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Patent 2378898 Summary

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(12) Patent Application: (11) CA 2378898
(54) English Title: METHOD FOR MEASURING COAGULANT FACTOR ACTIVITY IN WHOLE BLOOD
(54) French Title: PROCEDE DE MESURE DE L'ACTIVITE DU FACTEUR COAGULANT
Status: Deemed Abandoned and Beyond the Period of Reinstatement - Pending Response to Notice of Disregarded Communication
Bibliographic Data
(51) International Patent Classification (IPC):
  • A61K 38/00 (2006.01)
  • A61K 38/48 (2006.01)
  • C12Q 1/56 (2006.01)
  • G01N 33/86 (2006.01)
(72) Inventors :
  • MACKMAN, NIGEL (United States of America)
  • MCDONNELL, JOHN J. (United States of America)
(73) Owners :
  • THE SCRIPPS RESEARCH INSTITUTE
  • COAGULATION DIAGNOSTICS, INC.
(71) Applicants :
  • THE SCRIPPS RESEARCH INSTITUTE (United States of America)
  • COAGULATION DIAGNOSTICS, INC. (United States of America)
(74) Agent: MBM INTELLECTUAL PROPERTY AGENCY
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2000-07-24
(87) Open to Public Inspection: 2001-02-01
Availability of licence: N/A
Dedicated to the Public: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2000/020118
(87) International Publication Number: WO 2001007070
(85) National Entry: 2002-01-17

(30) Application Priority Data:
Application No. Country/Territory Date
60/145,140 (United States of America) 1999-07-23

Abstracts

English Abstract


The present invention is directed to methods to rapidly assess the overall
coagulant properties of a patient's blood sample by measuring and comparing
clotting time with and without an added inhibitor of a procoagulant or an
anticoagulant. When the sample is whole blood, the resulting clotting time
represents the overall coagulant activity of the plasma and cellular
components of the blood, which is indicative of existing or impending
pathology arising from abnormal coagulability. The invention also provides a
method for measuring the risk of a patient for a thrombotic event by
determining functionally current levels of one or more procoagulants or
anticoagulants in whole blood. In addition, a method for measuring the
effectiveness of anticoagulant therapy is provided. Kits for performing the
method of the invention are also provided.


French Abstract

L'invention concerne des procédés d'évaluation rapide des propriétés coagulantes globales d'un échantillon sanguin d'un patient, qui consistent à mesurer et à comparer le temps de coagulation avec et sans inhibiteur ajouté d'un procoagulant ou d'un anticoagulant. Lorsque l'échantillon est constitué de sang entier, le temps de coagulation résultant représente l'activité coagulante globale du plasma et des composants cellulaires du sang, laquelle est représentative d'une pathologie existante ou imminente découlant d'une coagulabilité anormale. L'invention porte également sur un procédé de mesure du risque chez un patient d'événement thrombotique, par la détermination des taux courants au plan fonctionnel de procoagulant(s) ou d'anticoagulant(s) dans le sans entier. Par ailleurs, une méthode de mesure de l'efficacité d'une thérapie anticoagulante est décrite ainsi que des kits pour la mise en oeuvre de la méthode de l'invention.

Claims

Note: Claims are shown in the official language in which they were submitted.


We claim:
1. A method for determining the presence and functional measurement of
a procoagulant in whole blood, comprising:
(a) collecting a sample of whole blood;
(b) dividing the sample of whole blood into at least two aliquots;
(c) adding at least one inhibitor of the procoagulant to one of the
aliquots;
(d) incubating the aliquots;
(e) measuring a clotting time for each of the aliquots; and
(f) comparing the respective clotting times of the aliquots.
2. The method of claim 1, wherein the procoagulant is selected from the
group consisting of a-2-antiplasmin, fibrinogen, high molecular weight
kininogen,
kallekrein, prekallekrein, tissue factor, Factor II, Factor IIa, Factor Va,
Factor VIIa,
Factor VIIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, and Factor
XIIIa.
3. The method of claim 1, further comprising adding a contact activation
inhibitor to the sample of whole blood or collecting the sample or whole blood
in the
presence of contact activation inhibitor.
4. The method of claim 3, wherein the contact activation inhibitor is corn
trypsin inhibitor.
5. The method of claim 1, wherein the inhibitor of the procoagulant is an
antibody or an analogue of a procoagulant substrate .
6. The method of claim 5, wherein the inhibitor is an antibody.
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7. The method of claim 5, wherein the analogue is a peptide.
8. The method of claim 6, further comprising adding a control antibody to
an aliquot that does not contain the antibody inhibitor of the procoagulant,
wherein
the control antibody is added in an amount that is substantially equivalent to
the
antibody inhibitor of the procoagulant.
9. The method of claim 1, wherein the procoagulant is not tissue factor.
10. A method for determining the presence and functional measurement of
an anticoagulant in whole blood, comprising:
(a) collecting a sample of whole blood;
(b) dividing the sample of whole blood into at least two aliquots;
(c) adding an inhibitor of the anticoagulant to one of the aliquots;
(d) incubating the aliquots;
(e) measuring a clotting time for each of the aliquots; and
(f) comparing the respective clotting times of the aliquots.
11. The method of claim 10, wherein the anticoagulant is selected from the
group consisting of .alpha.-1-antitrypsin, activated protein C, antithrombin
III, C1 esterase
inhibitor, heparin, protein C, protein S, thrombomodulin, and tissue factor
pathway
inhibitor.
12. The method of claim 10, further comprising adding a contact activation
inhibitor to the sample of whole blood or collecting the sample or whole blood
in the
presence of contact activation inhibitor.
13. The method of claim 12, wherein the contact activation inhibitor is
corn trypsin inhibitor.
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14. The method of claim 10 wherein the inhibitor of the anticoagulant is an
antibody.
15. The method of claim 14, further comprising adding a control antibody
to an aliquot that does not contain the antibody inhibitor of the
anticoagulant, wherein
the control antibody is added in an amount that is substantially equivalent to
the
antibody inhibitor of the anticoagulant.
16. The method according to claim 1, wherein the sample of whole blood
is collected into a solution comprising citrate and before step (e) the blood
is
recalcified.
17. The method according to claim 10, wherein the sample of whole blood
is collected into a solution comprising citrate and before step (e) the blood
is
recalcified.
18. The method according to claim 16, wherein the sample of whole blood
is collected into a solution comprising citrate and before step (e) the blood
is
recalcified.
19. A kit for determining the presence and functional measurement of a
procoagulant in whole blood, comprising:
(a) a vial containing an inhibitor of a procoagulant;
(b) a vial containing a non-inhibitory control reagent; and
c) a vial containing liquid citrate anticoagulant.
20. The kit of claim 19, wherein the vial containing liquid citrate
anticoagulant further comprises corn trypsin inhibitor.
21. The kit of claim 20, wherein the procoagulant is tissue factor.
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22. The kit of claim 21, wherein the inhibitor is at least one antibody.
23. A kit for determining the presence and functional measurement of an
anticoagulant in whole blood, comprising:
a) a vial containing an inhibitor of an anticoagulant;
b) a vial containing a non-inhibitory control reagent; and
c) a vial containing liquid citrate anticoagulant.
24. The kit of claim 23, wherein the vial containing liquid citrate
anticoagulant further comprises corn trypsin inhibitor.
25. The kit of claim 24, wherein the inhibitor is at least one antibody.
26. A method for determining the presence and functional measurement of
a procoagulant in whole blood, comprising:
(a) collecting a sample of whole blood;
(b) dividing the sample of whole blood into at least four aliquots;
(c) adding an immunomodulator to two of the aliquots;
(d) adding at least one inhibitor of the procoagulant to one of the
aliquots without the immunomodulator and to one of the
aliquots with the immunomodulator;
(e) incubating the aliquots;
(f) measuring a clotting time for each of the aliquots; and
(g) comparing the respective clotting times of the aliquots.
27. The method of claim 26, wherein the whole blood sample further
comprises an anticoagulant and corn trypsin inhibitor.
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28. The method of claim 27, wherein the immunomodulator is endotoxin.
29. The method of claim 27, wherein the inhibitor is an antibody.
-41-

Description

Note: Descriptions are shown in the official language in which they were submitted.


