JPS63273061A - Diagnostic probe for detecting antithronbin modified by elastase and treating method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Antithrombin modified with elastase (elas
tase modified antithrombi
A diagnostic probe for detecting the presence or absence of a coagulopathic condition consisting of
5tates) to provide a means for early detection. Once diagnosed, treatment includes anticoagulant therapy, fibrinolytic therapy or anti-elastase therapy, or treatment with recombinant antithrombin that is not subject to elastase inactivation.

Acquired antithrombin (AT-I), such as disseminated intraluminal coagulation (DIC), sepsis and other inflammatory diseases.
II) Acquired antithromb
in(A7'-m) deficiencies
) earthquakes are associated with pre-thromboti
c 5tate) has been hampered by the inability to diagnose the early onset of the disease. Treatment of acquired defects with AT-11 [or other elastase inhibitors] has been delayed until clinical symptoms appear.

Delayed treatment yielded equivocal results. These equivocal results are believed to reflect the unpredictable severity of symptoms and speed of onset of the disease state and the possibility of inappropriate treatment doses.

Recently, functional plasma antithrombin (functi)
anal plasma antithrombin)
The determination of AT-III consumption or replacement (t
constitutes the only diagnostic test to confirm (unover). These are major thrombotic episodes.
Although it can provide some information during or after thrombotic episodes), it is insensitive to pre-thrombotic conditions. vascular inner layer
AT- in a limited or local reaction zone such as ining)
Consumption of III will generally have little effect on the total plasma pool of antithrombin. Local events within the vasculature, even locally severe, have little effect on the normally high (2 micromolar) overall antithrombin levels. Current clinical tests that measure antithrombin levels are inadequate to diagnose the potentially small changes that occur in prethrombotic conditions.

Applicant's recent studies that inactivation of AT-III is heparin-dependent suggests that under conditions the nonthrombotic nature of the vasculature
provides a mechanism by which re) is neutralized. Based on this mechanism, detecting the presence or absence of elastase-modified antithrombin provides early diagnosis and therefore treatment of prothrombotic and thrombotic conditions.

The present invention relates to a diagnostic glove for detecting the presence or absence of elastase-modified antithrombin. Once the diagnosis is made, anticoagulant, fibrinolytic or anti-elastase therapy can be instituted.

Direct means for detecting elastase-modified antithrombin include monospecific (monospec) for elastase-modified antithrombin.
Hic) Includes the use of polyclonal antibodies or monoclonal antibodies specific for antithrombin modified with elastase.

Indirect means for detecting elastase-modified antithrombin include separating the smaller cleaved peptide and quantifying the amount of the separated smaller cleaved peptide.

Publications and other documents which further describe the invention are set forth below in bibliographical form and are incorporated herein by reference.

Under normal conditions, endothelium-bound heparin (endothelin)
Bound heparin forms a complex with antithrombin and imparts non-thrompogenic properties to the inner surface of blood vessels.
ogenic nature). Under certain conditions such as DIC, inflammation, and sepsis, the non-thrompogenic nature of the inner lining of blood vessels is compromised.
d) result in life-threatening thrombosis. Recent studies have shown that heparin inhibits neutrophil elastase, which acts to override heparin's normal anticoagulant function.
It was suggested that it has an affinity for (elastase).

Heparin is an accelerator cofactor for antithrombin, a circulating inhibitor of clotting enzymes.
factor). The presence of this anticoagulant in the blood vessel wall contributes to the non-thrompogenic nature of its surface. Apparently contrary to this function, heparin was found to greatly enhance the inactivation of antithrombin by neutrophil elastase.

The inactivation rate was enhanced several hundred times in vitro by heparin fractions with anticoagulant activity. Neutrophil elastase also showed considerable affinity for immobilized heparin.

These results suggest a mechanism that, if operative in vivo, could lead to localized neutralization of heparin function.

