade or better.
Methods: FPB Antiserum: An immunoconjugate of human FPB was
prepared by coupling 5 mg of N-cysteinyl-FPB to 10 mg of maleimide-activated
keyhole limpet hemocyanin according to a protocol provided by Pierce Chemical
Company. Polyclonal antiserum to FPB was raised in New Zealand White
rabbits. Three rabbits each received a primary subcutaneous injection of
immunoconjugate (one mg) in complete Freund's adjuvant followed by two
subcutaneous booster injections (one mg each) in incomplete Freund's adjuvant)
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given at four week intervals. Rabbits were bled at two-week intervals starting
two weeks after the primary injection. After allowing the blood to clot
overnight
at 4 C, antiserum was collected by centrifugation (2000xg, 10 min) and stored
at
-20 C until analyzed. The FPB antibody titer of each antiserum was determined
in a direct-binding ELISA. Briefly, microplate wells were coated with
synthetic
FPB and blocked with BSA. Wells were then incubated with serial dilutions of
FPB antiserum followed by peroxidase-conjugated goat anti-rabbit IgG detecting
antibody. Wells were developed with o-phenylenediamine (OPD) substrate
solution, and the absorbance (490 nm) of each well was measure in a microplate
reader (Molecular Devices, Sunnyvale, CA). The antibody titer was arbitrarily
defined as the antiserum dilution resulting in an absorbance of 1Ø The
antiserum yielding the highest titer (1:2500) was used in the FPB assay
described below.
FPB Assay: A competitive ELISA was developed for determination of
the FPB concentration in plasma and urine ultrafiltrates. Unless otherwise
noted,
all steps were performed at room temperature, and the microplates were washed
three times in between each step with 0.02 M NaH2P04, 0.15 M NaCI,
1 mM EDTA, pH 7.4 (PNE) containing 0.1 % tween-20. Microplate wells were
first coated with synthetic FPB (2 pg/ml in 0.2 M NaHC03, 100 ~l/well)
overnight at 4 C, and then "blocked" with 1% BSA in PNE (PNEA) for one
hour. Meanwhile, 20 ~1 of ultra-filtered samples and synthetic FPB standards
(1.56-100 ng/ml) were pre-incubated separately with 200 p1 of FPB antiserum
(1:2500) in polypropylene microfuge tubes for one hour. Dilutions of the FPB
standard and antiserum were prepared in dilution buffer (PNEA containing
0.1% tween-20). At the end of the pre-incubation period, 100 p1 of each
mixture
were applied to duplicate FPB-coated wells and incubated for one hour. After
washing, 100 ~1 of peroxidase-conjugated goat anti-rabbit IgG detecting
antibody (1:5000 in dilution buffer) were added to each well and incubated for
one hour. After a final wash, each well was developed with 100 p1 of OPD
substrate solution for exactly five minutes. The reaction was stopped by
addition
of an equal volume of 3 N HzS04, and the absorbance (490 nm) of each well was
measured in a microplate reader. The concentration of FPB in the samples was
interpolated from a standard curve obtained by plotting the FPB concentration
of
each standard versus the corresponding absorbance. If sample values exceeded
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the upper limit of the standard curve, those samples were re-tested after
appropriate dilution with dilution buffer. The intra- and inter-assay
coefficients
of variation for the FPB assay were 14.1 % and 5.0%, respectively. The
competitive ELISA was also used to assess the cross-reactivity of the FPB
antiserum with human fibrinogen, fibrinopeptide A (FPA), des-arg FPB, and
canine FPB.
Fibrinogen Assay: Fibrinogen levels in plasma were determined using a
commercial kit (Sigma, St. Louis, MO). Briefly, 251 of sample (or standard)
diluted in buffer were mixed with SO~tI of thrombin solution provided with the
kit, and the clotting time was recorded. The clotting time was taken as the
first
appearance of a fibrin "string" from repeated dipping of a wooden applicator
stick into the sample (or standard) and thrombin mixture. All assays were
performed in triplicate. The fibrinogen concentration of each sample was
determined by interpolation from a standard curve obtained by plotting log
(fibrinogen concentration) versus log (clotting time). The fibrinogen
concentration of samples containing heparin, or samples with low fibrinogen
(less than 20 mg/dL) could not be determined due to an inability to form a
fibrin
clot in the assay.