CA 02378898 2002-O1-17
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METHOD FOR MEASURING COAGULANT FACTOR ACTIVITY
IN WHOLE BLOOD
This invention was made with Government support under Grant No. HL
48872 awarded by the National Institutes of Health. The Government has certain
rights in this invention.
FIELD OF THE INVENTION
This invention relates generally to the fields of medical diagnostics and
disease prevention. More specifically, it relates to diagnostic methods and
test kits for
rapidly assessing the coagulation activity of blood by measuring the rate of
blood
clotting using whole blood samples in the presence and absence of at least one
inhibitor of a procoagulant or anticoagulant. The coagulation activity in the
samples
of an individual's blood, and the difference in activity between the samples,
is an
indicator of the existence or potential development of certain pathological
conditions.
BACKGROUND OF THE INVENTION
The propensity for blood to clot too rapidly is an important predictor of the
development, progression, and recovery from a number of serious pathological
conditions. These conditions arise either directly from the clotting process,
or are
modulated by it. Examples of such conditions include heart attack, stroke,
coronary artery disease, deep vein thrombosis, and pulmonary embolism, among
others. Of these diseases, coronary artery disease is a leading cause of
mortality in the
United States.
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Furthermore, certain clinical conditions, such as vascular disease, surgery,
trauma, malignancy, prosthetic vascular devices, general anesthesia,
pregnancy, the
use of oral contraceptives, systemic lupus erythematosus, and infection may
predispose individuals to undergo adverse clotting events. Often, patients
with acute
conditions suspected of resulting from clotting abnormalities appear in the
emergency
room. A method for rapidly detecting, in a whole blood sample, the patient's
current
risk for clot formation would help rule in or rule out thrombotic events and
coagulopathies. This would also improve the delivery of emergency health care
to
those who need it, while offering early identification of patients whom may
progress
to potentially lethal clotting pathology.
Blood may also clot too slowly, or not at all, which can lead to bleeding or
other blood coagulation disorders. The hemophilias are examples of inheritable
bleeding disorders. In addition, diseases affecting the liver, such as
alcoholic cirrhosis
and acute and chronic hepatitis, are associated with numerous clotting
abnormalities,
because this organ synthesizes many of the coagulation factors.
The best known of the inherited disorders of coagulation are hemophilia A and
B, associated with a decrease in the activity of Factor VIII and IX,
respectively. The
severity of the disorder depends on the extent of depletion of the respective
clotting
factors. Severe cases are manifested early in life, and children with
hemophilia
usually show easy bleeding in large joints, such as the knees, and marked
defects in
clot formation. In milder forms, hemophilia may not be evident until later in
life.
Treatment of hemophiliac generally consists of transfusions of concentrates of
blood products in which there is a large amount of coagulation Factors VIII or
IX.
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While many hemophiliacs can lead a relatively normal life, extra precautions
must be
taken in engaging in sports and during surgery or dental care. Unfortunately,
10
percent of people with hemophilia develop antibodies to Factor VIII and become
difficult to treat.
The condition in which blood clots too quickly (i.e., hypercoagulability) is
also a pathological condition. Disseminated intravascular coagulation (DIC) is
an
example of an acquired coagulation disorder characterized by pathologically
fast
blood clotting.
Blood clotting is a complex process involving multiple initiators, cascades of
activators, enzymes, and modulators, ultimately leading to the formation of
fibrin,
which polymerizes into an insoluble clot. The intrinsic and extrinsic blood
clotting
pathways are described in, for example, Davie et al., The Coagulation Cascade:
Initiation, Maintenance, and Regulation, Biochemistry, vol. 30(43):10363-70
(1991),
which is incorporated herein by reference.
Classically, the propensity for blood to clot is determined, either manually
or
automatically, by measuring the time needed for a sample of plasma or blood to
form
insoluble fibrin strands or a clot. For example, clot formation may be
detected
visually by observing the formation of fibrin strands, or by automated
methods, such
as by detecting changes in viscosity by measuring mechanical or electrical
impedance,
or by photo-optical detection.
The measurement of clotting time may be made immediately on freshly drawn
blood without added anticoagulants. Alternatively, one can use blood
containing a
calcium ion-binding anticoagulant such as citrate. In this case, the clotting
time
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CA 02378898 2002-O1-17
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measurement is initiated by adding a calcium salt to reverse the effect of the
anticoagulant. This latter determination is referred to as the recalcification
time.
Typical methods for the measurement of blood coagulation time that have been
conventionally employed include those relying on the measurement of
prothrombin
time (PT), the measurement of activated partial thromboplastin time (APTT),
the
measurement of thrombin time, and the fibrinogen level test. Detection of a
thrombotic event also may be performed by measuring the level of soluble
fibrin or
fibrin degradation products in the circulation.
Determination of the coagulation time has been most commonly used for the
diagnosis of diseases such as hemophilia, Von Willebrand disease, Christmas
disease
and certain hepatic diseases, wherein abnormally prolonged clotting times
typically
have diagnostic utility. Although there are many serious conditions involving
abnormally fast blood coagulation, current measurement methods are not
sensitive
enough to be diagnostically valuable in identifying all but the most abnormal
of these
fast clotting pathological conditions.
The PT and APTT tests do not have utility in the detection of clinically
pathological hypercoagulable states. In general, these tests are used to
detect
conditions with prolonged clotting times, that is, conditions of
hypocoagulability.
These tests are usually performed on plasma, which does not contain activated
platelets and monocytes, both of which may contribute significantly to altered
coagulation states. Furthermore, these tests utilize reagents added to the
sample that
are themselves procoagulants and reduce the clotting time of plasma from about
six
minutes to values of about 10-13 seconds, and 25-39 seconds, for PT and APTT,
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respectively. Laboratory Test Handbook, 4th ed., Lexi-Comp Inc., 1996, pp. 227
(APTT) and 262 (PT). By excluding the influence of the cellular components of
whole blood, such as monocytes, these popular plasma-based methods for
measuring
clotting time do not fully provide maximum predictive and diagnostic value for
thrombotic events modulated by the cellular components of blood.
Furthermore, the monitoring of anticoagulant therapies such as heparin and
warfarin would be improved if the coagulability of whole blood, rather than
plasma
alone, were measured. The presence of these therapeutically-administered
anticoagulants modulates coagulability through cellular as well as soluble
(plasma)
blood constituents.
A number of important initiators and modulators of the blood clotting process
are present in whole blood. One such molecule is a procoagulant protein called
tissue
factor, also known as Factor III, which is a transmembrane glycoprotein
present on the
surface of circulating cell known as monocytes. Tissue factor is also found in
phospholipid vesicles within the blood plasma. Elevated levels of circulating
tissue
factor have been linked to many thrombotic disorders and pathologic states.
For
example, tissue factor has been found on circulating cells and vesicles in
plasma from
patients with cancer, infections, and thrombotic disorders such as heart
attack and
stroke. The level of tissue factor activity in whole blood is a diagnostically
useful
parameter for identifying patients at risk of undergoing thrombotic events.
Tissue factor (TF) must form an active complex with a plasma clotting factor,
Factor VII, or its activated form, Factor VIIa. The TF:Factor VIIa complex
then
activates zymogens Factor IX and Factor X to their enzymatically active forms
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Factors IXa and Xa, respectively. Factor Xa combines with Factor Va to yield
the
prothrombinase complex (active procoagulant), which then cleaves prothrombin
to
thrombin. Thrombin, in turn, cleaves fibrinogen to produce fibrin, which forms
a clot.
Methods for the direct measurement of tissue factor have been described. In
addition to immunoassay procedures, such as that described in U.S. Patent No.
5,403,716, the exposure of whole blood to endotoxin, as described in U.S.
Patent No.
4,814,247 and by Spillert and Lazaro, 1993, J. Nat. Med. Assoc. 85:611-616,
provides
an assessment of TF levels within several hours. In the method using
endotoxin, a
modified recalcification time is measured for a blood sample. This assessment
represents the tissue factor expression present when the endotoxin or other
immunomodulator creates a condition that simulates disease or trauma, thus
measuring the patient's propensity to clot when experiencing such conditions.
The
present invention provides another important assessment of tissue factor by
providing
a simple method to determine the current actual value of circulating tissue
factor
1 S activity in whole blood, thus measuring the patient's current risk of clot
formation.
For example, one can measure fibrin formation using a Sonoclot Coagulation
and Platelet Function Analyzer (Sienco, Wheat Ridge, CO), which uses a
disposable
vibrating probe inmmersed in whole blood to measure the viscous drag of fibrin
strands. Alternatively, one can use the HEMOCHRONT"" system (International
Technidyne Corp.), which uses a precision aligned magnet within a test tube
and a
magnetic detector located within the instrument to detect clot formation.
Currently, there are no standard clinical assays for measuring tissue factor
(TF) or tissue factor pathway inhibitor (TFPI) functional activity. In a
research
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setting, however, assays measuring the activity of certain procoagulants or
anticoagulants do exist. For example, the percentage of Factor XII activity
present in
plasma can be determined by the degree of correction obtained when the plasma
is
added to severely Factor XII deficient plasma. This assay is a modification of
the
APTT test and measures the ability of the patient's plasma to "correct" the
APTT of
plasma containing less than 1% Factor XII. The amount of correction achieved
by
dilution of the patient's plasma is compared to the correction obtained by
known
concentrations of Factor XII. Normal plasma is considered to give 100%
correction.
One can also determine the percentage of thrombin (Factor II) activity present
in plasma by the degree of correction obtained when the plasma is added to
severely
Factor II deficient plasma. This assay is a modification of the prothrombin
time test
and measures the ability of the patient's plasma to "correct" the PT of plasma
containing less than 1% Factor II. The amount of correction achieved by
dilution of
the patient's plasma is compared to the correction obtained by known
concentrations
of Factor II. Normal plasma is considered to give 100% correction. However,
current
coagulation factor assays of the type discussed are only useful in a clinical
setting to
detect conditions of hypocoagulability (i.e. pathologically slow blood
clotting).
There are immunoassays for several markers of the activation of the blood
coagulation cascade such as F 1.2 prothrombin fragment, D-dimer, soluble
fibrin and
the thrombin/antithrombin III complex. In general, these coagulation
immunoassays
have enjoyed limited acceptance outside the research setting since these kits
involve
slow and relatively labor intensive ELISA procedures.

