WO1992018539A1

WO1992018539A1 - METHOD FOR EVALUATING CONTRIBUTIONS OF EXTRINSIC AND INTRINSIC COAGULATION FACTORS TO A FACTOR Xa ASSAY

Info

Publication number: WO1992018539A1
Application number: PCT/US1992/002813
Authority: WO
Inventors: Harvey B. Pollard
Original assignee: The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
Priority date: 1991-04-15
Filing date: 1992-04-14
Publication date: 1992-10-29
Also published as: AU1879692A

Abstract

The present invention relates, in general, to a method for evaluating the contribution of an extrinsic or intrinsic coagulation factor to a Factor Xa assay. In particular, the present invention relates to a test kit for evaluating the contribution of an extrinsic or intrinsic coagulation factor to a Factor Xa assay.

Description

METHOD FOR EVALUATING CONTRIBUTIONS OF EXTRINSIC AND INTRINSIC COAGULATION FACTORS TO A FACTOR Xa ASSAY BACKGROUND OF THE INVENTION Field of the Invention The present invention relates, in general, to a method for evaluating the contribution of an extrinsic or intrinsic coagulation factor to an assay. The present invention also relates to a test kit for evaluating the contribution of an extrinsic or intrinsic coagulation factor to an assay for Factor Xa.

Backσround Information I. THE MECHANISM OF BLOOD COAGULATION

ON THE ENDOTHELIAL CELL SURFACE A. The first stage of hemostasis occurs after injury to the endothelial lining of a blood vessel.

Endothelial cells line blood vessels throughout the body, and must therefore present to the blood a surface with profoundly anti-coagulant properties. However, the endothelial cells cover, and contribute biosynthetically to, the collagen rich subendothelium, the exposure of which is the primary molecular signal for the initiation of hemostasis. For example, a slight injury to the endothelium can lead to exposure of the subendothelial collagen. As shown in Figure 1, this exposed collagen immediately activates platelets to form an adherent plug by means of specific platelet/collagen contacts mediated by vWF (von Willibrand's Factor, also referred to in older literature as Factor VIII:R. It is secreted from platelets and endothelial cells when appropriate stimulus occurs) . B. The second stage of hemostasis strengthens the primary repair event.

The second stage of hemostasis occurs when fibrin is formed from fibrinogen to cross-link and strengthen the platelet/collagen complex. The pro- coagulant processes which lead to the formation of fibrin occur on the surfaces of the endothelial cells at the site of injury, and depend on numerous clotting factors, including Factor VIII. The endothelial cell surface is also the site of a number of negative feedback proteins which further act to restore the anti-coagulant state before too much hemostasis occurs. Finally, it should be appreciated that the equilibrium between the pro-coagulant and anti-coagulant state is dynamic and physiologic. Injury and repair are always occurring. For example, merely climbing five flights of stairs significantly and transiently elevates the Factor VIII activity in the blood. (Indeed, this fact was once used as a basis for increasing the Factor VIII activity of plasma collected for transfusion into hemophiliac patients.)

II. ASSAY FOR FACTOR VIII ACTIVITY IN PLASMA A. Intrinsic and extrinsic pathways promote pro- coagulant activity. The formation of fibrin proceeds by two main

Processes. One of these is intrinsic to the blood, and involves Factor VIII and Factor IXa, which together activate Factor X to Factor Xa (see Figure 2) . Factor Xa, also termed pro-coagulant activity, then converts prothrombin to thrombin, by proteolysis. This process depends on Factor V. Thrombin then converts fibrinogen to fibrin (see Figure 3) . Activation of Factor X requires not only Factor VIII and Factor IXa, but also calcium and acidic phospholipids, which are contributed by activated platelets and broken endothelial cells. A defective or missing Factor VIII, or the existence of an anti-Factor VIII antibody, can cause the hemostatic disorder. Hemophilia A. Similarly, a defective or missing Factor IX can also cause a hemostatic disorder, which is termed Hemophilia B.

