AU6369700A - Method for measuring coagulant factor activity in whole blood - Google Patents

Description

WO 01/07070 rn/uuu/ZUin 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 5 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 10 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. 15 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, 20 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. -1- WO 01/07070 PCT/USOU/20118 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 5 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 10 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, 15 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 20 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 IX. -2- WO 01/07070 PCT/USOO/20118 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. 5 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 10 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. 15 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, 20 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 -3- WO 01/07070 rUi/UoUU/ZU11? 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 5 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 10 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 15 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 20 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, -4- WO 01/07070 PCT/US00/20118 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 5 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) 10 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 15 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 20 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 -5- WO 01/07070 PCT/US00/20118 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 5 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 10 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 15 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

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M system (International 20 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 -6- WO 01/07070 PrI/UsuiuI11 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 5 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 10 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 15 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 Fl .2 prothrombin fragment, D-dimer, soluble fibrin and 20 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. -7- WO 01/07070 1l1/lUoU/118 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 5 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 10 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 15 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 20 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. -8- WO 01/07070 PCT/US0U/20118 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 5 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 10 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 15 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. 20 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 -9- WO 01/07070 PCT/US00/20118 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). 5 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 37oC. 10 Figure 3 shows the effect of anti-TF antibodies on re-calcified whole blood clotting times. Incubation of whole blood with LPS (10 gg/mL) for 2 hours at 37oC 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 15 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 20 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. - 10- WO 01/07070 r1/u1UU/izwi Figure 5b shows the effect of an inhibitory anti-Factor XI antibody (100 gg/mL) added to blood before incubation at 37 0 C for 10 minutes (mean ± standard deviation, n = 3). Addition of the anti-Factor XIa antibody prolonged the clotting 5 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 pig/mL) added to blood before incubation at 37oC for 2 hours with or without LPS stimulation (mean ± 10 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. 15 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. 20 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 9 5 th percentiles of the clotting times. -11- WO 01/07070 PCT/USOU/20118 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 5 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 10 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 15 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 20 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 -12- WO 01/07070 PCT/US00/20118 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 5 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 10 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 15 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 20 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. -13- WO 01/07070 'CT/U1USUU/IZUllS 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 5 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 10 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 15 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. 20 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 -14- WO 01/07070 PCT/US00/20118 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. 5 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. 10 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 15 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. 20 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 - 15- WO 01/07070 r'cl/uuIUiti1 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 T M Coagulation Analyzer, available from 5 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 10 computerized thrombelastograph (CTEG), from Haemoscope Corp. The

SONOCLOT

T M 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. 15 Other instruments such as the HEMOCHRONTM measure clotting time but do not provide a graph of the change in a clotting parameter as a function of time. The