CA 02378898 2002-O1-17
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Therefore, it is desirable to provide a rapid and simple in vitro assessment
of
the overall coagulability of blood, which correlates with the risk of blood
clotting in
vivo, as well as the contributory effect of a particular effect of a
procoagulant or
anticoagulant on coagulation. This would provide health care professionals
with
diagnostically and clinically useful data for: (1) assessing the patient's
condition; (2)
selecting the proper course of therapy; and (3) monitoring the rate and
effectiveness of
surgical and non-surgical therapies. A rapid assessment method of overall
blood
coagulability that specifically evaluates the contributions of tissue factor
and other
procoagulants and anticoagulants was not previously available. The detection
of
elevated levels of procoagulants and anticoagulants will permit earlier
therapy,
thereby improving prognosis. Currently, there is no whole blood clotting assay
to
accurately assess this hypercoaguable or hypocoaguable state. The instant
method
measures the hypercoaguable or hypocoaguable state by comparing the patient's
whole blood clotting time with and without the presence of at least one
inhibitor of
procoagulant or anticoagulant activity.
SUMMARY OF THE INVENTION
The present invention provides a method to rapidly assess the overall
coagulant properties of a patient's blood sample by measuring and comparing
clotting
time with and without an added inhibitor of a procoagulant or an
anticoagulant. When
the sample is whole blood, the resulting clotting time represents the overall
coagulant
activity of the plasma and cellular components of the blood, which is
indicative of
existing or impending pathology arising from abnormal coagulability.
_g_