Fibrinogen Replacement and Thrombosis Model.' Eight healthy male
mongrel dogs (20-24 kg) were used in the study. Each animal was anesthetized
with intravenous Propofol (6-10 mg/kg), intubated, and mechanically
ventilated.
Anesthesia was then maintained throughout the study period with Halothane
(1-2% in room air). Native canine fibrinogen was depleted by intravenous
infusion of ancrod (2-3 u/kg) over a four-hour period. Each animal was allowed
to recover from anesthesia and returned to quarters. Two days later, each
animal
was anesthetized, intubated and ventilated as before, and a Foley catheter was
placed for urine collection. A catheter was placed in the dorsalis pedis
artery for
continuous blood pressure monitoring. Normal saline (0.9% NaCI) was
administered intravenously to maintain urinary output at approximately 50
mL/h.
Each animal was then transfused with six grams of purified human fibrinogen
(dissolved in 250 mL of normal saline) over a 90-minute period. Following
fibrinogen replacement, tranexamic acid was administered intravenously
(110 mg/kg every six hours) to inhibit fibrinolysis. Cut-downs were performed
bilaterally on the saphenous veins for placement of special double-balloon
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3~
catheters, which were subsequently advanced under fluoroscopy into the right
and left femoral veins. After balloon inflation, which created a sealed intra-
vascular chamber, thrombosis was induced by injecting bovine thrombin (200
units) through a small port in the catheter between the inflated balloons.
During
a four-hour clot-aging period, the balloons were partially deflated to allow
for
restoration of blood flow and clot propagation. After the clot-aging period,
pulmonary emboli were created by passive motion of one of the legs containing
a femoral thrombus (see below).
Allocation of subjects: Three of the eight animals were arbitrarily
selected to receive intravenous heparin (300 units/kg bolus followed by 90
units/kg/hr infusion) one hour prior to embolization of the femoral thrombus.
At
the conclusion of the study (five hours after embolization), each animal was
given a bolus intravenous injection of heparin (3000 units) to prevent post-
mortem blood coagulation, followed by intravenous nembutal (120 mg/kg) to
induce cardiac arrest. An autopsy was carefully performed to determine the
precise location of all clots. Finally, all femoral vein thrombi and pulmonary
emboli were collected separately and weighed. The protocol was approved by
the University of California, San Diego Animal Subjects Committee. Care and
handling of experimental animals conformed to the standards established by the
University of California, San Diego Department of Veterinary Services, which
are in compliance with Federal recommendations.
Sample Collection and Processing: Blood samples (4.5 mL) were drawn
at various times during the study and added to vacutainer tubes containing
0.5 mL of 0.129 M buffered sodium citrate with protease inhibitors (500 units
each of aprotinin and heparin). Protease inhibitors were omitted from blood
samples used for fibrinogen assay. Plasma was obtained by centrifugation at
2500xg for 10 minutes (4 C). Plasma for fibrinopeptide B (FPB) assay was
diluted with an equal volume of 0.9% (w/v) NaCI and subjected to
ultrafiltration
using Ultrafree (Biomax-100) centrifugal filter devices (Millipore, Bedford,
MA)
according to the manufacturer's recommendations, and the ultrafiltrate was
saved for analysis. Urine was collected in 60-minute intervals throughout the
study, starting with the onset of human fibrinogen transfusion. The volume of
urine collected during each 60-minute time interval was recorded, and a sample
(4.5 mL) for FPB assay was added to a vacutainer tube containing sodium
citrate
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38
with protease inhibitors, clarified by centrifugation (2500xg, 10 minutes, 4
C),
and then subjected to ultrafiltration as described above. All samples were
stored
at -70 C until analyzed.
Statistics: Data are presented as mean~standard error of mean (SEM)
unless otherwise noted. Group comparisons were performed on log
transformation of raw data using the unpaired t test (two-sided). Differences
between means were deemed significant forp<0.05.
