EP1212073A1

EP1212073A1 - Method for measuring coagulant factor activity in whole blood

Info

Publication number: EP1212073A1
Application number: EP00950617A
Authority: EP
Inventors: Nigel Mackman; John J. Mcdonnell
Original assignee: Coagulation Diagnostics Inc; Scripps Research Institute
Current assignee: Coagulation Diagnostics Inc; Scripps Research Institute
Priority date: 1999-07-23
Filing date: 2000-07-24
Publication date: 2002-06-12
Also published as: WO2001007070A1; EP1212073A4; KR20020042622A; AU6369700A; CN1368886A; JP2003505678A; CA2378898A1

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.

Description

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. 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 hemophilias generally consists of transfusions of concentrates of

blood products in which there is a large amount of coagulation Factors VIII or LX. 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 measurement is initiated by adding a calcium salt to reverse the effect of the

anticoagulant. This latter determination is referred to as the recalcifϊcation 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, 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 Vila. The TF:Factor Vila complex then

activates zymogens Factor IX and Factor X to their enzymatically active forms 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

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 HEMOCHRON™ 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 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. 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. 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 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 Vila, 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 μg/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 5a shows the clotting times for cells isolated from blood and mixed

with various plasmas. Figure 5b shows the effect of an inhibitory anti-Factor XI antibody (100

μg/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 5c shows the effect of corn trypsin inhibitor (CTI) (32 μg/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^th and 95^th percentiles of the clotting times. 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 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 recalcifϊcation 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. 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

Vila complex. In one embodiment, two antibodies, designated VD10 and VIC 12 are

used in combination. The concentration of the antibody or combination of antibodies 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 VIlai, which is a

catalytically inactive version of Factor Vila.

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 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 SONOCLOT™ 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

SONOCLOT™ 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 HEMOCHRON™ measure clotting time but do

not provide a graph of the change in a clotting parameter as a function of time. The

HEMOCHRON™ 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 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 lO 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. 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 carried out on the Hemochron instrument. It uses an anti-TF antibody

inhibition test to assess endogenous circulating tissue factor levels in whole blood. 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 μL 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 μL of the lipidated recombinant TF stock solution to 0.99 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 μL 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 μl 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 from each test vial to a Hemochron P213 sample tube containing 50 μl 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. 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 com trypsin

inhibitor (CTI)

Hemochron P213 tube preparation

Prior to performing the stimulated tissue factor clotting time test, pre-load the

Hemochron P213 tubes with 50 μL 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 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 μl 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 Sonoclot™ 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 discard tube (blue-top Vacutainer) first, and then draw into a plastic Vacutainer tube

containing 50 ug/ml com 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 0.1M (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. Sonoclot™ Analyzer 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 com trypsin inhibitor

11. 19mm 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 any 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 Sonoclot™ 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 μl of 0.1M 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 μl of sample

into cuvette in the Sonoclot™ 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 Sonoclot™ 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 Sonoclot™ Analyzer. The Sonoclot™

Analyzer automatically calculates the clotting time. The test should be allowed to 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 diagnostic research use only.

QUALITY CONTROL

1. The viscosity oil control provided with the Sonoclot™ 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 μl 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 Sonoclot™ 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 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 drags (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™ Coagulation and Platelet Function Analyzer (Sienco, Inc., Wheat Ridge,

CO), which uses a disposable vibrating probe immersed in 300 μl 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 5ml Vacutainer glass test 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 (1ml) was incubated in a plastic tube at 37°C

for 10 minutes or 2 and 4 hours with and without bacterial lipopolysaccharide (LPS)

(lOμg/ml) (E. coli 055:B5 Westphal, Difco; Detroit, MI). During the incubation at

37°C there was no agitation. After incubation blood was remixed and 300 μl 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 stirring 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, 5G9, 6B4) at a final concentration of 10 g/ml. A noninhibitory murine IgG,

antibody (10H10) 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) (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 com trypsin inhibitor, which

inhibits Factor Xlla; and iii) we used an inhibitory anti-Factor Xla antibody to inhibit

Factor Xla 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 Xl-deficient, Factor XII-

deficient and Factor Vll-deficient (Sigma). For experiments analyzing Factor Xlla

inhibition, we used com trypsin inhibitor (32 g/ml) (Haematologic Technology, Essex

Junction, VT). For experiments analyzing Factor Xla inhibition, goat anti-human

Factor Xla 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. 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. 5 A),

which may reflect partial activation of monocytes and platelets during isolation of the

cells. Cells reconstituted in normal plasma or Factor Vll-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 Vll-deficient plasma may be due to differences between fresh plasma versus

frozen/lyophilized plasmas. In contrast to the results with Factor Vll-deficient

plasma, clotting times were significantly prolonged when cells were reconstituted

with both Factor XI- and Factor Xll-deficient plasmas, which suggested that the

contact activation pathway contributed to the clotting times of unstimulated blood.

Similarly, an inhibitory anti-Factor Xla antibody significantly prolonged the clotting

time of unstimulated whole blood (Fig. 5B).

We used com trypsin inhibitor to block Factor Xlla activity (4). Again, we

observed consistently prolonged clotting times of unstimulated blood in the presence

of com trypsin inhibitor (Fig. 5C). The mean difference in clotting times with and

without com trypsin inhibitor was 141±88 seconds (mean±SD, n=28). Importantly,

com trypsin inhibitor did not block clotting times of LPS-stimulated blood (Fig. 5C),

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- 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

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 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.

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 1995; 75:771-776.

3. Morrissey 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, Gaffhey 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; 81(l):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.

Claims

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 α-2-antiplasmin, fibrinogen, high molecular weight kininogen,

kallekrein, prekallekrein, tissue factor, Factor II, Factor Ila, Factor Va, Factor Vila,

Factor Villa, Factor IXa, Factor Xa, Factor Xla, Factor Xlla, and Factor Xllla.

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 com

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.

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 α-1-antitrypsin, activated protein C, antithrombin III, Cl 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

presence of contact activation inhibitor.

13. The method of claim 12, wherein the contact activation inhibitor is

com trypsin inhibitor.

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

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

recalcified.

18. The method according to claim 16, wherein the sample of whole blood

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 com trypsin inhibitor.

21. The kit of claim 20, wherein the procoagulant is tissue factor.

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 com 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 com trypsin inhibitor.

28. The method of claim 27, wherein the immunomodulator is endotoxin.

29. The method of claim 27, wherein the inhibitor is an antibody.