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It is an object of the invention to provide a method for measuring the risk of
a
patient for a thrombotic event by determining functionally current levels of
one or
more procoagulants or anticoagulants in whole blood.
It is a further object of the invention to provide a method for measuring the
effectiveness of anticoagulant therapy, such as that of warfarin or low
molecular
weight heparin, by measuring the coagulant activity in a sample of whole blood
by
first exposing a sample of whole blood to inhibitor, followed by measuring the
clotting time of the blood sample by standard methods. The value of the
clotting time
or the differences between the control value and that of the inhibitor-treated
sample, is
useful in monitoring anticoagulation therapy.
It is a further object of the invention to provide a method to monitor the
recovery of a patient from a condition related to adverse blood coagulation by
monitoring the clotting of blood in accordance with the methods described
herein.
It is yet another object of the invention to provide diagnostic kits for the
measurement of the clotting time of whole blood and plasma in the presence and
absence of at least one inhibitor of a procoagulant or anticoagulant.
These and other aspects of the present invention will be better appreciated by
reference to the Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a diagram showing the central role of monocyte TF during the
initiation of fibrin clot formation in whole blood. The TF:VIIa complex
activates
Factor X to Xa and Factor IX to IXa. Thrombin (Factor II) activates platelets,
which
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CA 02378898 2002-O1-17
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form a thrombogenic surface for the prothrombinase complex (Xa:Va). Fibrinogen
is
cleaved to yield a fibrin clot. This diagram was adapted from Kjalke et al.,
Active
site-inactivated Factors VIIa, Xa, and IXa inhibit individual steps in a cell-
based
model of tissue factor-initiated coagulation, Thromb. Haemost., 80:578-84
(1998).
Figure 2 shows the detection of exogenously added TF in whole blood through
comparison of clotting times in the presence and absence of anti-TF antibody
after 10
minutes of incubation at 37°C.
Figure 3 shows the effect of anti-TF antibodies on re-calcified whole blood
clotting times. Incubation of whole blood with LPS (10 ~g/mL) for 2 hours at
37°C
caused a shortening in clotting time due to induction of TF expression.
Addition of
anti-TF antibodies blocked the LPS-mediated reduction in the re-calcified
whole
blood clotting time. In contrast, addition of a non-inhibitory control
antibody had no
effect on the LPS clotting time.
Figure 4 shows the effect of recombinant TF on the re-calcified whole blood
clotting time. Recombinant lipidated TF added to whole blood shortened the re-
calcified whole blood clotting time in a dose-dependent manner over a range of
0 to
80 pg/mL (mean ~ 95% confidence interval of mean, n = 11 replicates at each
point).
Figure Sa shows the clotting times for cells isolated from blood and mixed
with various plasmas.
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Figure Sb shows the effect of an inhibitory anti-Factor XI antibody (100
pg/mL) added to blood before incubation at 37°C for 10 minutes (mean ~
standard
deviation, n = 3). Addition of the anti-Factor XIa antibody prolonged the
clotting
time. In contrast, addition of a corresponding amount of control antibody did
not
affect the clotting time.
Figure Sc shows the effect of corn trypsin inhibitor (CTI) (32 g,g/mL) added
to
blood before incubation at 37°C for 2 hours with or without LPS
stimulation (mean ~
standard deviation, n = 3).
Figure 6a shows the effect of unfractionated heparin (0-0.1 U/ml) on the
clotting time of LPS-stimulated blood.
Figure 6b shows the effect of low molecular weight heparin (LMWH; 0-0.25
U/ml) on the clotting time of LPS-stimulated blood.
Figure 6c shows the effect of hirudin (0-0.1 U/ml) on the clotting time of LPS-
stimulated blood.
Figure 7 compares the re-calcified whole blood clotting times of patients with
unstable angina (n = 8) with healthy normals (n = 37). Circles represent
individuals
outside the 5'h and 95'h percentiles of the clotting times.
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DETAILED DESCRIPTION OF THE INVENTION
Abnormalities of blood coaguability causes a range of pathologies. In
particular, factors that increase the coagulability or prothrombotic potential
of blood
are in most instances highly undesirable and may lead to serious pathologic
states, for
example, heart attack, stroke, coronary artery disease, deep vein thrombosis,
and
pulmonary embolism. Furthermore, certain clinical conditions, such as vascular
disease, surgery, trauma, malignancy, the presence of prosthetic vascular
devices,
general anesthesia, pregnancy, use of oral contraceptives, systemic lupus
erythematosus, and infection, may predispose patients to undergo adverse
clotting
phenomena. These conditions alter the coagulation state of the blood to cause
the
prothrombotic pathways to predominate and intensify, as compared with the
protective anticoagulant pathways.
The overall coagulability of blood is governed by factors contributed by both
the soluble (plasma) portion of blood as well as that provided by the cellular
portion.
Traditional measures of clotting or blood coagulability, for example,
prothrombin
time (PT) and active partial thromboplastin time (APTT), among others,
generally use
plasma to measure blood coagulability. These plasma-based methods, however,
omit
contributions to blood coagulability provided by the cellular components. One
example is the contribution of tissue factor to blood coagulability. As
described
above, tissue factor is an initiator and modulator of blood coagulation, and
may be
present in the blood. Elevated levels are associated with pathologic states.
In addition
to tissue factor, other components present in or on the cellular components of
blood
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may also modulate blood coagulability and also contribute to the propensity
for blood
to clot in vivo.
In one embodiment, the method of the invention involves measuring whole
blood clotting with or without an inhibitor of a procoagulant or
anticoagulant. The
magnitude of difference between the clotting times with or without the
inhibitor is
proportional to the amount of procoagulant or anticoagulant present in the
sample, i.e.,
a larger difference represents more of the factor being measured. In addition
to the
difference in clotting times, the absolute clotting times are important
because a patient
may be hypercoagulable due to an abnormality other than elevated procoagulant
levels.
With respect to performing the assay of the invention with an inhibitor of
tissue factor, in contrast to the above-mentioned modified recalcification
time test
described by Spillert and Lazaro wherein endotoxin incubated with the whole
blood
sample induces the synthesis of tissue factor, which in turn influences the
coagulant
properties of the blood sample, the method of the present invention does not
measure
the effect of tissue factor synthesis on blood coagulability. Instead, it
measures the
influence of existing tissue factor present in the whole blood sample on blood
coagulability. See, for example, Santucci et al., Measurement of Tissue Factor
Activity in Whole Blood, Thromb. Haemost., vol. 83(3):445-54 (2000), which is
incorporated by reference herein.
The method of the present invention may be performed with fresh whole
blood, to which an inhibitor is added, followed by measurement of the
coagulability
of the blood sample and a sample without the inhibitor by standard methods.
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Alternatively, a blood sample may be collected in the presence of an
anticoagulant,
such as citrate, oxalate, EDTA, etc. This does not include an anticoagulant
that blocks
the intrinsic pathway of clot formation, that is, the anticoagulant will block
the
extrinsic or common pathways. Subsequently, the inhibitor may be added, and
then
the coagulability of the blood determined by standard methods. Any known
procedure for measuring blood coagulability may be used in the methods of the
invention.
In the instance where the blood is collected with an anticoagulant, the effect
of
the anticoagulant in the blood sample must be reversed at the time that blood
coagulability or clotting time is measured. This is accomplished by the
addition of a
calcium salt, such as, for example, calcium chloride. The measurement of
clotting
time on a sample of anticoagulated blood by the addition of a calcium salt to
reactivate the clotting process is referred to as the recalcification time.
Any inhibitor of a procoagulant or anticoagulant is suitable for use in the
methods of the invention, so long as it is specific for a particular
procoagulant or
anticoagulant. Suitable inhibitors include, among other things, antibodies or
analogues of substrates for procoagulants or anticoagulants. In one preferred
embodiment, the analogue is a peptide. Suitable inhibitors are known to those
skilled
in the art and are commonly available from commercial sources.
In a preferred embodiment, inhibitory antibody or combination of antibodies
exhibiting sufficient affinity for tissue factor may be used as the inhibitor
of TF:Factor
VIIa complex. In one embodiment; two antibodies, designated VD10 and VIC12 are
used in combination. The concentration of the antibody or combination of
antibodies
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in the reagent is provided so that it may be easily added to the blood sample
to
provide the proper final concentration in order to carry out the method of the
present
invention. In another embodiment, the inhibitor is Factor VIIai, which is a
catalytically inactive version of Factor VIIa.
S Determination of clotting time by the methods of the invention may also be
performed in the presence of certain additional compounds, which provide
useful