The second process which can contribute to formation of the fibrin polymer is extrinsic to the blood, and is due to the synthesis and expression of Tissue Factor on the endothelial cell surface. Tissue Factor and Factor VII join together with acidic phospholipids and calcium to produce activated Factor Vila, which then converts Factor X to Factor Xa. This process is shown in Figure 4. Tissue Factor also occurs in a pre-expressed form on some non-endothelial cells.

B. The Factor Xa assay can be used to measure pro-coagulant activity. In order for Factor Xa to proteolyze prothrombin to thrombin. Factor Xa must further interact with Factor V on the endothelial cell surface. However, the Factor Xa protease activity, per se, can be measured directly in the absence of Factor V with a chromogenic substrate, and is the basis of a clinically useful test for Factor VIII. This test is marketed by Kabi-Vitrum as the COATEST (an acronym for CO-factor Eight TEST) . As shown in Figure 5, the test is carried out by adding human plasma, with an unknown amount of Factor VIII, to a mixture of bovine Factor IXa, calcium, phospholipids, and bovine Factor X. Any active Factor Xa that is generated under these conditions is due to Factor VIII in the human plasma. The Factor Xa assay (COATEST assay) works well with plasma from a variety of species, including human, bovine, and rat. For the cases of human and bovine, a titration of Factor Xa activity as a function of different concentrations of diluted plasma is shown (Figure 6) . C. Specific antibodies distinguish intrinsic from extrinsic mechanisms for activation of Factor X. The Factor Xa Assay can also detect activation of Factor Xa due to Tissue Factor/Factor vr a in the extrinsic pathway. However, the two pathways can be clearly distinguished from each other by the fact that the intrinsic pathway requires Factor IX. Indeed, virtually no activity due to the extrinsic pathway occurs in plasma. However, it is always possible that activity measured in cell extracts could have an extrinsic pathway component.

The present invention provides a method to analyse Factor Xa activity in terms of inhibition of the reaction by specific antibodies against Factors VII, VIII and IX. It was found that a rabbit anti- Factor IX antibody completely abolished Factor Xa activity when Factor VIII in plasma or cells was being measured. A monoclonal antibody to Factor VIII

(Hybritech) abolished Factor Xa activity in plasma, and to different extents in cell extracts or cell surfaces. Finally, anti-Factor VII inhibited only that Factor Xa activity which was dependent upon the Tissue Factor/ Factor VII system. The details of this assay will become more apparent from a reading of the description that follows. SUMMARY OF THE INVENTION It is a general object of this invention to provide a method for evaluating the contribution of factors to a Factor Xa assay. It is a specific object of this invention to provide a test kit for evaluating the contribution factors to a Factor Xa assay.

Further objects and advantages of the present invention will be clear from the description that follows.

In one embodiment, the present invention relates to a method for evaluating the contribution of an extrinsic or intrinsic coagulation factor to a Factor Xa assay. In another embodiment, the present invention relates to a test kit for evaluating the contribution of an extrinsic or intrinsic coagulation factor to a Factor Xa assay.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. The First Stage of Hemostasis.

Exposure of the subendothelium leads to platelet activation and formation of platelet plugs mediated by chains of von Willibrand's Factor Molecules. Fibrin cross-links later reinforce the plug. Figure 2. Activation of Factor X By Factor

IXa and Factor VIII.

Figure 3. Activated Factor Xa and Factor V Activate Prothrombin to Thrombin. Thrombin cleaves fibrinogen to fibrin, which cross-links the platelet- subendothelium plug.

Figure 4. Mechanism of Activation of Factor Xa By The Extrinsic Pathway. Tissue Factor activates Factor VII, and together both molecules activate Factor Xa.

Figure 5. Molecular Basis of the Factor Xa Assay for Procoagulant Activity. Factor VIII mediates the activation of Factor X to Xa by activated Factor IXa. Factor Xa cleaves a chromogenic substrate to yield a yellow color.