Results
Specificity of the FPB Antiserum: The specificity of the FPB antiserum
was assessed in the competitive ELISA described above. As shown in Figure
1A, native human FPB in solution was an effective competitive inhibitor of
antibody binding to surface-bound synthetic human FPB. In contrast, native
canine FPB in solution did not compete for antibody binding to surface-bound
synthetic human FPB. Competitive ELISA was also used to assess the cross-
reactivity of the FPB antiserum with human fibrinogen as well as several
synthetic human fibrinopeptides. As shown in Figure 1B, the competition curves
for FPB and des-arg FPB were nearly identical. The cross-reactivity of
des-arg FPB, defined as ICS° of FPB (6.7 nM) divided by ICS° of
des-arg FPB
(8.9 nM) times 100, was 75%. Thus, the FPB assay is sensitive to both FPB and
its primary metabolite, des-arg FPB. Although FPA exhibited essentially no
cross-reactivity (<0.1%), parent fibrinogen showed significant cross-reaction.
The ICS° of fibrinogen (2.3 nM) was about one-half the ICS°
of FPB, not
unexpected since each fibrinogen molecule harbors two potentially cross-
reacting FPB sequences. Cross-reacting fibrinogen molecules (340,000 kD)
were effectively removed from study samples by centrifugal ultrafiltration
with
Biomax-100 membranes (100,000 kD cut-off) during sample processing (data
not shown).
Fibrinogen Replacement: Because the FPB antiserum did not cross-react
with canine FPB in the FPB assay, it was necessary to replace canine with
human fibrinogen in the thrombosis model. This was accomplished by pre-
treating each animal with ancrod, which effectively depleted autologous
fibrinogen from the circulation. Plasma fibrinogen levels were determined
before and after treatment with ancrod, and then again before and at various
times after transfusion with purified human fibrinogen. During a two-day rest
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period in between ancrod treatment and heterologous fibrinogen transfusion,
ancrod was cleared from the circulation (data not shown) and fibrinogen levels
remained below the limit of detection. Fibrinogen levels after ancrod
treatment
and before replacement with human fibrinogen were below the limit of detection
S (20 mg/dL) in all animals. Following transfusion with purified human
fibrinogen, the plasma fibrinogen level was restored to approximately
400 mg/dL and remained within normal limits (200-400 mg/dL) throughout the
remainder of the study period.
Clot Characteristics: A femoral thrombus and pulmonary embolus was
recovered from each animal at autopsy. Emboli were sometimes lodged in
multiple lobar and/or segmental pulmonary arteries. Clots ranged in weight
from
0.37-2.50 grams for thrombi and 0.29-0.78 grams for emboli (combined weight
of all pieces). The mean~SEM clot weights for thrombi (0.820.26 g) and
emboli (0.540.06 g) were not significantly different (p=0.31).
1 S Plasma FPB Levels During Thromboembolism: FPB levels in plasma
were measured before, and at various times after induction of femoral vein
thrombosis. As shown in Figure 3, the mean~SEM plasma level rose sharply
from baseline (3.71.1 ng/mL) to a peak level of 25.48.9 ng/mL (p<0.005) ten
minutes after induction of thrombosis. FPB levels then fell but remained
significantly elevated (p<0.05) for the first hour after thrombosis before
returning to near baseline. Four hours from induction of thrombosis, one of
the
two femoral clots in each animal was embolized. One hour prior to
embolization, three of the animals were treated with intravenous heparin as
described in the Methods section. After embolization, FPB again rose sharply
to
a peak level of 20.27.7 ng/ml, and then returned to baseline within one to two
hours. However, the rise in FPB following embolization was completely
suppressed in the animals treated with heparin prior to embolization (Figure
2).
Urinary FPB Levels During Thromboembolism: Urine was collected
hourly from each animal before and after induction of femoral vein thrombosis.