information of diagnostic and clinical utility in the identification and
monitoring of
certain disease states related to thrombosis. Compounds such as homocysteine,
tissue
factor, Russell's viper venom, and other procoagulant venoms are contemplated.
Other modulators of the clotting process contemplated for use in the present
invention
include procoagulants such as thrombin, platelet activating factor,
fibrinogen, kaolin,
celite, adenosine diphosphate, arachidonic acid, collagen, and ristocetin.
Factors with
anticoagulant activity useful as modulators of the clotting process of the
present
invention include protein C, protein S, antithrombin III, thrombomodulin,
tissue
plasminogen activator, urokinase, streptokinase, tissue factor pathway
inhibitor and
Von Willebrand Factor. The addition of therapeutic drugs, which may modulate
the
coagulant activity of blood, may also be used as modulators in the invention.
In
addition, cancer cell extracts and amniotic fluid may serve as modulators.
The invention is not limited to any particular method of measuring clotting.
Any number of available procedures for measuring blood clotting may be used in
the
present invention, including manual, semi-automated, and automated procedures,
and
their corresponding equipment or instruments. Instruments suitable for this
purpose
include, for example, all instruments that measure mechanical impedance caused
by
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initiation of a clot. The reagents that initiate clotting or affect clotting
times may be
presented in various forms, including but not limited to solutions,
lyophilized or air-
dried forms, or dry card formats.
For example, the SONOCLOTT"" Coagulation Analyzer, available from
Sienco, Inc., measures viscoelastic properties as a function of mechanical
impedance
of the sample being tested. Such analysis is very sensitive to fibrin
formation, thereby
providing improved sensitivity and reproducibility of results.
Another device, the thrombelastograph (TEG), can also be used for measuring
viscoelastic properties. An example of this type of instrumentation is the
computerized thrombelastograph (CTEG), from Haemoscope Corp. The
SONOCLOTT"" and CTEG are capable of recording changes in the coagulation
process by measuring changes in blood viscosity or elasticity, respectively. A
complete graph of the entire process is obtained.
Other instruments such as the HEMOCHRONT"" measure clotting time but do
not provide a graph of the change in a clotting parameter as a function of
time. The
HEMOCHRONT"~ system (International Technidyne Corp.), which uses a precision
aligned magnet within a test tube and a magnetic detector located within the
instrument to detect clot formation.
In one embodiment of the invention, where the assays are performed on an
emergent basis, for example, in the emergency room on a patient suspected of
having
an acute thrombotic event such as a heart attack or stroke, no anticoagulant
need be
used and the assays may be performed directly with a fresh blood sample. The
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necessary reagents, such as an antibody, may be preloaded into the coagulation
analyzer, and the clotting times determined, along with that of a control
sample
without the addition of antibody. Alternatively, the blood is first collected
with an
anticoagulant that binds calcium ions, such as citrate, oxalate, EDTA, etc.,
and the
clotting times made subsequently under traditional laboratory conditions. In
order to
initiate clotting in a sample containing one or more of these anticoagulants,
calcium
salt must be added. The time required for the formation of fibrin polymers is
referred
to in this instance as the recalcification time. In another embodiment of the
invention,
improved sensitivity and specificity in the detection of coagulation
procoagulants or
anticoagulants may result when blood collection is performed in the presence
of a
specific inhibitor of the intrinsic contact activation coagulation pathway,
like corn
trypsin inhibitor.
As an example of the performance of the assays on anticoagulated blood, one
milliliter of citrated blood is combined with inhibitory monoclonal anti-
tissue factor
antibodies (final concentration 10 microgram/milliliter). Another aliquot of
one
milliliter of citrated blood is prepared with control antibody or protein
control. After
mixing the samples, they are placed in a 37 C incubator. After a given
incubation
period, 300 microliters of each sample is mixed with 40 microliters of 0.1 M
calcium
chloride, and the recalcification time (the time necessary for fibrin to form)
measured
using automated instrumentation. The difference between the recalcification
time of
the control versus the sample containing inhibitor (antibody) is used
diagnostically to
indicate whether the patient has abnormal blood coagulability due to elevated
tissue
factor and is in need of medical intervention.
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In a further embodiment of the invention, a test kit is provided for
determining
coagulability in which inhibitors at the proper concentrations are provided in
order to
determine the clotting or recalcification time according to the methods of the
invention.
Tissue factor (TF), also known as Factor III, is responsible for initiating
the
extrinsic coagulation pathway. Tissue factor is primarily present in the
monocytes of
circulating blood. Certain disease states may predispose a person's monocytes
to be
primed with tissue factor, and thus have the propensity for an abnormally fast
whole
blood clotting time. Such patients could be at risk for thrombosis or other
events.
Currently there is no method to accurately assess this hypercoaguable state.
The present invention may be used to assess hypercoagulability by detecting
circulating TF levels through comparison of the unstimulated clotting times in
the
presence and absence of an anti-TF antibody. In another form of the invention
thought
to measure the physiological potential for hypercoaguability by comparing the
patient's LPS-stimulated whole blood clotting time in the presence or absence
of anti-
TF antibody.
Example 1
The tissue factor whole blood assay described in this example involves a test
procedure carned out on the Hemochron instrument. It uses an anti-TF antibody
inhibition test to assess endogenous circulating tissue factor levels in whole
blood.
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Materials and reagents needed to assess TF in blood circulation
Hemochron P213 sample tubes
0.01 M calcium chloride stock solution
control reagent vials containing non-inhibitory antibody
reagent vials containing dried anti-tissue factor antibody
Assay Quality Control Reagents
Hemoliance RecombiPlasTin Stock Solution (lipidated recombinant tissue
factor)
TF diluent solution (20 mM HEPES 150 mM sodium chloride, pH 7.4 with
0.10 mg/mL bovine serum albumin)
Blood collection tubes containing liquid citrate anticoagulant and corn
trypsin
inhibitor (CTI)
Hemochron P213 tube preparation
Prior to performing the unstimulated and clotting time test, pre-load the
Hemochron P213 tubes with 50 ~.L of the 0.10 M calcium chloride solution.
Store
tubes at room temperature with stoppers closed.
Quality control sample preparation for the unstimulated while blood clotting
time
Negative control = TF diluent solution
High positive TF control:
Make a 1:100 dilution of the lipidated recombinant TF stock solution.
i.e., Add 10 ~L of the lipidated recombinant TF stock solution to 0.99
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mL of TF diluent solution.
Low positive TF control:
Make a 1:7 dilution of the high positive TF control solution, i.e., to
0.60 mL of TF diluent add 100 ~,L of the high positive TF control.
Blood draw
After discarding the first few mL of the blood draw, draw blood into the 5 mL
citrate/CTI blood collection tubes. Blood should be analyzed within 4 hours of
the
blood draw.
Unstimulated clotting time test for circulating TF (10 minute incubations)
Dedicate one of the citrate/CTI blood collection tubes per 8 test vials for
the
unstimulated clotting time test.
Place 10 ~1 of either the negative or positive TF control reagents in the 4
test
vials listed below:
control vial + negative TF control
anti-TF antibody vial + negative TF control
control vial + positive TF control
anti-TF vial + positive TF control
Transfer 0.50 ml of citrate/CTI anticoagulated blood to each test vial. Mix by
gentle tube inversion several times.
Place in a 37 degree water bath for ten minutes.
After the ten minute incubation is completed, transfer 0.40 ml of the blood
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from each test vial to a Hemochron P213 sample tube containing 50 ~l calcium
chloride.
On the Hemochron 8000, select test = "ACT" and tube = "P214/5". Press
"Start" to initiate clot timer. Gently swirl Hemochron tube to mix the blood
with the
calcium chloride solution. Place tube in Hemochron sample well. Turn tube in
sample well until green light stays on.
Typical unstimulated clotting times
Control vial + negative TF control: >850 seconds
TF antibody vial + negative TF control: >850 seconds
Control vial + high positive TF control: 250 to 350 seconds
Control vial + low positive TF control : 650 to 750 seconds
TF antibody vial + low or high positive control: >850 sec
To illustrate the use of the present invention in the detection of circulating
TF
to assess hypercoagulability, Figure 2 shows the detection of exogenously
added TF
in whole blood through comparison of clotting times in the presence and
absence of
anti-TF antibody after 10 minutes of incubation at 37°C.
Example 2
This example describes a lipopolysaccharide-stimulation test procedure on the
Hemochron instrument that assesses the production of TF in whole blood in
response
to endotoxin (lipopolysaccharide) stimulus.
Materials and reagents needed to assess production of TF in whole blood in
response to LPS stimulus.
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Hemochron P213 sample tubes
0.10 M calcium chloride stock solution
Reagent vials containing dried LPS