Figure 6. Tests on Linearity of Factor Xa Assay For Pro-Coagulant Activity in Bovine and Human Plasmas.

Figure 7. Factor Xa Activity in Endothelial Cell Extracts. A. A linear relationship exists between Factor Xa activity and the amount of extract in assay, if an internal standard of human plasma is used to correct for naturally occurring inhibitors. B. Naturally occurring inhibitor of Factor Xa in endothelial cell extracts.

Figure 8. Inhibition of Clotting Activity in Plasma and Endothelial Cell Extracts by Different Concentrations of Anti-Factor IX.

Figure 9. Inhibition of Factor Xa Activity in Human Plasma by Endonexin II. A. Change in absorbance at 405 nm as a function of endonexin II concentration. B. Percent change in maximal Factor Xa activity, calculated from data in A.

Figure 10. Inhibition of Factor Xa Activity in Endothelial Cell Extracts by Endonexin II. A. Inhibition of Coatest activity at 405 nm by increasing concentrations of endonexin II. B. Percent change in Coatest activity, calculated from data in A.C. Percent maximum change in activity as a function of endonexin II concentration, calculated from B. Figure 11. Inhibition of Factor Xa Activity in Human Plasma by Endonexin II occurs only if Protein is Added Before Activation of Factor X to Xa. A. Factor Xa activity in different concentrations of endonexin II, when protein is added before (filled circles) or after (solid circles) activation. B. Percent change in maximum activity, calculated from data in A.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates, in one embodiment, to a method for evaluating the contribution of an extrinsic or intrinsic coagulation factor to a Factor Xa assay. The method comprises:

(1) incubating a biological sample with antisera containing anti-Factor VII; anti-Factor VIII; anti- Factor IX; or anti-Factor VII, anti-Factor VIII, and anti-Factor IX under conditions such that antibody/antigen binding can occur,

(2) adding Factor IXa and Factor X to (1) and incubating under conditions such that Factor Xa may be formed,

(3) adding chromogenic substrate to (2) , and

(4) assaying for change in said chromogenic substrate. Biological samples that can be used in the present method include cell extracts and plasma. The samples can be obtained from animals, preferably mammals, including humans, cattle and pigs. In a preferred embodiment, calcium and/or phospholipids are added to the incubation mixture resulting from step (1) in the above-described method. For example calcium can be added to a final concentration in the range of 2 to 10 mM. Phospholipids, for example phosphatidyl- serine, can be added to a final concentration of 10 to 20 μg/ml.

The amount of biological sample and antisera with anti-Factor VII; anti-Factor VIII; anti-Factor IX; or anti-Factor VII, anti-Factor VIII, and anti-Factor IX to be used can readily be determined by one skilled in the art. The amount of biological sample to be added is a volume of up to 50 μl of various dilutions of sample, while the amount of antisera to be added is a volume of up to 50 μl of various dilutions of antisera. Likewise, the amounts of Factor IXa and X can be readily determined by one skilled in the art. Preferably, the amount optimized for human plasma in the COATEST assay is employed. Chromogenic substrates suitable for use in the present invention include substrates that are hydrolyzed by Factor Xa, for example, S2222 (50% N- benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p- nitroanilide hydrocholoride and 50% of its methyl ester) . Changes in the substrate can be detected by an increase in the optical density at 405 nm.

In another embodiment, the present invention relates to a test kit for evaluating the contribution of an extrinsic or intrinsic coagulation factor to a Factor Xa assay. The kit can include a container means having disposed there within Factor IXa; a container means having disposed there within Factor X; a container means having disposed there within a chromogenic substrate; and at least one container means having disposed there within at least one of anti- Factor VII; anti-Factor VIII; and anti-Factor IX.

