CA2070647C

CA2070647C - Use of an anticoagulant as a diagnostic agent

Info

Publication number: CA2070647C
Application number: CA002070647A
Authority: CA
Inventors: Christiaan Reutelingsperger
Original assignee: Boehringer Ingelheim International GmbH
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 1989-12-27
Filing date: 1990-12-19
Publication date: 2001-04-10
Anticipated expiration: 2010-12-19
Also published as: DE4040817A1; AU642202B2; KR0181520B1; KR920703121A; PT96385B; NO922528D0; NZ236620A; EP0806670A3; PT96385A; EP0806670B1; CA2070647A1; WO1991009628A1; DE59010946D1; NO922528L; EP0509026B1; FI922719A0; AU7071191A; HU9202149D0; HU209650B; ES2113372T3

Abstract

The invention relates to agents, in particular annexins, which are labelled with a detectable substance and their use for di-agnostic purposes.

Description

S012878J.32 Use of an anticoagulant as a diagnostic agent The present invention relates to agents, particularly annexines, which are labelled with a detectable substance and the use thereof for diagnostic purposes.
Blood consists of special unbound cells dispersed in a plasma medium. The contents of the cell is separated from the surrounding plasma by so-called plasma membranes. These membranes are made up of phospholipids in the form of a double layer and associated proteins which partially penetrate this double layer or protrude from it.
The various phospholipids are not randomly distributed over the outer and inner shell of the double layer but are held by the cell in an asymmetric configuration (7,8). Whereas phosphatidyl choline (PC) and sphingomyelin (SPH) are the dominating species of the outer shell, phosphatidyl serine (PS), phosphatidyl ethanolamine (PE) and phosphatidyl inositol (PI) are located predominantly in the inner coat, facing the cytosol. This energy-consuming state of asymmetry is of exceptional physiological importance. PC and SPH are the most inert species of the phospholipid family and in stark contrast to the other species show exceptionally neutral behaviour in the presence of the plasma components. This reactive inertia of the outer coat relative to the plasma proteins is the absolute prerequisite for ensuring that the blood remains liquid.
Special plasma proteins which belong to the coagulation cascade, namely the so-called coagulation factors, are in fact able to convert liquid blood into a solid state when they are activated (9). These coagulation factors can be activated by phospholipids such as phosphatidyl serine.
In many cases, e.g. after injury to a blood vessel, it is necessary, not to mention crucial to survival, for the coagulation factors to be activated. In such a situation, a special blood cell, the platelet, can give up its membrane asymmetry by activation mechanisms which transport phosphatidyl serine to the outer coat, where it aids the activation of the coagulation factors {10).
This systematic change in the phospholipid composition of the outer coat of the platelet plasma membrane is of major physiological importance in haemostasis, as indicated for example by Scott syndrome (11) .
However, physiology also includes pathology; thus, the homoeostasis of the blood may in some cases slip into a pathological state, as occurs in arterial, coronary and venous thrombosis.
These haemostatic disorders are usually iodiopathic and make it impossible for doctors to predict their occurrence and develop preventive treatment.
The aim of the present invention was to provide agents which would help in the early recognition of haemostatic disorders.
Unlike the development of the symptoms, the development of the disorders usually occurs slowly.
During this phase of symptomless progression, the prothrombotic state, continuous activation of the coagulation system is occurring locally. Connected with this local activation there is, in the periphery, an occurrence of platelets which are at an early stage of activation. These platelets have already begun to change the phospholipid composition of their outer plasma membrane coat. Phosphatidyl serine is present in the outer coat. Agents which could diagnose this so-called prothrombotic state would therefore be of extremely great clinical importance.
A further object of the invention was to provide adjuvants which are capable of specifically distinguishing phosphatidyl serine from phosphatidyl c 2~'~~~4~1 choline.
In addition to the occurrence of weakly activated platelets in the periphery, fully activated platelets will accumulate anywhere where the wall of the blood vessel is in a pathological condition. This location can be regarded as the trigger for the activation of the haemostatic system. Locating this pathological site and the thrombus forming at this point would be of major therapeutic value.
A further aim of the invention was therefore to provide an agent by means of which the starting point of the activation of the haemostatic system and/or a thrombus could be located.
The blood coagulation mechanism constitutes a cascade of enzymatic reactions at the end of which is the formation of thrombin which finally converts fibrinogen into fibrin. Various procoagulant reactions such as, for example, the activation of prothrombin by factors Xa and Va are catalysed by phospholipid surfaces to which the clotting factors bind.
The proteins which bind to phospholipids and interfere with processes dependent on phospholipid surfaces constitute a family which are Ca2'-dependent in their binding to phospholipids.
This family, also known as the annexines, includes, in addition to lipocortin i, calpactin I, protein II, lipocortin III, p67-calelectrin, the vascular anticoagulant protein (VAC) and IBC, PAP, PAPI, PP4, endonexin II and lipocortin V.
The structural features common to the annexines are presumably the basis for their similar Caz' and phospholipid-binding properties. Although this general property applies to all annexines, there is clear individuality with regard to their affinity for Ca2'" and the various types of phospholipid.
The physiological functions of the annexines relate to membrane-associated processes. The basic mechanism L' 2a~0~~'~