Reagent vials containing dried LPS and dried anti-tissue factor antibody
Blood collection tubes containing liquid citrate anticoagulant and corn
trypsin
inhibitor (CTI)
Hemochron P213 tube preparation
Prior to performing the stimulated tissue factor clotting time test, pre-load
the
Hemochron P213 tubes with SO ~L of the 0.10 M calcium chloride solution. Store
tubes at room temperature with stoppers closed.
Blood draw
After discarding the first few ml of the blood draw, draw blood into the 5 ml
citrate/CTI blood collection tubes provided in the kit. Blood should be
analyzed
within 4 hours of the blood draw.
LPS-stimulated clotting time test (2 hour incubation)
Transfer 0.50 ml of the citrate/CTI anticoagulated specimen blood to each test
vials listed below:
Vial containing dried LPS and control reagent
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Vial containing both dried LPS and dried anti-TF antibody
Mix samples by gentle inversion of the test vials several times. Place on a 37
degree water bath for 2 hours
After 2 hour incubation period is completed, mix test vials by gentle tube
inversion. Transfer 0.40 ml of the blood from each test vial to a Hemochron
P213
sample tube containing 50 p,1 calcium chloride.
On the Hemochron 8000, select test = "ACT" and tube = "P214/5". Press
"Start" to initiate clot timer. Gently swirl Hemochron tube containing
recalcified
blood. Place on Hemochron. Twist tube in sample well until green light stays
on.
Typical clotting times after 2 hour LPS stimulation
LPS vial: 200 to 350 seconds
LPS with anti-TF antibody vial: >850 seconds
Example 3
The tissue factor whole blood assay described in this example involves a test
procedure carried out on the SonoclotT"" instrument. It uses an anti-TF
antibody
inhibition test to assess tissue factor levels in LPS-stimulated whole blood.
SPECIMEN REQUIREMENTS
Using a 19 mm gauge needle, follow phlebotomy procedures as detailed, for
example, in Collection, Transport and Processing of Blood Specimens for
Coagulation Testing and Performance of Coagulation Assays (National Committee
for
Clinical Laboratory Standards document # H21-A-2, Volume XI, No. 23). Collect
a
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discard tube (blue-top Vacutainer) first, and then draw into a plastic
Vacutainer tube
containing 50 ug/ml corn trypsin inhibitor (CTI) and 0.5 ml 3.2% sodium
citrate to
use as the actual test specimen. The Vacutainer tube should contain at least
4.5 ml of
blood. If it does not, do not proceed with the test. If you do not obtain a
good blood
flow during the specimen collection, discard the specimen and attempt another
venipuncture in the patient's other arm. Invert Vacutainer gently 5-7 times
after
drawing. Note: the specimen is to remain at room temperature after collection.
REAGENT PREPARATION AND STORAGE
1. Vials (2.0-ml polypropylene) with caps and lyophilized reagents provided by
Sienco. Vials are color-coded by their cap: either a clear cap (for control
mAb), a
white insert (for TF mAb cocktail), yellow insert (contains no reagent), or a
blue
insert (contains 20 microliters of 0.5 mg/ml of lyophilized Difco endotoxin).
Each
patient sample will require four tubes, one of each color. Store at 2-
8° C.
2. Calcium chloride O.1M (Analytical Control Systems, Inc., Fishers, Ind.)
Store at
2-8° Centigrade.
1. Anti-osteocalcin monoclonal antibody in tris buffered saline (pH 7.4) 1
mg/ml
BSA. Store at 2-8°
2. Tissue factor monoclonal antibody in tris buffered saline (pH 7.4) 1 mg/ml
BSA.
Store at 2-8°
EQUIPMENT REQUIRED
1. SonoclotT"" Analyzer
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2. Instruction Manual
3. Cuvettes provided by Sienco. For each patient sample, you will need 5
cuvettes (4
to hold the blood samples and one to warm the calcium chloride).
4. Magnetic stir bars provided by Sienco
5. Probes provided by Sienco
6. Probe extractor provided by Sienco
7. Thermal heating block or water bath set to 37 degrees Centigrade
8. Timer
9. Eppendorf pipette and pipette tips (40 microliter, 300 microliter, and 1000
microliter)
10. 13 X 100mm Haematologic Technologies Inc. Vacutainer containing 0.5 ml of
3.2% buffered citrate and 50 ug/ml of corn trypsin inhibitor
11. l9mm gauge needle
PROCEDURE
1. Sample preparation: Place one clear vial, one white vial, one yellow vial,
and one
blue vial in test tube rack. Mix the Vacutainer by hand 6-10 times to ensure
homogeneity. Open Vacutainer under a hood or behind a splash guard.
2. Pipette 1.0 ml of blood into each of the clear and white vials. Add 5
microliters of
control antibody to the clear vial. Add 5 microliters of TF mAb cocktail to
the
white vial. Cap each vial, invert gently 6-10 times, and place vials in the
water
bath. Set a timer for 10 minutes.
3. Pipette 1.0 ml of blood into each of the yellow and blue vials. Do not add
anv
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reagents to these vials. Cap each vial, invert gently 6-10 times, and place
vials in
the water bath. Set a timer for 2 hours.
4. Sample testing.'
a) Warm the calcium chloride to 37 degrees Centrigrade by pipetting 300
microliters
into a cuvette that has been placed in one of the side wells in the Sonoclot
Analyzer.
b) Place probe on SonoclotT"" in appropriate position using a slightly
twisting motion.
Place cuvette firmly down in the receptacle, also using a slight twisting
motion.
c) Insert one magnetic stir bar into cuvette.
d) Add 40 ~l of O.1M calcium chloride to the cuvette. Close head.
e) Remove appropriate color vial from water bath after correct incubation time
and
invert gently 6-10 times. Remove cap and immediately pipette 300 ~1 of sample
into cuvette in the SonoclotT"" Analyzer. Avoid formation of bubbles.
f) Gently reaspirate the sample once only (to avoid platelet activation) to
mix it well
with the calcium chloride already in the cuvette. Once the sample is delivered
into
the cuvette and mixed with the calcium chloride, immediately press the metal
toggle switch on the SonoclotT"" Analyzer to the "Start" (down) position to
activate
the magnetic stirrer. Wait for the audio tone and written instructions on
screen to
close head. Close the port head. This will introduce the probe into the sample
and
which will begin the test.
g) An audio tone will sound when the test is complete. Read the values on the
data
panel and the chart recording of the SonoclotT"" Analyzer. The SonoclotT"~
Analyzer automatically calculates the clotting time. The test should be
allowed to
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run for at least 20 minutes to obtain additional raw data, even though the
tone will
sound earlier.
h) Promptly record the whole blood clotting time together with identifying
information, including vial type (clear, white, yellow, or blue) patient
identification number and date and time the test was performed.
i) Dispose of cuvette and probe with the probe extractor.
j) Repeat steps a-i above for each sample vial once it has incubated for its
appropriate time period.
3. Safety Precautions: Technicians should take universal precautions to
eliminate the
possibility of contracting disease through blood borne pathogens. These
precautions should, at a minimum, include eyewear, gloves, and appropriate
gown. Proper disposal techniques of pipette, tips, vials, and sample
containers
should be utilized.
PROCEDURAL COMMENTS
1. Check reagent dates. Reagents should not have reached their expiration
date.
2. Assure incubation times and temperatures are accurate. These steps are
critical.
3. Handle patient samples with appropriate precautions.
4. Use plasticware for all pipette tips. Under no circumstances can glass come
in
contact with blood.
5. Use appropriate disinfection procedures to remove spilled blood.
6. Store test kit materials at 2-8° C. Do not use after expiration
date. This test is for
in vitro
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diagnostic research use only.
QUALITY CONTROL
1. The viscosity oil control provided with the SonoclotT"~ Analyzer should be
performed as stated in the instruction manual.
2. Technicians should perform periodic duplicate readings of samples (at least
once
for each shift or every 25 readings, whichever is more frequent). To do so,
draw
two 300 ~l samples from the same incubation vial and place in two instruments
to
record whole blood clotting times readings simultaneously. Use a new pipette
tip
for each sample drawn from the vial. The duplicate values should be within 10
percent of the mean value.
3. The instruments should be evaluated daily for quality control according to
the
manufacturer's specifications in Chapter 4 of the SonoclotT"' Instruction
Manual.
SOURCES OF ERROR
The errors that can occur while performing the assay are those attributed to
technician errors, instrument or supply problems, and recording errors. The
major
sources of error for each group include:
1. Technician Error - Poor or traumatic venipuncture, less than full drawn
sample, sample not properly mixed and inverted, inappropriate volume of
sample or calcium, incubation time error, instrument use error, data
transfer error, or sample mix up
2. Instrument Errors - Failure to follow instructions, failure to perform
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instrument manufacturer's quality control procedures, failure to perform
coagulation quality control procedures, temperature error, and persistent
lack of agreement between duplicate samples.
3. Recording Errors - Failure to keep accurate and timely notes, data transfer
errors, failure to record lot numbers of the vials.
SUBJECT POPULATION
Blood donor subjects were drawn from a healthy, normal population of the
General Clinical Research Center (GCRC) at The Scripps Research Institute in
La
Jolla, California. Subjects took no medications chronically, and could not use
nonsteroidal anti-inflammatory drugs (NSAIDS) in the 20 days before blood
donation.
Use of human blood samples was approved and governed by the Human Subjects
Research Committee.
DETERMINATION OF CLOTTING TIMES
Whole blood clotting times were determined as described above using a
Sonoclot'~"'t Coagulation and Platelet Function Analyzer (Sienco, Inc., Wheat