The activity of coagulation Factor VIII in human plasma is assessed clinically by the use of the COATEST, a kit manufactured by Kabi-Vitrum (now Kabi- Pharmacia) , which contains Factor IXa, Factor X, and the chromogenic substrate, S2222. In the presence of Factor VIII in plasma. Factors VIII and IXa form a complex which activates Factor X to Factor Xa. Factor Xa then hydrolyzes S2222 to a yellow product. This kit is used to follow the clinical course of hemophiliac patients. However, many clinicians run more tedious biological assays because of the possibility that extrinsic pathway coagulation factors, such as Factor VII and Tissue Factor, might contribute to the signal. This is because Factor VII and Tissue Factor together can activate Factor X directly. The present invention provides a method of blocking the intrinsic pathway (VIII and IX) by monoclonal anti-VIII antibody, or by polyclonal rabbit anti-IX antibody. The possible contribution by the extrinsic pathway can be blocked by rabbit anti VII antibody. Using this new positive method, the clinical COATEST kit (or any other such combination) can be expanded to provide maximum information for the clinician without recourse to biological assays.

The present invention is described in further detail in the following non-limiting examples. EXAMPLE 1

Ouantitation of Factor Xa activity in extracts of cultured bovine endothelial cells It was necessary to determine whether the antigens detected by anti-Factor VIII antibody in adrenal endothelial cells were indeed associated with pro-coagulant activity. Therefore, pro-coagulant activity in cell extracts was measured with the COATEST, a clinical kit made by Kabi-Vitrum. Extracts of bovine, human and porcine endothelial cells were prepared (The first description of the bovine endothial cells is Banerjee, D.K. , et al. (1985) Proc. Natl. Acad. Sci. USA 82: 4702-4706). It was determined that the extracts contained substantial amounts of Factor Xa-positive activity. However, as shown in Figure 7B for the case of bovine cells, it was also found that the cell extracts contained a powerful inhibitor of Factor Xa activity. Indeed, progressive dilution of the endothelial cell extract resulted in increased levels of Factor Xa activity.

To correct for the inhibitory activity in the extracts, human plasma, as an internal control with known amounts of authentic Factor VIII activity, was added. Thus, from these data, the true activity of the sample could be estimated. Using this method it was possible to compute the total activity in the extract, and to generate a linear relationship between the amount of extract and the measured activity (see Figure 7A) . Pre-treatment with the monoclonal antibody against Factor VIII from Hybritech was found to reduce the total Factor Xa activity of this bovine cell extracts by 18.9%. In the same experiment the antibody inhibited an aliquot of human plasma by 95.2%. It was therefore possible that a fraction of the Factor Xa activity was indeed due to Factor VIII.

EXAMPLE 2 Total Factor Xa activity in adrenal medullary endothelial cells from different species, and in some non-endothelial cells.

Endothelial cell cultures were also prepared from adrenal medulla tissue of other species, including human and pig. The cells grew in a manner quite similar to that which was observed for the bovine cells, and therefore, these cells from other species were tested for Factor Xa activity. As shown in Table I, bovine endothelial cells were the most active, followed by porcine and human cells. In each case, care was taken to ensure that the extract was prepared from the same number of cells. This was a relatively simple task since a confluent T75 flask of endothelial cells from each species routinely contained 5 - 6 million cells.

For comparison human I38 and WI38V fibroblasts were examined because of their reported high levels of Tissue Factor and Tissue Factor mRNA. Indeed, the Tissue Factor cDNA had been cloned from a I38 library (10) . However, the Factor Xa activity in these cells was ca. 20-fold lower than even the human endothelial cells. Substantial Factor Xa activity could only be elicited from the I38 or I38V extracts if Factor VII was added. These data thus appeared to support the hypothesis that the Factor Xa Assay could be used to delineate an authentic extrinsic pathway system for activation of Factor X from one involving the intrinsic pathway.

Factor Xa activity was also tested in extracts of rat PC12 cells, and the level was found to be zero. As a positive control it was verified that rat plasma Factor VIII could be easily assayed by the COATEST method. The PC12 cells were of specific interest because they represented a transformed state of the chromaffin cells with which the endothelial cells co-existed in the adrenal medulla. RELATIVE +TOTAL

CELLS ACTIVITY ACTIVITY

25.0 15.6 3.25

0.138

0.125

RAT PC12

Optical density units in coatest assay /5x10⁶ cells.