of the anti-coagulant effect of VAC was recognised as an inhibition of the catalytic capacity of the phospholipids by the binding of VAC to their surface, thereby preventing the formation of the coagulation-promoting complex on their surface.
Studies of binding have shown that VAC associates reversibly with procoagulatory phospholipids in calcium-dependent manner.
Other bivalent cations from the series Cd2+, Znz+, Mn2+ and Co2+ also have a positive effect on association, but not to the same extent as Caz+.
Furthermore, it has surprisingly been found that VAC absorption on phospholipids is positively influenced to an exceptional degree in the presence of CaZ+ and Zn2+
ions.
Surprisingly, it has been found that, at plasma-calcium concentrations, VAC binds to phosphatidyl serine but not to phosphatidyl choline and sphingomyelin. VAC
will therefore specifically recognise and bind peripheral platelets with PS in their outer membrane coat. Furthermore, VAC will also specifically recognise those locations in the vascular system which are presenting PS to the blood.
This differentiation among phospholipids makes VAC, like the other annexines, an ideal reagent for achieving early recognition of the prothrombotic state as described above.
By means of the present invention it is, surprisingly, possible for the first time to recognise the prothrombotic state of the vascular system. This diagnosis is made possible by the specificity of substances, of agents which are capable of recognising the prothrombotic state of the platelets, which is different from the normal state. Since the prothrombotic state differs from the normal state of the platelets in that the outer coat of only the prothrombotic platelet shows phosphatidyl serine, this ~~7o~4rr principle can be exploited according to the invention by any agent capable of specifically distinguishing phosphatidyl serine from phosphatidyl choline. The agents which may be used according to the invention are characterised by their specificity for phosphatidyl serine, which can be determined by the binding tests described in the specification.
By making use of this specificity of the agents according to the invention it is also possible to locate the starting point for the activation of the haemostatic system and/or the thrombus.
Consequently, the present invention provides, for the first time, agents which make it possible, by early diagnosis of a state which might possibly develop into a health-threatening condition, to adopt suitable therapeutic measures.
Preferred agents according to the invention are anticoagulant polypeptides provided that they have the necessary specificity for the phospholipid phosphatidyl serene.
The family of the annexines, particularly VAC, is particularly preferred.
In order to be able to use the agents according to the invention, particularly VAC or the other annexines as a diagnostic agent, they are labelled in a manner known per se. Suitable labelling may be achieved, for example, by labelling with fluorescent groups or by radioactive labelling. A fluorescent marker which may be used to advantage is fluorescein isothiocyanate, whilst radioactive markers which may be used to advantage are the radioisotopes of the halogens, particularly those of iodine, for example '3'I or ~Z~I or lead, mercury, thallium, technetium or indium (2o3Pb, ~9aHg~ 2o~Tl ~ ~r~,c~ ii~In) .
Fluorescein isothiocyanate (Serva) may be used for labelling VAC in a manner known per se (13). Labelling is also possible by means of a paramagnetic contrast c.' agent which is detectable in a MRI (magnetic resonance imaging) system. It is possible to use gadolinium, cobalt, nickel, manganese or iron complexes by means of which conjugates may be provided as diagnostic agents which are detectable in a MRI system. A strong magnetic field is used in such systems in order to adjust the nuclear spin vectors of the atoms in the organism. Then the field is destroyed which causes the nuclei to return to their initial state. This process is observed and recorded.
The anticoagulant polypeptide thus provided with a detectable marker is then administered by the intraarterial or intravenous route. The quantity applied has to be such that it suffices for the subsequent measurement after a sufficient incubation period.
In order to diagnose a prothrombotic state, the polypeptide or agent labelled according to the invention is added extracorporally to the blood to be examined, optionally in the presence of a further anticoagulant which does not decrease the plasma calcium concentration such as, for example, heparin, and then the labelling associated with specific types of cells is analysed.
The polypeptide, labelled according to the invention, may be used for the purpose of the invention in blood-isotonic aqueous solution or with adjuvants.
Adjuvants may, for example, include TWEFN 80* arginine, phosphate buffers and physiologically compatible preservatives. Other substances are well known to the skilled man and may also be used.
The radioactive labelling is carried out using, for example, the known iodogen method (12) or the conventional chloramine-T method. In view of its half-life of 8 days, '3'I is recommended for in vivo diagnosis. The radioactively labelled agent is taken up in blood-isotonic aqueous solution. After sterile filtration it is injected. The whole body scintigraphs Trade-mark - 7 _ are taken with a gamma camera, e.g. for '3'I, 1, 2, 4 and 7 days after the injection.
As well as the genuine forms of the annexines it is also possible to use altered forms for the purposes of the invention.
Reference should be made in particular to the mutants described in EPA 0 293 567. Furthermore, the fragments or chemically modified derivatives of the annexines may also be used which are specific to the phospholipids phosphatidyl serine/phosphatidyl choline and are therefore capable of recognising the prothrombotic state of the platelets involved.
The present invention relates specifically to:
- An anticoagulant polypeptide from the family of the annexines, preferably VAC which has a detectable label.
An anticoagulant polypeptide which, as the detectable labelling, contains fluorescent labelling, preferably fluorescein isothiocyanate, a radioisotope of a halogen, technetium, lead, mercury, thallium or indium, particularly preferably '3'I or ~25I or a paramagnetic contrast agent.
- An anticoagulant polypeptide as described above for distinguishing phosphatidyl serine from phosphatidyl choline.
- An anticoagulant polypeptide as described above for use as a diagnostic agent.
- A process for locating the starting point for the activation of the haemostatic system, in which _ g _ a) an anticoagulant polypeptide or agent, provided with a detectable label, from the family of annexines, preferably VAC, is administered to the system, preferably by intraarterial or intravenous route and b) after an incubation period the distribution of said polypeptide is observed, either extracorporally with a gamma-scintillation camera or by magnetic resonance measurement.
- A process for diagnosing the prothrombotic state, in which a) the blood to be examined is mixed extracorporally with an anticoagulant polypeptide or agent from the family of annexines, preferably VAC which has a detectable marker and b) the labelling associated with specific cell types is analysed.
An agent which besides an anticoagulant polypeptide from the family of the annexines, preferably VAC
which has a detectable marker, contains additionally an adjuvant such as, for example, a physiological solution of sodium chloride, TWEEN
80* arginine and/or phosphate buffer, an anticoagulant, preferably heparin, which does not reduce the plasma calcium concentration optionally being used.
- A kit for the diagnostic detection of the prothrombotic state or the starting point of activation of the haemostatic system and/or a thrombus containing an agent or an anticoagulant Trade-mark 2a'~~64'~
polypeptide of the invention which is capable of distinguishing phosphatidyl serine from phosphatidyl choline.
- Use of an anticoagulant polypeptide or agEnt of the invention for distinguishing phosphatidyl serine from phosphatidyl choline.
- Use of an anticoagulant polypeptide or agent of the invention for the diagnosis of the prothrombotic state or the starting point of disorder of the haemostatic system or the thrombus.
Materials and Methods VAC is produced analogously to either EPA 0 181 465 or EPA 0 293 567. The following experiments were carried out with VACa, but the results can also be applied to the other annexines, particularly VAC/3.
Lipids Dioleoyl-phosphatidyl choline (DOPC, No. P-1013) Dioleoyl-phosphatidyl ethanolamine (DOPE, No. P-0510), Cardiolipin (CL, No. C-5646), Dioleoyl-phosphatidyl glycerol (DOPG, No. P-9664), Phosphatidyl inositol (PI, No. P-0639), Dioleoyl-phosphatidic acid (DOPA, No. P-2767), Stearylamine (SA, S-6755) and egg yolk sphingomyelin (S-0756) were obtained from the firm Sigma Chemical Co.
The purity of DOPC and DOPE was tested by thin layer chromatography. Dioleoyl-phosphatidyl serine (DOPS) was produced by conversion of DOPC according to (1). ~4C-labelled DOPS (specific activity 100,000 dpm/~.g) was obtained from Amersham.
c°