Total FPB excreted in the urine during each time interval is presented in
Figure
3. The meantSEM total urinary FPB for all animals was significantly elevated
(3.30.7 fig) from baseline (0.5~ 0.1 fig) within one hour of thrombosis
(p<0.001) and remained significantly elevated throughout the study period. The
initial peak of urinary FPB (20.37.3 pg) occurred four hours after induction
of
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thrombosis as opposed to the initial peak of plasma FPB, which occurred ten
minutes after induction of thrombosis. The initial peak of urinary FPB was
suppressed in animals treated with heparin prior to embolization. Total
urinary
FPB was on the rise again in all animals during the last time interval of the
study
(five hours after embolization). Besides total FPB, the concentration of FPB
in
each hourly urine collection following induction of thrombosis was also
significantly elevated compared to baseline for each group of animals (data
not
shown).
Conclusions
Plasma FPB rapidly rises during active thrombosis, but is also rapidly
cleared from the circulation. The peptide appears in the urine after a delay
of
one to several hours from the onset of thrombosis and remains elevated even if
thrombus propagation is suspended by systemic anticoagulants. Thus, urine FPB
levels can provide an excellent indication of recent thrombotic activity even
when the thrombus propogation is no longer occuring at the time the patient is
tested or seeks medical assistance. It was also noted that pulmonary
embolization itself causes an acceleration of thrombosis, which can be
completely inhibited with heparin. Therefore, DVT patients with a high risk of
embolization should be promptly anticoagulated.
Example 4: Elevation of FPB in Clinical Samples Taken from Patients with
DVT and PE
The objectives of this experiment was to determine if plasma and urine
levels of FPB (using the newly created ELISA assay) are higher in patients
with
acute pulmonary embolism or deep venous thrombosis than they are in normal
volunteers and in those with other medical conditions.
VTE commonly occurs in the setting of other medical illnesses
(Goldhaber et al. 1983, Hirsch et al. 1995), and it would be helpful to
include
these complex patients in the current project. Unfortunately, many medical
conditions involve diffuse fibrin polymerization at sites of inflammation,
which
may alter the plasma levels of thrombosis markers. Therefore, the next step in
the project is to determine whether FPB,o~ levels can distinguish patients
with
VTE from patients with other illness.
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A clinical study was performed at UCSD testing urinary FPB levels in
patients suspected of having DVT and/or PE. The results demonstrated that FPB
levels increased sufficiently during DVT and PE to distinguish patients with
thrombosis from those with other medical conditions (Eckhardt et al. 1981)
Methods: Consecutive inpatients at the UCSD medical center referred by
their treating physicians to the Acute Venous Thrombo-embolism Service, and
suspected of having either a DVT or PE were asked to participate in the study.
Informed consent was obtained by one of the investigators.
Upon entry into the study, blood and urine samples were collected from
the patient (prior to the initiation of systemic anticoagulation, if
possible). These
samples were processed for ex-vivo analysis with the newly created FPB ELISA
assay.
The subjects completed diagnostic workup for venous thromboembolic
disease (as indicated clinically). Without knowledge of the laboratory test
results, the PI reviewed the objective tests used to confirm or refute the
diagnoses of DVT (lower extremity compression ultrasound exams, venograms)
and PE (ventilation/perfusion scans, contrast-enhanced helical CT scans,
pulmonary angiograms). According to accepted criteria, the patients were
classified as: 1) YTE positive - thromboembolic disease confirmed by objective
testing; 2) VTE negative - thromboembolic disease refuted; or 3) YTE
indefinite
- definitive testing not completed.
In those patients who received therapy for VTE, a second blood and urine
sample was obtained once adequate levels of anticoagulation have been achieved
(assessed by clinical parameters),. Again, these samples were processed for ex-
vivo analysis with the FPB ELISA assay.
Results:
The urine FPB levels were markedly higher in the patients with DVT
than in patients without DVT and in controls (figure 4). Ten patients in whom
the diagnosis was conformed by objective testing had mean (+/- SEM) levels of
FBP in the urine of 96 (+/- 41) ng/ml. Three patients in whom the diagnosis
was
refuted had urinary FPB levels of 2.7 (+/- 1.9) ng/ml. The mean (+/- SEM)
urinary FPB level in nine healthy control patients was 2.15 (+/- 1.9) ng/ml.