WI38 and WI38V are non -transformed and transformed lines, respectively, of a human fibroblast line used to clone tissue factor from a cDNA library.

Table I . PRO-COAGULANT ACTIVITIES IN EXTRACTS OF ENDOTHELIAL

CELLS, AND IN NOMINALLY NON- ENDOTHELIAL CONTROL CELLS. The WI38 cells are human fibroblasts enriched for tissue Factor. WI38V is virally transformed line of WI38.

EXAMPLE 3 Influence of antibodies to Factors VII . VIII and IX on Factor X activation activities of plasma and of endothelial cells. The Factor Xa assay depends on the presence of Factor VIII to combine with Factor IXa and Factor X, thereby activating Factor X to Xa. It was expected that anti-Factor VIII antibody would inhibit endogenous Factor VIII directly, and thereby reduce Factor Xa activity. Indeed, the monoclonal antibody that was chosen did so in plasma. However, only a 10-20% inhibition of total Factor Xa activity was observed when cell extracts were used. The problem was whether it was valid to conclude that the level of inhibition in cell extracts indeed represented a quantitative estimate of the fraction of Factor Xa activity due to Factor VIII. Previously, lipids had been reported to interfere with the ability of anti-Factor VIII to block the pro-coagulant activity of Factor VIII (11) . In fact, a lipid rich formulation including Factor VIII has been marketed in Europe by ImmunoAG as a strategy to circumvent endogenous inhibitors (antibodies) present in some hemophiliac patients. A less direct, but nonetheless specific approach might be to remove Factor IXa from the Factor Xa assay, or to inactivate Factor IX with a specific antibody. The antibody approach was chosen in order to retain the Factor Xa Assay, as marketed, as our basic test system. It was determined whether anti-Factor IX would abolish any activity contributed by Factor VIII because Factor IX was the obligatory medium through which Factor VIII caused activation of Factor X. As a control, a separate experiment was also prepared with a reaction mixture missing added Factor IXa.

The other possible source of activation of Factor X could come through the intrinsic pathway, which depends on Tissue Factor and Factor VII. Anti- Factor IX would have no inhibitory effect on the intrinsic pathway. However, inclusion of anti-Factor VII could abolish whatever fraction of the Factor Xa activity was due to Factor VII.

To test these concepts, samples of cell extract or plasma were pre-incubated with 1:30 dilutions of antisera containing either anti-Factor

VII, anti-Factor VIII or anti-Factor IX, or a mixture of all three for 5 minutes at 37°C. Immediately thereafter calcium, phospholipids, and the mixture of Factors IXa and X were added. Activation was allowed to proceed for 10 minutes and the chromogenic substrate added. As shown in Table II, bovine plasma Factor Xa activity was essentially abolished by anti-Factor IX, substantially reduced by anti-Factor VIII, and even reduced somewhat by anti-Factor VII. By comparison, when the same protocol was employed with bovine endothelial cell extract, anti-Factor IX reduced Factor Xa activity to 37% of control. By contrast, anti- Factor VIII reduced Factor Xa activity by 12% and anti- Factor VII was essentially without effect. A similarly treated porcine endothelial cell extract was inhibited by anti-Factor IX to approximately 54% of the control activity, while anti-Factor VIII and anti-Factor VII appeared to have no effect. These initial data indicated that the anti-clotting factor antibodies, in concert with the COATEST, could prove useful for differentially evaluating the contributions of intrinsic pathway clotting activity to pro-coagulant activity in plasma and cells.