Antithrombin, the major plasma inhibitor of blood clotting enzymes, is a member of a family of related proteins that includes several serine protease inhibitors (serpins) (1). These proteinase inhibitors participate in a well-known mechanism of forming covalently bound, essentially irreversible complexes with target enzymes (2). Interestingly,
Some of these inhibitors are themselves targets of attack by proteinases of mammalian and non-complementary origin. In the case of antithrombin, specific and inactivating cleavage is catalyzed by neutrophil elastase near the active site sequence of this inhibitor (3). This may contribute to the development of life-threatening thrombosis with widespread activation of neutrophils in inflammation and sepsis, as proposed (4°5).

Maintaining the plasma concentration of antithrombin at or near the normal level of about 2 micromolar is clearly essential to avoid the tendency to blood clotting in inherited defects and diseases. Disease-related reductions, which can be more than 50% in extreme cases of sepsis, have often been attributed to the consumption of antithrombin in the process of inhibiting clotting enzymes.

The recently described in vitro inactivation of antithrombin by neutrophil elastase (4) suggests another possible explanation. Nevertheless, extrapolation of this observation to physiological settings must take into account the very high plasma levels of specific elastase inhibitors. This inhibitory factor,
σ-I proteinase inhibitor inhibits elastolytic activity in plasma.
ty). To address this conceptually problematic situation, we explored the potential influence of heparin on the elastase/antithrombin interaction.

The anticoagulant factor heparin is a functionally heterogeneous substance that has the ability to bind to antithrombin and promote the rate of inhibition of antithrombin's clotting enzymes.
It is a sulfated carbohydrate (heterogenous).

Heparin does not normally circulate in the blood, but rather is an integral component of the vascular endothelial cell wall (7).

The anticoagulant activity of heparin purified from animal tissues and administered therapeutically is derived from a limited subtraction (35%) of heparin molecules with affinity for antithrombin (8). Despite its central role in the normal function of antithrombin, a similar enhancement by heparin of antithrombin inactivation by elastase has not been detected in previous studies (
4). We were inspired to re-examine this possibility in view of the profound influence of heparin on antithrombin inhibitory function and its potential ability to localize events to surfaces in vivo.

A study of the kinetics of inactivation of highly purified human antithrombin by neutrophil elastase is shown in Figure 1. We have found that it is essential to use a prepared antithrombin preparation. This is because heparin and often platelet factor 4, a heparin-neutralizing component, are often contaminants of antithrombin purified by affinity chromatography on immobilized heparin supports. The absence of these contaminants was ensured in this study by a specific separation step (9) and confirmed by specific assays for these components (10).

As shown in Figure 1, inactivation of antithrombin occurred at a negligible rate in the absence of heparin. However, small amounts of anticoagulantly active heparin (11)
, a gradual and eventually complete loss of antithrombin inhibitory activity occurred. 50ng/mQ
In the curve representing the heparin concentration, the approximate calculated molar ratios of heparin, elastase and antithrombin were 1/1.5/600, respectively. The rate of inactivation was dependent on heparin concentration at low levels, but reached an apparent maximum rate at approximately 0.5 ug/mo of active heparin species. Therefore, to reach the maximum inactivation rate,
2 of the concentration required for stoichiometric equivalence with antithrombin.
Heparin concentrations of less than % were required. These studies indicate that both heparin and elastase act catalytically to inactivate antithrombin. This catalytic role for elastase distinguishes the mechanism of this inactivation phenomenon from inhibition of clotting enzymes by antithrombin. In the latter case, heparin catalyzes the formation of a covalent stoichiometric complex of antithrombin and the enzyme (12).

To determine the magnitude of the effect of the heparin of the present invention, we compared the rates of inactivation in the presence or absence of saturating levels of heparin (loug/m12) and in each case calculated the first order rate. I guessed that it would be hatched. Approximately 400-fold speed enhancement was observed using 5 nM elastase and 500 nM antithrombin. This enhancement value is 650 nM/min in the presence of active heparin species and 1.0 nM/min in the absence.
Derived from a rate rate of 6 nM/min. Although the determination of this rate enhancement reflects a completely arbitrary set of conditions and reactant concentrations, its magnitude emphasizes that heparin greatly enhances this inactivation phenomenon.