Ridge,
CO), which uses a disposable vibrating probe immersed in 300 ~l whole blood to
measure the viscous drag of fibrin strands (1, 2). The clotting time is
derived by
calculating the number of seconds until the impedance of the recalcified
sample rises
6 units above the baseline using software (Sienco) modified to use a custom
onset
algorithim (Coagulation Diagnostics Inc., Bethesda, MD). As mentioned, whole
blood samples for testing were collected atraumatically into Sml Vacutainer
glass test
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tubes containing 0.5 ml 3.2% sodium citrate (Becton Dickinson, Franklin Lakes,
NJ).
The time from blood draw to performance of the assay was less than 4 hours.
Tubes
containing blood were inverted several times to remix the whole blood before
aliquoting into incubation tubes. Blood (1m1) was incubated in a plastic tube
at 37°C
for 10 minutes or 2 and 4 hours with and without bacterial lipopolysaccharide
(LPS)
(10~g/ml) (E. coli OSS:BS Westphal, Difco; Detroit, MI). During the incubation
at
37°C there was no agitation. After incubation blood was remixed and 300
~1 was
aliquoted into warmed cuvettes that were preloaded with 40 microliters of 0.1
M
CaCl2 and a magnetic stir bar. Following a ten second stirnng sequence, the
clotting
time was determined.
EFFECT OF INHIBITION OF TF ACTIVITY ON CLOTTING TIME
The effect of inhibitory anti-TF antibodies on the clotting time was
determined
by adding a cocktail of inhibitory murine IgG, MAb against human tissue factor
(3)
(9C3, SG9, 6B4) at a final concentration of 10 g/ml. A noninhibitory murine
IgG,
antibody ( 1 OH 10) was used as a control.
To determine the contribution of TF to the clotting times of whole blood, we
examined the effects of a cocktail of inhibitory anti-TF antibodies on
clotting times.
Inhibitory anti-human TF monoclonal antibodies significantly prolonged the
clotting
times of LPS-stimulated blood but not unstimulated blood from healthy
volunteers.
Control non-inhibitory antibodies had no effect on stimulated or unstimulated
blood
(Figure 3). Further studies on 19 healthy individuals demonstrated that
inhibitory
anti-TF antibodies had no effect on mean base line clotting times (10 minutes)
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(478~78 with antibody versus 437~ 113 without antibody, mean ~SD). These data
showed that the assay measured TF-dependent fibrin strand formation in LPS-
stimulated whole blood. Recombinant lipidated TF added to whole blood
shortened
the re-calcified whole blood clotting time in a dose-dependent manner over a
range of
0 to 80 pg/mL (Figure 4).
Example 4
ROLE OF THE CONTACT ACTIVATION PATHWAY ON CLOTTING
TIMES OF UNSTIMULATED BLOOD
To assess the contribution of the contact activation pathway to the
coagulation
of whole blood, we employed 3 approaches: i) we clotted blood reconstituted in
Factor XII, XI, or VII deficient plasmas; ii) we employed corn trypsin
inhibitor, which
inhibits Factor XIIa; and iii) we used an inhibitory anti-Factor XIa antibody
to inhibit
Factor XIa activity. To determine the effect of deficient plasmas, cells were
separated
from plasma by centrifugation at 850 x g for 10 minutes, washed and
resuspended in
the following plasmas: autologous, normal pooled, Factor XI-deficient, Factor
XII-
deficient and Factor VII-deficient (Sigma). For experiments analyzing Factor
XIIa
inhibition, we used corn trypsin inhibitor (32 g/ml) (Haematologic Technology,
Essex
Junction, VT). For experiments analyzing Factor XIa inhibition, goat anti-
human
Factor XIa antibody ( 10 g/ml) (kindly provided by Dr. K. Mann) or control non-
immune goat antibody was added to the whole blood prior to incubation at 37
degrees
C. In all cases, blood was incubated for 10 minutes at 37 degrees C before
determining the whole blood clotting time.
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Cells isolated from whole blood were reconstituted with various plasmas
before determining clotting times. Cells reconstituted in autologous plasma
exhibited
a slightly faster clotting time than the clotting time of unmanipulated blood
(Fig. 5A),
which may reflect partial activation of monocytes and platelets during
isolation of the
cells. Cells reconstituted in normal plasma or Factor VII-deficient plasma
exhibited
clotting times that were similar to those observed with autologous plasma,
which is
consistent with the anti-TF antibody studies that showed no TF activity in
unstimulated blood. The small difference between autologous plasma and normal
or
Factor VII-deficient plasma may be due to differences between fresh plasma
versus
frozen/lyophilized plasmas. In contrast to the results with Factor VII-
deficient
plasma, clotting times were significantly prolonged when cells were
reconstituted
with both Factor XI- and Factor XII-deficient plasmas, which suggested that
the
contact activation pathway contributed to the clotting times of unstimulated
blood.
Similarly, an inhibitory anti-Factor XIa antibody significantly prolonged the
clotting
time of unstimulated whole blood (Fig. 5B).
We used corn trypsin inhibitor to block Factor XIIa activity (4). Again, we
observed consistently prolonged clotting times of unstimulated blood in the
presence
of corn trypsin inhibitor (Fig. SC). The mean difference in clotting times
with and
without corn trypsin inhibitor was 141~88 seconds (mean~SD, n=28).
Importantly,
corn trypsin inhibitor did not block clotting times of LPS-stimulated blood
(Fig. SC),
which we have shown is TF-dependent (see Fig.3). Taken together, these studies
indicate that the contact activation pathway contributes to clotting times of
unstimulated blood but does not significantly affect the shorter clotting
times of LPS-
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stimulated blood.
Example 5
EFFECT OF INHIBITING ANTICOAGULANTS ON WHOLE BLOOD
CLOTTING TIMES
The effect of unfractionated heparin, low molecular weight heparin (LMWH)
and hirudin anticoagulants on the clotting times of LPS-stimulated blood was
examined. The results are shown in Figures 6A-6C, respectively. Unfractionated
heparin and LMWH inhibit both thrombin and Factor Xa. However, unfractionated
heparin shows greater antithrombin activity relative to its anti-Factor Xa
activity (5).
In contrast, the LMWHs have antithrombin activity that is low, compared with
their
anti-Factor Xa activities (5). Hirudin selectively inhibits thrombin (6).
Administration of clinically relevant doses of any of these three
anticoagulants to
1 S LPS-stimulated blood prolonged the clotting times in a dose-dependent
manner.
These data indicate that this clotting time assay could be used in a clinical
setting to
monitor anticoagulant therapy.
Example 6
CLOTTING TIMES OF BLOOD FROM UNSTABLE ANGINA PATIENTS
For studies on unstable angina, we analyzed blood from patients admitted to
the emergency room of The John Hopkins Hospital, Baltimore, with admitting
diagnosis of unstable angina (n= 8). Patients taking anticoagulants were
excluded. A
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CA 02378898 2002-O1-17
WO 01/07070 PCT/US00/20118
group of healthy volunteers (n=37) from the same site were used as a control
group.
Use of human blood samples was approved and governed by the Human Subjects
Research Committee. Levels of TF protein have been reported in the literature
to be
elevated in blood from patients with unstable angina (7, 8, 9, 10). We
examined
clotting times of unstimulated blood from patients admitted to the emergency
room
with unstable angina. Clotting times of unstimulated blood from unstable
angina
patients were significantly faster than clotting times from a group of healthy
volunteers (Figure 7). These results indicate that patients with unstable
angina had
elevated levels of circulating TF activity.
While the foregoing specification teaches the principles of the present
invention,
with examples provided for the purpose of illustration, it will be appreciated
by one
skilled in the art from reading this disclosure that various changes in form
and detail
can be made without departing from the true scope of the invention.
1 S All cited patents and publications are hereby incorporated by reference in
their
entirety.
REFERENCES
1. Chandler WL, Schmer G. Evaluation of a new dynamic viscometer for
measuring the viscosity of whole blood and plasma. Clin Chem 1986;(32):505-
507.
2. Hett DA, Walker SN, Pilkington SN, Smith DC. Sonoclot analysis. Br J
Anaesth
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WO 01/07070 PCT/US00/20118
1995;75:771-776.
3. Mornssey JH, Fair DS, Edgington TS. Monoclonal antibody analysis of
purified
and cell-associated tissue factor. Thromb Res 1988; 52:247-261.
4. Rand MD, Lock JB, van't Veer C, Gaffney DP, Mann KG. Blood clotting in
minimally altered whole blood. Blood 1996; 88:3432-3445.
5. Canton, MM in Clinical Hematology and Fundamental of Hemostasis, Second
Edition, Harmening DM, editor; 1992, F.A. Davis Company, Philadelphia, pp.
510-512.
6. Bates SM, Weitz JI. Direct thrombin inhibitors for treatment of arterial
thrombosis: potential differences between bivalirudin and hirudin. Am. J.
Cardiol. 1998; 82: 12P-8P.
7. Leatham E, Bath P, Tooze J, Camm A. Increased monocyte tissue factor
expression in coronary disease. Br Heart J 1995; 73:10-13.
8. Suefuji H, Ogawa H, Yasue H, Kaikita K, Soejima H, Motoyama T, Mizuno Y,
Oshima S, Saito T, Tsuji I, Kumeda K, Kamikubo Y, Nakamura S. Increased
plasma tissue factor levels in acute myocardial infarction. Am Heart J 1997;
134:253-259.
9. Misumi K, Ogawa H, Yasue H, Soejima H, Suefuji H, Nishiyama K, Takazoe K,
Kugiyama K, Tsuji I, Kumeda K, Nakamura S. Comparison of plasma tissue
factor levels in unstable and stable angina pectoris. Am J Cardiol 1998;
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WO 01/07070 PCT/US00/20118
81 ( 1 ):22-26.
10. Agraou B, Corseaux D, McFadden EP, Bauters A, Cosson A, Jude B. Effects of
coronary angioplasty on monocyte tissue factor response in patients with
stable
or unstable angina. Thromb Res 1997; 88(2):237-243.
S
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Event History