These experiments were repeated a number of times on different preparations of cell extracts and plasma samples. The results are summarized in Table III, for human and bovine plasma, and for bovine and porcine endothelial cells. From these data it is apparent that the general patterns of sensitivities observed in the initial experiment are typical. Secondly, approximately 50% of the activity found in extracts appeared sensitive to anti-Factor IX. These studies had been performed in one specific dilution of antisera, it was therefore essential to repeat these experiments in the context of a titration. Because the COATEST contained Factor IX in vast excess, the dilution of antibody chosen, although small, might still not have been enough to inactivate all Factor IXa molecules. An additional advantage of a titration would be to allow testing of whether the mechanisms of inhibition in both plasma and cells were similar. For example, if the mechanisms were similar, a similar inhibition constant and slope for the inhibition curves would be present. As shown in Figure 8, increasing concentrations of anti-Factor IX antibody inhibited activation of Factor Xa activity in human and bovine plasma, and in bovine endothelial cell extracts in a similar and complete manner. Thus it can be concluded that coagulant activity in these samples are similarly dependent upon Factor IX. By contrast, approximately 25% of the Factor Xa activity in porcine endothelial cells is apparently resistant to inhibition by anti-Factor IX antibody. Nonetheless, for the 75% which was sensitive to anti-Factor IX, the apparent inhibition constant was not substantially different from that observed with the other samples.

Thus, bovine endothelial cells contain activity in the Factor Xa assay that is strongly suggestive of a similarity to Factor VIII activity found in plasma. It is also apparent that the porcine cells also have such activity, although apparently proportionately less. Finally, the activity, at whatever level, does not appear to be due to contributions from the extrinsic pathway. This conclusion is valid, not only because of the dependence on Factor IX, which was verified in a separate assay with Factor IX omitted. but also because of the insensitivity of the reaction to anti-factor VII. Table II. INFLUENCE OF ANTIBODIES TO FACTOR VII, FACTOR VIII AND FACTOR IX

ON PROCOAGULANT ACTIVITIES OF PLASMA AND ENDOTHELIAL CELLS.

Table III. SUMMARY OF INFLUENCE OF ANTIBODIES TO FACTOR VII, VIII AND IX ON FACTOR Xa ACTIVITY IN BOVINE AND PORCINE ENDOTHELIAL CELLS, AND IN BOVINE AND HUMAN PLASMA

BOVINE 82 + 18 (2) 35.8 + 13.3 (3) 5.8 + 5 (4) 0(1) HUMAN 84 (1) 6.5 + 0.1 (2) 5.7 + 3.3 (n=2)

(n) = number of separate experiments; + = S.E.M.(n>2) + = range (n=2)

EXAMPLE 4 Sensitivity of Factor Xa activity to the sulfhvdryl reaσent. N-ethyl-maleimide. Sulfhydryl groups on proteins involved in the activation of Factor X by the intrinsic pathway can be blocked by the sulfhydryl reagent N-ethyl-maleimide (NEM) . It thus appeared that a further test of the relationship between Factor Xa activity in plasma and in cell extracts might be approached by testing both samples for sensitivity to NEM. Plasma was therefore treated from humans and cows, and endothelial cell extracts from cows and pigs, with 1 mM NEM for 30 minutes on ice prior to initiating the Factor Xa assay. As shown in Table IV, treatment with NEM blocked the Factor Xa activity of bovine plasma and bovine endothelial cell extract by approximately 58% and 61%, respectively. Porcine endothelial cell extracts were blocked by 73%, and human plasma was blocked by 94%. These data indicate that the type of activity contributed by plasma, believed to be nearly exclusively Factor VIII, seemed to be present to some degree in the cell extracts.

To test the nature of the residual activity in plasmas and cell extracts following treatment with NEM, plasma and cell samples were treated with NEM, and then added the different anti-clotting factor antibodies prior to starting the Factor Xa assay. The consequences of adding anti-Factor VII, VIII or IX, was that the residual activities were approximately as sensitive to these reagents as were the initial activities measured in the absence of NEM pre-treatment (see Table V) . Therefore, it was concluded that the residual activity following NEM treatment was a surviving representative of the original activity, and not some new or different activity. Furthermore, the NEM inhibition experiment did not seem to distinguish between Factor Xa activity measured in either plasma samples or cell extracts.