Figure 1 also shows that the inactive heparin fraction did not promote the cleavage of antithrombin by elastase.

Other glycosaminoglycans, including dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and dextran sulfate, also had no significant stimulatory effects at similar concentrations (data not shown). In contrast to the inactive heparin fraction and other sulfated carbohydrates, the distinguishing property of active heparin is its ability to bind with high affinity and specificity to antithrombin.

The apparently stringent requirement for active heparin species in this reaction strongly suggests that the heparin-antithrombin complex is the substrate for elastase attack.

Previous studies using cross-immunoelectrophoresis showed that, in contrast to functional antithrombin, elastase-cleaved antithrombin has little or no affinity for heparin (4). . We confirmed this result using affinity chromatography on immobilized heparin. Figure 2 shows that under conditions where functional antithrombin binds tightly to the heparin column (9), antithrombin inactivated with elastase is not adsorbed.

During the course of this study, we also observed tight binding interactions between heparin and neutrophil elastase itself. The main peak of elastase activity eluted at a sodium chloride concentration of approximately 0.5M. No elastase activity was detected in the unadsorbed eluate region containing elastase-cleaved antithrombin. The avidity of this enzyme for heparin is only slightly less than the avidity of functionally active antithrombin itself under similar conditions of chromatography, which is associated with highly cationic elastase and this acidic affinity matrix. This binding interaction of may be primarily of electrostatic nature. Neutrophil elastase has previously been purified based on its affinity for chromatography gels containing sulfonate residues. Surprisingly, a similar interaction with the physiologically relevant sulfated carbohydrate, heparin, has not been previously reported.

Heparin on the kinetics of antithrombin inactivation/
The outcome of neutrophil elastase interactions is uncertain.

Although these two components work together as a catalyst to inactivate antithrombin, the data of this study did not suggest any interspecificity between them. Rather, inactivation of antithrombin occurs only in the presence of an anticoagulantly active heparin fraction that specifically binds antithrombin. Furthermore, the association of antithrombin is apparently tighter than that of heparin and elastase.

Despite these considerations, the interaction of elastase with endothelium-bound heparin in vivo localizes this enzyme and brings it into close proximity with antithrombin molecules bound at its surface. It may be sequestered from plasma inhibitory factors. From this perspective, the affinity of neutrophil elastase for heparin may have a profound effect on the rate of antithrombin inactivation and be an essential component of this mechanism.

Inhibition of coagulation enzymes by the antithrombin-heparin system is accepted as a crucial regulatory mechanism of the coagulation process. Recent demonstration of anticoagulantly active heparin in vascular endothelium (7) suggests that procoagulant
ulant) and the antithrombin/heparin system as a key component in the balance between the anticoagulant pathway. However, the results of the present in vitro study suggest that under certain conditions, binding of antithrombin by heparin may lead to quite different results.

The rapid and specific inactivation of heparin-bound antithrombin by elastase represents a novel and unexpected function for heparin. A similar occurrence at in vivo heparin sites could result in a localized reversal of the non-thrompogenic properties of the inner lining of the blood vessel.

Reference 1, R. W. Carrel and D. R. Postwell, Proteinase Inhibitors, edited by N. Palette and Hischy Salvesen, (Elastase, Amsterdam, 1986) pp. 403-420 [R, W, Ca.
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Proteinase Inhibitb'rs,
A, Barrett and G, 5alvesen
, Eds, (Elsvier, Amster
dam, 1986) pp 403-4201.

2. J. Travis and G. Nis Salvesen,
Annual Review of Biochemistry, Volume 52,
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9. Human antithrombin was isolated by the method described above using heparin-agarose affinity chromatography [M. Miller-Anderson, H. Borg and L. O. Anderson, Thrombosis Research, 439]. 1974) (
M., Miller-Andersson.