Preparation of the phospholipid double layers on silicon plates Phospholipid double layers were applied using a "Langmuir-film balance" (Lauda type FW-1) as described in Corsel et al. (2). Hydrophilic silicon plates were treated for 24 hours in 30% chromosulphuric acid and water and stored in 50% ethanol/water. Before use they were thoroughly washed with detergent and water. The film balance was filled with demineralised water and 50 ~M CaCl2. 20 ~cl of a solution containing about 2 g/1 of phospholipid in chloroform were applied to this substrate. The DOPS fractions in the double layers were tested with '4C-labelled DOPS mixed with DOPC. The double layers filled up were removed from the silicon plates with the scintillation detergent (Du Pont Formula 989) and the total radioactivity was measured in a scintillation counter.
Measurement of binding by ellipsometry The adsorption of VAC on the phospholipid double layers was measured by means of an automatic ellipsometer as described (2,3).
The binding tests were carried out in a hydrophilic cuvette containing 5 ml of a stirred buffer (0.05 M
Tris/HC1; 0.1 M NaCl; pH=7.5; T=20°C). The divalent cations were added stepwise as chlorides.
At VAC-concentrations of <0.1 ~,g/ml, the buffer which contained the specific VAC concentration was added continuously in order to create an adequate buffer capacity for VAC.
The refractive index and the thickness d of the adsorbed film were determined from the combined polarising and analysing data (4). The quantity t of the adsorbed protein layer was determined from the refractive index and the thickness using a modified Lorentz-Lorenz equation [1] (3,5):
c [ 1 ] r=3 d ( nZ-nbz ) / [ ( nz+2 ) ( r ( nb2+2 ) -v ( nb2-1 ) ) ] ;
nb is the refractive index of the buffer. The values r=0.254 and v=0.71 were used for the specific molar refractivity and the partial specific volume (3).
Results The effect of divalent cations on the binding of the VAC
to ~hosphol ipids VAC binds to phospholipid membranes consisting of 20% DOPS/80% DOPC, depending on the calcium concentration. The subsequent addition of EDTA resulted in immediate and total desorption (Fig. 1). By varying the free Ca2+ concentration it was possible to initiate adsorption several times, without any noticeable change in the quantity adsorbed or the rate of adsorption.
Irreversible changes to the VAC molecule or the phospholipid double layers caused by adsorption or desorption are therefore improbable. The binding was also totally reversible when the cuvette was rinsed out with Ca2+-free buffer.
The Ca2+ dependency of the VAC binding to phospholipids is shown in Fig. 2. The Ca2'-dosage-activity curve shows quite clearly a CaZ+ concentration at which half the maximum VAC adsorption is achieved:
[Caz+] i~Z. The [Ca2+] »z value depends on the composition of the phospholipid surface. With phospholipid surfaces containing 100%, 20%, 5% and to DOPS, [CaZ+]»Z values of 36 ~,M, 220 ~,M, 1.5 mM and 8.6 mM were measured, respectively (Table 1). These results accord particularly well with the [Caz+]»Z value of 53 ~M, which was measured for the endonexin II (=VAC) binding to equimolar mixtures of PS/PC vesicles (6). The maximum quantity of protein adsorbed (rmax) was independent of the DOPS fraction of the membrane and amounted to ~o~os~~