EXAMPLE 5 Blockade of Factor Xa activity in plasma and endothelial cells by endonexin II (placental anticoagulant protein) . Human endonexin II, also known as placental anticoagulant protein, PAP (12) , is enriched in endothelial cells, and has been shown to function as an inhibitor of the intrinsic pathway for activation of Factor X. The exact mechanism of action of endonexin II is not known. However, this protein might be used as a reagent to compare, and perhaps distinguish, procoagulant activities in plasma and cell extracts. As shown in Figure 9A, endonexin II can completely inhibit Factor VIII-dependent Factor Xa activity in human plasma, with a k(l/2, app.) = 12.5 nM (Figure 9 B).

TABLE IV. INFLUENCE OF N- ETHYL MALEIMIDE ON CLOTTING ACTIVITY IN PLASMAS AND ENDOTHELIAL CELL EXTRACTS FROM BOVINE AND PORCINE SOURCES.

SAMPLE ACTIVITY % INHIBITION

58.2

BOVINE ENDO'S:

CONTROL 0.567 ± .027

+NEM 0.205 ± .002 60/9

PORCINE ENDO'S:

CONTROL 1.563 ± .007

+NEM 0.419 + .007 73.2

94-

SAMPLES WERE PRE-TREATED WITH ImM NEM ON ICE FOR 30 MIN, AND THEN ASSAYED BY THE FACTOR Xa ASSAY.

% MAXIMAL ACTIVITY IN ImM NEM

+ NUMBERS IN ( )'s ARE ACTIVITIES IN PRESENCE OF 1 mM NEM.

TABLE V. NEM-TREATED HUMAN AND BOVINE PLASMAS, AND PORCINE AND BOVINE

ENDOTHELIAL CELLS: SENSITIVITY OF NEM-RESISTANT ACTIVITY TO ANTIBODIES TO FACTOR VII, VIII AND IX.

A similar titration of endonexin II was performed with bovine endothelial cell extracts. As shown in Figure 10A, approximately 70% of the total Factor Xa activity in the extract could be inhibited over the same concentration range (Figure 10 A and B) . However, if the total % change occurring in this titration were compared, rather than the fraction of total activity inhibited, as in Figure IOC, the k (1/2, ^aPP«) — 14.1 nM. These data thus indicate that endonexin II inhibits substantial amounts of Factor Xa activity in both samples, and that the inhibition constants are similar for both inhibition processes. An important control for these studies was the observation that the site of action of the endonexin II in the Factor Xa assay was on the activation step for Factor X, rather than on the activity of activated Factor Xa. As shown in Figures 11 A and B, a dose-dependent inhibition of the Factor Xa activity found in plasma was only observed when endonexin II was added before activation of Factor X to Factor Xa. However, when endonexin II was added after activation of Factor X, no inhibition was observed. Therefore, a common mechanism of inhibition by endonexin II for both plasma and cell extract Factor Xa activity strongly indicates that both samples act at a common site for the activation of Factor X. Based on these and the previous pieces of data the possibility that Factor VIII is the common mediator cannot be excluded. EXAMPLE 6 Expression of procoaσulant activity on the endothial cell surface Endothelial cells express procoagulant activity on their surface in response to injury, or in response to monocyte-derived cytokines such as Interleukin-1 (IL-1) or Tumor Necrosis Factor (TNF) . Similar expression occurs in reponse to gram negative bacterial cell walls (liposaccharide, LPS) <Bevilacqua, M.P. (1984) J. Exp. Med. 160:618-623). As shown in

Table VI, Factor Xa activity on both bovine and porcine cells was substantial, but insensitive to anti-Factor VII. By contrast, anti-Factor IX reduced the activity by 20-30%. These data thus indicated that surface procoagulant activity was not likely to be due to

Tissue Factor/Factor Vll-activation of Factor X (see Figure 4 for details) .