Description Date
Inactive: IPC from MCD 2006-03-12
Application Not Reinstated by Deadline 2004-07-26
Time Limit for Reversal Expired 2004-07-26
Deemed Abandoned - Failure to Respond to Maintenance Fee Notice 2003-07-24
Letter Sent 2002-10-07
Letter Sent 2002-10-07
Inactive: Single transfer 2002-08-14
Inactive: Courtesy letter - Evidence 2002-07-23
Inactive: Cover page published 2002-07-22
Inactive: Notice - National entry - No RFE 2002-07-18
Inactive: First IPC assigned 2002-07-18
Application Received - PCT 2002-05-01
National Entry Requirements Determined Compliant 2002-01-17
Application Published (Open to Public Inspection) 2001-02-01

Abandonment History

Abandonment Date Reason Reinstatement Date
2003-07-24

Maintenance Fee

The last payment was received on 2002-07-05

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Fee History

Fee Type Anniversary Year Due Date Paid Date
Basic national fee - standard 2002-01-17
MF (application, 2nd anniv.) - standard 02 2002-07-24 2002-07-05
Registration of a document 2002-08-14
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
THE SCRIPPS RESEARCH INSTITUTE
COAGULATION DIAGNOSTICS, INC.
Past Owners on Record
JOHN J. MCDONNELL
NIGEL MACKMAN
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Abstract 2002-01-17 1 59
Claims 2002-01-17 5 126
Drawings 2002-01-17 11 243
Cover Page 2002-07-22 1 37
Description 2002-01-17 36 1,245
Reminder of maintenance fee due 2002-07-18 1 114
Notice of National Entry 2002-07-18 1 208
Courtesy - Certificate of registration (related document(s)) 2002-10-07 1 109
Courtesy - Certificate of registration (related document(s)) 2002-10-07 1 109
Courtesy - Abandonment Letter (Maintenance Fee) 2003-08-21 1 176
PCT 2002-01-17 4 175
Correspondence 2002-07-18 1 25