H, Borg and L, -0, Andersso
n, Thromb, RES, 5°439 (19
74))], sodium chloride gradient elution (0°4-1
．． Repurification in heparin-agarose using 5M) served to more tightly separate antithrombin from the bound platelet factor 4 [R.P. Well, and R. D. Rosenberg, Journal of Biological Chemistry 257.4.
00 (1982) (R.E., Jordan, L.
V, Favreau, E, H, Braswell a.
nd R, D, Rosenberg, L, Biol.
, Chew, 25z, 400 (1982)]. Residual contaminating heparin derived from the heparin affinity matrix is removed from QAE-Sepharose (QAE-5e
pharose (Pharmacia) twice.

The functional activity level of heparin, N.N.T. and M.G., Thrombosis Research.
1dan, 399' (1977) [A, N, Te1e
-n and M, Lie, Thromb,
Res, 10.399 (1977)] by a chromogenic substrate assay using Factor Xa. Levels of platelet factor 4 were measured using a commercially available competitive radioimmunoassay (competitive radioimmunoassay).
e radioimmunoassay) kit (Abbott Laboratories, Chicago ■).

11. Active heparin is commercially available porcine mucosal heparin with anticoagulant activity [Scientific Protein Laboratories, Waunekee, Rice Consin (Sci
Entific Protein Laborator
ies.

Waunakee, Wisconsin)]. Active heparin was isolated by adsorption onto a column of immobilized human antithrombin as described above [M. Hook, I. Bjouk, J. Hopwood, and N. Lindahl, FE.
BS Letters 11.90 (1976) (M, Hoo
k, 1. Bjork, J., Hopwood and
U, Lindahl.

FEBS Letters 66, 90 (1976)
) ]. The anticoagulant activity of this substance is approximately 550 units/mg compared to 150 units/mg for the unfractionated substance.
Met.

The anticoagulantly inactive heparin fraction was prepared by removal of the active component by adsorption onto an immobilized antithrombin column and had an activity of less than 5 units/mg.

12, R.D. Rosenberg and B.N. Damas, Journal of Biological Chemistry 248.6490 (1973) [R.D., Ros.
enberg and P, S, Damus J, B
iol, Chem.

擲, 6490 (1973)].

13. D. Y. Toumashi and I. E. Lienner, Journal of Biological Chemistry.
252.1917c1977) [D, Y, Twumas
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, Chem, 252, 1917 (1977)]
; R.G. Bauch and J. Travis, Biochemistry 15, 836 (1976) [R
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chemistry 15.836 (1976)]
.

14, K. Nakajima, J.C. Powers, B.M.Ashe and M.E. Zimmerman, Journal.
of Biological Chemistry-254,402
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J, C, Powers, B, M, Ashe and
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m, 254.4027 (1979)].

15. We thank Dr. James Travis for providing human neutrophil elastase and for helpful suggestions. We also thank our colleagues M, Fournel and T, Em Tsung for important discussions during these studies.
erson), Schroeder ([), [)
,, Chroeder) and M. Mosen (M,M.

I would also like to thank everyone at zen.

FIG. 2 shows heparin-Sepharose chromatography of antithrombin inactivated with elastase. Antithrombin 5mg and elastase 150ug
Start the reaction mixture containing 1 with traces of heparin.
initiate), and antithrombin functional activity was monitored until inactivation was complete. Before adding the reaction mixture to the column, free heparin is removed by adsorption on QAE-Sepharose (9). The reaction mixture was heated to 0.
It was applied to a heparin-Sepharose column (1.1 x 8.8') pre-equilibrated in a buffer consisting of 25 M NaCl, 0.02 M Tris-HCl, pH 7.5.

Antithrombin (Δ) inactivated with elastase
is one independent peak under the conditions of application.
It was eluted as unbound peak).