A positive control was needed for this important result, and therefore, cells known to express Tissue Factor were tested for sensitivity to anti- Factor VII and IX. These cells included a human adrenal medullary endothelial cell culture (IM42-12) , the human fibroblast line WI38, from which Tissue Factor had been cloned, and WI38V, a virally transformed line of WI38. As shown in Table Via, all three cell types had low endogenous Factor Xa activities, which were insensitive to anti-Factor VII, but activated by anti-Factor IX. However, a mixture of anti-Factors VII and IX suppressed the activity. These data indicate that the anti-Factor IX antiserum contained Factor Vila, which, in concert with cellular Tissue Factor, was able to activate Factor X directly. It can be concluded that bovine and porcine endothelial cell cultures do not express Tissue Factor, and thus the Factor Xa/antibody assay was clearly able to distinguish intrinsic (Factor VIII and Factor IX) from the extrinsic coagulation pathway.

TREATMENT BOVINE CELLS PORCINE CELLS PCA % INHIBITION PCA % INHIBITION

+ % OF MAXIMAL ACTIVITY = 109%. + + % OF MAXIMAL ACTIVITY = 117%.

TABLE VI. SENSITIVITY OF SURFACE PROCOAGULANT ACTIVITY IN BOVINE AND PORCINE ENDOTHELIAL CELLS TO ANTI-FACTOR VII AND ANTI-FACTOR IX.

ACTIVATION OF TISSUE-FACTOR-DRIVEN

FACTOR Xa ACTIVITY BY RABBIT SERUM CONTAINING

ANTI-FACTOR IX ON THE CELL SURFACE OF HUMAN

WI38 AND HUMAN WI38V FIBROBLASTS, AND

ON A PASSAGE OF HUMAN ENDOTHELIAL CELLS

CELLS AND CONDITIONS ACTIVITY

HUMAN ENDO'S (IM42-12) CONTROL 0

+ANTI-FACTOR VII 0.018 ± .002

+ANTI-FACTOR VIII 0.012 ± .002

+ANTI-FACTOR IX 0.288 + 0.18

HUMAN WI38

CONTROL 0.077 ± .012

+ANTI-FACTOR IX 0.719

HUMAN WI38V CONTROL 0

+ANTI-FACTOR IX 0.587

TABLE VIA. SURFACE PROCOAGULANT ACTIVITY DUE TO

TISSUE FACTOR/FACTOR VII-DRIVEN ACTIVATION OF FACTOR X IN HUMAN CELLS

CONTAINING TISSUE FACTOR. Human endothelial cells and human fibroblasts (WI38 and WI38V) express large amounts of Factor Xa activity when exposed to rabbit serum containing anti-Factor IX.

The antibody inactivates Factor IX, but the serum also contains Factor Vila, which together with Tissue Factor in the cells directly activates Factor X to Xa. * * * * * All publications mentioned hereinabove are hereby incorporated in their entirety by reference. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for evaluating the contribution of an extrinsic or intrinsic coagulation factor to an assay for Factor Xa activity comprising: (1) incubating a biological sample with antisera containing anti-Factor VII; anti- Factor VIII; anti-Factor IX; or anti-Factor VII, anti-Factor VIII, and anti-Factor IX under conditions such that antibody/antigen binding can occur,

(3) adding chromogenic substrate to (2) , and (4) assaying for change in said chromogenic substrate.

2. The method according to claim 1, wherein said biological material is animal cell extract or animal plasma.

3. The method according to claim 1, wherein calcium and phospholipids are added with Factor IXa and

Factor X to (1) .

4. The method according to claim 1, wherein said chromogenic substrate is S2222.

5. The method according to claim 1, wherein said animal is a mammal.

6. The method according to claim 5, wherein said animal is selected from the group consisting of humans, cattle, and pigs.

7. A test kit for evaluating the contribution of an extrinsic or intrinsic coagulation factor to a Factor Xa assay comprising a container means having disposed there within Factor IXa; a container means having disposed there within Factor X; a container means having disposed there within a chromogenic substrate; and at least one container means having disposed there within at least one of anti- Factor VII, anti-Factor VIII, and anti-Factor IX.