In these experiments, there was no difference in plasma FPB,ot levels
between normal volunteers and patients with DVTs (results not shown) despite
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~a
the strong dependence observed with urine FPB levels. Although the few
patients with pulmonary emboli tended to have higher plasma levels of FPB~o~,
the differences were not great and the population tested in these preliminary
experiments was small. Therefore, although the invention includes FPB tests
for
both plasma and urine, the focus for the next examples was on the
determination
of FPB levels in the urine.
It was noted that we observed excellent correlation of FPB levels in urine
with the presence or absence of DVT even though no correction was made for
urine volume or rates of glomular filtration. In some applications it is
beneficial
to also measure a marker (e.g., creatinine, protein, albumin) in the urine
that is
indicative of urine volume or rates of glomular filtration. The concentration
of
this marker can be used to normalize the measured concentration of FPB so as
to
account for these effects.
Conclusions
Thus, urine FPB levels are much higher in patients with venous thrombo-
embolic disease than they are in other patients.
Example 5: Measurement of FPB in the Plasma and Urine of Hospitalized
Patients at Risk of DVT/PE.
Objective: To further delineate the specificity of FPB for acute
thrombosis, FPB plasma/urine levels are measured in hospitalized patients with
a
variety of other medical and surgical conditions, in whom the diagnosis of
venous thromboembolic disease is not suspected (and not suggested by screening
tests).
Methods:
Enrollment. Consecutive inpatients at UCSD Medical Center (n=765) in
whom compression stockings are requested for VTE prophylaxis, and in whom
the attending physicians give permission for entry were considered candidates
for the study. Informed consent is obtained by one of the investigators. The
investigators perform an initial screen for DVT (see below). Exclusion
criteria
include refusal of consent, DVT on initial CUS, arterial insufficiency,
contact
allergy to the cuff material, injury to the lower extremity precluding cuff
use, use
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y.3
of IPC devices for greater than 12 hours during the current admission and
previous episodes of VTE.
Plasma and Urine FPB Levels are measured in each patient as follows: At
each time point described below, blood samples (20 ml by fresh venupuncture)
and urine samples (20-40 ml) are collected into tubes containing
anticoagulants.
Plasma and urine are ultrafiltered to remove fibrinogen and tested for
fibrinopeptide B. The levels are compared to the results of the results of the
following anatomical screening tests for DVT and PE.
Time Points for Outcome Measurement. Patients are screened for VTE
at the following time points: upon entry into the study, two weeks after
application of the venous compression device, upon discontinuation of the
device and one week after discontinuation of the device. In those cases where
the patients have been discharged earlier than one week after discontinuation
of
the devices, they are offered a small monetary incentive for returning for a
final
examination. The details of the screening are discussed below.
Outcome - DVT. Compression ultrasonography (CUS) is used to
confirm the presence of DVT in symptomatic patients and to screen for DVT in
asymptomatic patients. The strength of this approach is that the specificity
of
CUS for asymptomatic DVT is 97%, roughly equal to the specificity in
symptomatic patients. Therefore, a positive test can be relied upon.
At each time point, patients are interviewed, examined and CUS is
performed. If no signs or symptoms of DVT are disclosed, and the CUS is
negative, no further work-up is performed. If CUS discloses non-compressible
proximal LE veins (popliteal and above), the patient is considered to have a
DVT, regardless of symptoms. If signs or symptoms of DVT are present, and
the initial the CUS is negative, it is repeated on day two and seven. If any
of the
serial CUS exams disclose non-compressible LE veins, the patient is considered
to have a DVT. If none are positive, the patient was considered to be free of
DVT.
Patients with signs or symptoms DVT upon entry into the study in whom
the first series of CUS studies disclose DVT are assumed to have had DVT prior
to application of IPC. These patients are dropped from the study and excluded
from further analysis.
WO 01/01150 _. --PCT/US00/17977
Outcome - PE. At each time point, patients are interviewed and
examined. If no signs or symptoms of PE are disclosed, no further work-up is
performed. If a PE is clinically suspected, the patients attending physician
is
notified. It is the standard of care at UCSD Medical Center to perform a work-
up that follows ATS guidelines. Briefly, a ventilation/perfusion (V/Q) scan is
performed, if possible. Normal V/Q scans are accepted as ruling out PE. Scans
disclosing two or more segmental or larger perfusion defects unmatched by
ventilation defects ("high probability" scans) are accepted as diagnostic of
PE.