HEMOCHRON

T M 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. 20 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 -16- WO 01/07070 PCTI/USUO/20118 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 5 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 10 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 15 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 20 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. -17- WO 01/07070 PCT/US00/20118 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. 5 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. 10 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 15 patient's LPS-stimulated whole blood clotting time in the presence or absence of anti TF antibody. Example 1 20 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. - 18- WU U1/UU/UU rk lUiUUkUlO 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 5 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 10 0.10 mg/mL bovine serum albumin) Blood collection tubes containing liquid citrate anticoagulant and corn trypsin inhibitor (CTI) Hemochron P213 tube preparation 15 Prior to performing the unstimulated and clotting time test, pre-load the Hemochron P213 tubes with 50 pL 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 20 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 pL of the lipidated recombinant TF stock solution to 0.99 - 19- WO 01/07070 r-L /UsUU/Zuno5 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 pL of the high positive TF control. 5 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. 10 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 pl of either the negative or positive TF control reagents in the 4 test 15 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 20 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 - 20 - WO 01/07070 r I/UUU/IUIIo 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 5 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 10 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 15 in whole blood through comparison of clotting times in the presence and absence of anti-TF antibody after 10 minutes of incubation at 37oC. Example 2 This example describes a lipopolysaccharide-stimulation test procedure on the 20 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. -21 - WU UI/U/U7U rI/U3uu/zu11a Hemochron P213 sample tubes 0.10 M calcium chloride stock solution Reagent vials containing dried LPS 5 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 10 Prior to performing the stimulated tissue factor clotting time test, pre-load the Hemochron P213 tubes with 50 iL of the 0.10 M calcium chloride solution. Store tubes at room temperature with stoppers closed. 15 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. 20 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 - 22 - WO 01/07070 I 1/uouu/i u15 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 5 inversion. Transfer 0.40 ml of the blood from each test vial to a Hemochron P213 sample tube containing 50 pl 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. 10 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 15 procedure carried out on the SonoclotTM instrument. It uses an anti-TF antibody inhibition test to assess tissue factor levels in LPS-stimulated whole blood. 20 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 25 Clinical Laboratory Standards document # H21-A-2, Volume XI, No. 23). Collect a - 23 - WO 01/07070 PCT/US00/20118 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 5 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 10 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 2-80 C. 15 2. Calcium chloride 0.1M (Analytical Control Systems, Inc., Fishers, Ind.) Store at 2-8o Centigrade. 1. Anti-osteocalcin monoclonal antibody in tris buffered saline (pH 7.4) 1 mg/ml BSA. Store at 2-8o 2. Tissue factor monoclonal antibody in tris buffered saline (pH 7.4) 1 mg/ml BSA. 20 Store at 2-8o EQUIPMENT REQUIRED 1. SonoclotTM Analyzer - 24 - WO 01/07070 rYtL/umUUult 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 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 10 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. 19mm gauge needle 15 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. 20 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 - 25 - WO 01/07070 PC/USU/UI ZU115 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 5 into a cuvette that has been placed in one of the side wells in the Sonoclot Analyzer. b) Place probe on SonoclotTM in appropriate position using a slightly twisting motion. Place cuvettefirmly down in the receptacle, also using a slight twisting motion. c) Insert one magnetic stir bar into cuvette. 10 d) Add 40 itl 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 pl of sample into cuvette in the SonoclotTM Analyzer. Avoid formation of bubbles. f) Gently reaspirate the sample once only (to avoid platelet activation) to mix it well 15 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 SonoclotTM 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 20 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 SonoclotTM Analyzer. The SonoclotTM Analyzer automatically calculates the clotting time. The test should be allowed to - 26 - WO 01/07070 PCT/US00/20118 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 5 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 10 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. 15 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 20 contact with blood. 5. Use appropriate disinfection procedures to remove spilled blood. 6. Store test kit materials at 2-8o C. Do not use after expiration date. This test is for in vitro -27- WO 01/U707U rUtl/UNUU/2U115 diagnostic research use only. QUALITY CONTROL 5 1. The viscosity oil control provided with the Sonoclot"M 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 pl samples from the same incubation vial and place in two instruments to 10 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 SonoclotTM Instruction Manual. 15 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 20 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 25 transfer error, or sample mix up 2. Instrument Errors - Failure to follow instructions, failure to perform -28- WU UI/UTU/U r 1/uauV/Zueo 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 5 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 10 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. 15 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 20 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 - 29 - WO 01/07070 PCT/US00/20118 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 (lml) was incubated in a plastic tube at 37 0 C 5 for 10 minutes or 2 and 4 hours with and without bacterial lipopolysaccharide (LPS) (10pg/ml) (E. coli 055:B5 Westphal, Difco; Detroit, MI). During the incubation at 37 0 C there was no agitation. After incubation blood was remixed and 300 pl was aliquoted into warmed cuvettes that were preloaded with 40 microliters of 0.1 M CaCl 2 and a magnetic stir bar. Following a ten second stirring sequence, the clotting 10 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) 15 (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 20 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) -30- WO 01/07070 IL 1/u"JuUIZU115 (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 5 0 to 80 pg/mL (Figure 4). Example 4 ROLE OF THE CONTACT ACTIVATION PATHWAY ON CLOTTING TIMES OF UNSTIMULATED BLOOD 10 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 15 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 20 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. -31 - WO 01/07070 r I/uauU/LU11 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 5 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 10 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 15 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. 5C). The mean difference in clotting times with and without corn trypsin inhibitor was 141±88 seconds (mean±SD, n=28). Importantly, 20 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 - 32- WO 01/07070 PCT/US00/20118 stimulated blood. Example 5 EFFECT OF INHIBITING ANTICOAGULANTS ON WHOLE BLOOD 5 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 10 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 15 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. 20 Example 6 CLOTTING TIMES OF BLOOD FROM UNSTABLE ANGINA PATIENTS 25 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 -33- WO 01/07070 r LI/uouu/zuti 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 5 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. 10 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. 15 All cited patents and publications are hereby incorporated by reference in their entirety. 20 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 25 507. 2. Hett DA, Walker SN, Pilkington SN, Smith DC. Sonoclot analysis. Br J Anaesth -34- WO I/UU7U Y4 1/UUU/ZlU115 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, Gaffney DP, Mann KG. Blood clotting in 5 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 10 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, 15 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 20 factor levels in unstable and stable angina pectoris. Am J Cardiol 1998; -35 - WO 01/07070 PCT/USUU/20118U 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. 5 -36-

Claims (29)

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. -37- WO 01/07070 PCT/US00/20118

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 co- 1-antitrypsin, activated protein C, antithrombin III, C 1 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. -38 - WO 01/07070 PCT/US00/20118

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. -39- WO 01/07070 PCT/US00/20118

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. - 40 - WO 01/07070 PCT/US00/20118

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

29. The method of claim 27, wherein the inhibitor is an antibody. -41-