Elastase was eluted with a 120 mff linear gradient of 0.25M-0.6M NaCl. Elastase activity (0) was determined as described above (14) Chromogenic substrate methoxysuccinyl-ala-ala-pro-va
It was detected by its ability to hydrolyze 1-balanitroanilide (Sigma). The activity contained in each column fraction was normalized to the amount of absorbance produced at 410 nm in 1 minute per mff light of column eluate. Heparin-Sepharose was prepared as previously described (9).

FIG. 1 shows the heparin dependence of antithrombin cleavage by neutrophil elastase. Human neutrophil elastase was a gift from Dr. James Travis and was purchased from Elastin Products, Inc.
A human sputum-derived preparation (human sputum derived from human
sputum-derived preparation
Reaction mixtures containing purified human antithrombin (9) at a concentration of 2 micromolar and various concentrations of heparin in O,15 M NaCl, 0,02 M Tris-HC1, pH 7.5, were incubated at 37°C. It was heated to The reaction was started by adding elastase to a concentration of 5 nanomolar. 1 μg of α-1 proteinase inhibitor (alpha-1pr) per 1 m2.

the second containing teinase 1nhibitor)
Phase assay buffer (second-phas, eass)
ay buffer) (0.15M NaCl, 0.0
The reactions were stopped at the indicated times by diluting an aliquot of the reaction mixture 20-fold in 5M) Lis-MCI, pH 8.4).

Antitumbin activity was then determined by the chromogenic substrate heparin cofactor assay as previously described (6). The individual reaction mixtures were as follows: No heparin added (·): inactive heparin lμg/m
Q (△); anticoagulantly active heparin species 50 (■), 1
00 (○), 400 (mu), or 700 (ro) nanograms/1 mff.

A profound heparin dependence was observed for the in vitro inactivation of purified human antithrombin by neutrophil elastase. In the absence of heparin or during incubation, platelet factor 4 or polybrene (pol
In the presence of heparin blocking agents such as ybrene, little or no inactivation of antithrombin occurred. Only catalytic amounts of heparin were required to cause complete inactivation of antithrombin in 5-10 minutes at an enzyme:inhibitor ratio of 1:400. The elastase-cleaved form of antithrombin loses its affinity for heparin as a result of limited cleavage at a site approximately 42 amino acids from the carboxy terminus. We isolated this inactive antithrombin species and determined its circulating half-life.
g half-1ife) was injected into rabbits for determination. Interestingly, antithrombin inactivated with elastase remains in the circulation for a long time (Tl/2-13 hours) compared to the thrombin-antithrombin complex ('r t/2 less than 10 minutes). may provide a better explanation for the remaining and circulating non-functional antithrombin and suggest a mechanism by which the normally non-thrompogenic properties of the vascular lining may be neutralized during inflammatory conditions. .

The rate of inactivation of the inhibition of coagulation enzymes by antithrombin ■ is greatly increased by the interaction of this inhibitor with a limited anticoagulantly active subfraction of heparin molecules. We also observed that this same heparin subfraction greatly stimulated the rate of antithrombin inactivation by neutrophil elastase. The inactivation rate was increased several hundred times by catalytic amounts of anticoagulantly active heparin species and was not affected by the inactive heparin fraction or other glycosaminoglycans. Preliminary kinetic analysis using elastase-catalyzed reversal of the heparin-induced increase in antithrombin fluorescence suggests a Km less than luM for the heparin-antithrombin complex substrate and hundreds of times suggested a turnover/min/enzyme molecule. Although the specificity of the heparin effect appears to be at the level of its interaction with antithrombin, the elastase-heparin interaction can also be detected in kinetic analyses. Chromatographic studies using immobilized heparin confirmed that elastase itself binds tightly to this carbohydrate, whereas the elastase-cleaved form of antithrombin shows no affinity for heparin. I let it happen. These results suggest a complex and highly physiologically relevant mechanism in which both heparin and elastase act cocatalytically to inactivate antithrombin. Since anticoagulantly active heparin species are present in endothelial cells, the above mechanisms can significantly influence the balance of clotting factor precursors and anticoagulant factors at the normally nonthrompogenic endothelial surface. .