All other scans are considered non-diagnostic. Patients with non-diagnostic
V/Q
scans received pulmonary angiography. Although the investigators recommend
that the work-up for PE follows these guidelines, diagnostic tests are ordered
as
indicated by the patients' attending physicians.
The attending physician of any patient diagnosed with DVT or PE is
notified of the results of the diagnostic tests. Treatment is at the
discretion of the
attending physician, although a consultation from the Acute Venous Thrombo-
Embolism service is usually offered.
Results.
The mean (+/- SD) levels of FBP in the urine of 209 patients without
DVT, being admitted to the hospital for various reasons, were 11.6 (+/- 25.4)
ng/ml (figure 5). At the time of discharge (after hospitalization for a least
48
hours), the urine FPB levels in those without DVT were 16.5 (+/- 34.3).
Conclusions
These results indicate that elevated levels of urinary FPB are uncommon
in hospitalized patients, which is not the case for the plasma D-dimer test.
The
specificity of the FPB is therefore higher than for other clinically available
markers for thrombosis, such as the D-dimer.
Example 6: FPB Levels Durin~Treatment
The objective of this experiment was to determine if urinary or plasma
FPB levels, measured during anticoagulation with "therapeutic doses" of
heparin, are reduced relative to the pre-treatment levels in VTE patients.
CA 02372700 2001-12-20
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L/
The validity of FPB,o~ for following anticoagulation efficacy is tested by
determining whether urine levels (or the plasma levels described above)
reliably
fall as VTE is treated with large doses of heparin.
Consecutive patients referred to the VTE service in whom the diagnosis
of DVT and/or PE is confirmed (based on the criteria described above) are
enrolled. Prior to therapy, blood and urine samples are collected for
measurement of FPB,o~ (and other FPB measurements described above). Patients
are then be treated with intravenous heparin (80 units/kg bolus, 18
units/kg/hr
infusion), with the dose adjusted per protocol to keep aPTT 1.5-2.5 times the
control values. As soon as steady-state "therapeutic doses" of heparin are
maintained, blood and urine are collected again and the same parameters are
measured. In addition, plasma aPTT, anti-Xa and anti-thrombin measurements
are made as previously described (Morns et al. 1998).
For each patient, the difference between urinary and plasma FPBto~ levels,
1 S measured before and after anticoagulation, is recorded. The mean reduction
in
FPB,ot levels after anticoagulation is calculated. The required sample size is
estimated based on the preliminary data described below: The mean (+/-SD)
decrease in urinary FPB~o~ during anticoagulation was 60 ng/ml (+/-85.7
ng/ml).
Assuming similar standard deviations for the VTE patients to be studied,
thirty-
seven pairs of samples are required in order to detect a 50% decrease in the
levels after anticoagulation, with an alpha of 0.01 and a power of 95%.
Preliminary Results: Urinary levels of FPB significantly decreased
during anticoagulation in all but one patient (figure). The sole patient in
whom
urinary FPB increased had sustained a crush injury to his leg and required
major
surgery on the day that his repeat samples were obtained. More importantly,
his
heparin dose, as estimated by his aPTT measurements, had not achieved
therapeutic range during the time interval between his first and second tests.
In
fact this patient also demonstrated larger elevations in his plasma FPB on his
second day of sampling than on his first day (results not shown).
Discussion: This portion of the study suggests that urine levels of FPB
have the unique property of decreasing as patients with VTE are
anticoagulated.
After further development of the test (and perhaps development of variations
in
the FPB plasma test - described above) they may be used as valid estimates of
WO 01/01150 PCT/US00/17977
y6
the short-term effectiveness of anticoagulation. These markers may represent
the
first test of actual anti-thrombotic effectiveness applicable in the clinical
setting.
All publications and patents are herein incorporated by reference to the
same extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by reference. It
will be
apparent to one of ordinary skill in the art that many changes and
modifications
can be made in the invention without departing from the scope of the appended
claims.
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