In contrast to current methods for determining functional plasma antithrombin levels, the use of diagnostic probes for the detection of elastase-modified antithrombin may provide a sensitive probe for the detection of prothrombotic conditions. Dew. Diagnostic probes for detecting elastase-modified antithrombin are directed to structural or conformational differences between antithrombin and elastase-modified antithrombin. Neutrophil elastase is approximately 78°0
Antithrombin modified with elastase is in a different conformation because it has a molecular weight of 00 Daltons cut or cleaved.

The presence or absence of elastase-modified antithrombin can be detected directly or indirectly. A direct means of detecting elastase-modified antithrombin is to use polyflonal antibodies monospecific for elastase-modified antithrombin to determine its presence. Polyclonal antibodies can be obtained by injecting elastase-modified antithrombin into a host, causing the host to produce polyclonal antibodies against the elastase-modified antithrombin, and recovering the polyclonal antibodies from the host. Enhanced injections of elastase-modified antithrombin can be used to increase the yield of polyclonal antibodies. Polyclonal antibodies that cross-react with antithrombin and elastase-modified antithrombin are removed. A monospecific polyclonal antibody against elastase-modified antithrombin is then used, and the polyclonal antibody is reacted with a sample suspected of containing elastase-modified antithrombin, and the monospecific antibodies bound to this antigen are reacted with a sample suspected of containing elastase-modified antithrombin. determining the amount or presence of target polyclonal antibodies.

A second direct means for detecting elastase-modified antithrombin is to provide a monoclonal antibody specific for the elastase-modified antithrombin to detect the presence of elastase-modified antithrombin. Or to measure the absence. Monoclonal antibodies can be derived from hybridomas selected from appropriate fusion partners such that monoclonal antibodies specific for elastase-modified antithrombin are expressed. Methods for obtaining hybridomas include murine myeloma cells of BALB/c or other origins (
polyethylene glycol (PEG)-mediated fusion of P3-X63Ag8 and SP210-Ag14 or derivatives thereof with tile cells from BALB/c mice immunized with elastase-modified antithrombin, Electrofusion of murine myeloma cells and tile cells as described above, non-BALB/
c myeloma and non-BAL B/c from mice immunized with elastase-modified antithrombin.
PEG-mediated fusion and electrofusion with tile cells, or murine myeloma cells as described above, and tile cells immunized ex vivo for 3-6 days after primary immunization without in vivo immunization with elastase-modified antithrombin. Electrofusion or PEG-mediated fusion with. It is within the scope of the invention that more than one monoclonal antibody specific for different epitopes can be used to detect the presence or absence of elastase-modified antithrombin. The monoclonal antibody is reacted with a sample suspected of having elastase-modified antithrombin and the presence or absence of monoclonal antibody bound to the antigen is determined.

An indirect means of detecting the presence or absence of elastase-modified antithrombin is to separate or purify the smaller polypeptide from the larger polypeptide. Initial studies indicate that the smaller polypeptide contains approximately 42 amino acid sequences and the larger polypeptide contains approximately 390 amino acids. Smaller polypeptides can be separated by reduction of linking disulfide bonds, followed by separation of larger polypeptides using methods such as selective precipitation, chromatography, or electrophoresis. Detection of smaller, isolated polypeptides can be performed using polyclonal or monoclonal antibody techniques or by fluorescence, colorimetry or radiolabeling (radiolab).
It can be carried out by more conventional chemical methods such as the (Elling) method.

The above immunochemical assay systems include radioimmunoassay (RIA), ELISA, dot plot or Western blot.
This can be accomplished using well-known methods such as s or Western blotting.

The diagnostic probes described above are used to diagnose pre-thrombotic conditions and monitor the effects of anticoagulant, anti-elastase, fibrinolytic or recombinant antithrombin (not inactivated by elastase) therapy. can do. The first sample before the start of treatment is reacted with a diagnostic probe and the amount of elastase-modified antithrombin is determined. Following initiation of treatment, one or more samples can be measured. Comparisons can be made between pre- and post-treatment samples, and the reduction in the level of elastase-modified antithrombin indicates the effectiveness of the treatment. Levels of elastase-modified antithrombin can be measured at multiple intervals and subsequent treatment doses.

Recombinant antithrombin, which is not subject to inactivation by elastase, offers an alternative treatment. Such recombinant antithrombin lacks the elastase cleavage site found in naturally occurring antithrombin.

Neutrophil elastase was observed to reverse heparin-induced enhancement of antithrombin fluorescence.

As shown in Figure 3, the rate of conversion of antithrombin to elastase-modified antithrombin is
can be shown as a function of time.

lX to demonstrate heparin-induced enhancement of antithrombin.
Similar to the sample containing 10-'M antithrombin and 1X10-'M heparin, the 1X10''.M sample of antithrombin was run on the spectrofluorometer. Neutrophil elastase 1×lθ″
"Added to the sample at a concentration of 1M.

Analysis at 10 minutes shows reversal of heparin-induced potentiation.

The rate of conversion can be determined as a function of time by setting the spectrometer at 330 nanometers and running samples as soon as elastase is added. All of the samples were excited at 280 nanometers and emission was read at 330 nanometers.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the negligible rate of antithrombin inactivation in the absence of heparin. FIG. 2 is a graph showing that antithrombin modified with elastase is unable to bind heparin. FIG. 3 is a graph comparing the activity loss and fluorescence decay due to inactivation of AT-III with human neutrophil elastase. A>-+ thrombin 3 tongues, 1 residence (X) Shiromitsu/i (
280n Turtle) Technique Rastazen Horoscope Jq (ΔA 刹0/Intermediate/xi) N^
C1, (M) ---

Claims (1)

[Claims] 1. A diagnostic probe for measuring the presence or absence of elastase-modified antithrombin. 2. The diagnostic probe of claim 1, wherein the elastase-modified antithrombin is characterized by cleavage of antithrombin at a site approximately 42 amino acids from the carboxy terminus of antithrombin. 3. The method according to claim 1, wherein a polyclonal antibody specific for the elastase-modified antithrombin is used to measure the presence or absence of the elastase-modified antithrombin. diagnostic probe. 4. At least one monoclonal antibody specific for the elastase-modified antithrombin is used to measure the presence or absence of the elastase-modified antithrombin. Diagnostic probes as described in section. 5. A patent for separating the cleaved portion of said elastase-modified antithrombin from said elastase-modified antithrombin and measuring it as an indirect measurement of the presence or absence of said elastase-modified antithrombin. A diagnostic probe according to claim 2. 6. (a) reacting a stiff sample containing elastase-modified antithrombin with a specific diagnostic probe for measuring elastase-modified antithrombin; (b) reacting a stiff sample containing elastase-modified antithrombin; characterized by comprising the step of determining the amount of antithrombin modified with elastase,
A method for measuring the presence or absence of elastase-modified antithrombin. 7. The method according to claim 6, wherein the diagnostic probe according to claim 3 is used in step (a). 8. A method as claimed in claim 6 using in step 8 (a) a diagnostic probe as claimed in claim 5. 9. (a) before starting treatment, reacting a sample suspected of containing elastase-modified antithrombin with a specific diagnostic probe for measuring elastase-modified antithrombin; b) determining the amount of elastase-modified antithrombin from the sample of step (a); and (c) determining the amount of elastase-modified antithrombin from the sample of step (a);
) and (b); and (d) comparing the amount of elastase-modified antithrombin in each of said samples, and determining whether the amount of elastase-modified antithrombin in said sample has decreased. A method for monitoring the effectiveness of treatment of prothrombotic and thrombotic conditions characterized by reflecting successful treatment. 10. The method of claim 9, wherein said treatment is selected from the group of anticoagulants, anti-elastase preparations, and recombinant antithrombin that is not subject to elastase cleavage.