approximately 0.217 ~,g/cmZ. No adsorption was detected with pure DOPC double layers up to a Ca2+ concentration of 3 mM.
The adsorption of VAC to the phospholipid double layers of different concentrations is shown in Figure 5.
It is shown quite clearly here that virtually no adsorption of VAC is found with pure DOPC double layers.
The adsorption of VAC to stearylamide (SA) is also weak.
In experiments with cations other than Ca2+, it was found that the binding of VAC to the phospholipids is strongly Ca2+-specific (Fig. 3) . Cd2+, Znz+, Mnz+ and Co2' showed little promotion of binding; Baz+ and Mg2+ had no influence. This property of the cations can to some extent be correlated with the ion radii thereof.
Zinc svneraism High concentrations of zinc ions (1 mM) promote VAC-adsorption only to a small extent (Fig. 3); 50 ~,M
have no effect whatsoever on adsorption. Surprisingly, this concentration does influence binding in the presence of Caz+; there is some synergism. The [Caz+) ~~Z
value fell from 8.6 to 2.7 mM for double layers having only 1% DOPS ( [Zn2+]=50 ~.M) (Fig. 4) . 50 ~M [Zn2'J is within the normal range of the plasma zinc concentrations.
Diagnostic methods 1. In vitro diagnosis a) VAC is labelled by methods known per se with the fluorescein group, e.g. with fluorescein isothiocyanate; in this way VAC-FITC is obtained.
b) A patient's blood is placed in a plastic test tube containing an anticoagulant which does not - 13 - 2~"~0~4'~
reduce the plasma-calcium concentration (e. g.
heparin) and VAC-FITC.
c) After mixing, the blood cells are analysed using an FACS (fluorescence activated cell sorter). This analysis determines the intensity of fluorescence, which is associated with specific types of cell.
d) The analysis profiles show the quantity of platelets with bound VAC-FITC, i.e. platelets with exposed PS, and therefore the presence of a prothrombotic condition. This health-threatening condition can be recognised early by this method and consequently treated at an early stage.
2. In vivo diagnosis a) VAC is labelled with a short-lived isotope, for example '3'I using methods known per se; '3'I-VAC
is obtained.
b) '3'I-VAC is administered intravenously to a patient.
c) After a certain incubation period the patient is exposed to whole or partial body scintigraphy. The distribution of radioactivity can be observed using a large-field-of-view gamma camera.
d) Intravascular sites with ~3~I-VAC accumulation mark the point where the thrombosis is progressing. Suitable thrombosis-preventing or thrombosis-alleviating measures can be taken early.
c:

1. Radioactive labellinct 1.1 Preparations 1.1.1. Preparation of a 500 mmol/1 sodium phosphate buffer 24.5 g of sodium dihydrogen phosphate monohydrate were dissolved in 1 litre of twice distilled water and added to a solution of 35.5 g of disodium hydrogen phosphate in 1 litre of twice distilled water until a pH of 7.5 was achieved.
1.1.2. Preparation of a 20 mmoljl sodium phosphate buffer + 150 mmol NaCl lelution bufferl 2.76 g of sodium dihydrogen phosphate monohydrate were dissolved in 1 litre of twice distilled water and added to a solution of 2.84 g of disodium hydrogen phosphate in 1 litre of twice distilled water until a pH of 7.2 was obtained. The elution buffer was prepared by adding 8.77 g NaCl (150 mmol) to 1 litre of the buffer.
1.1.3. Eguilibration of the purification column A PD-10 column (Sephadex G25, Messrs. Pharmacia) was equilibrated with about 30 ml of the elution buffer.
1.1.4. Preparation of the reaction vessel 2 mg of IODO-GEN (molecular mass: 432.09 g/mol) were dissolved in 50 ml of highly pure dichloromethane. 200 ~.1 of this solution were pipetted into a 1.5 ml Eppendorf*container and then the solvent was evaporated off at 37'C
(thermostatic block). In this way 8 ~.g (1.85 x 10'2 mmol) of IODO-GEN were finely distributed over the wall of the reaction vessel.
Txade-mark ~~~os4 1.1.5. VAC-a used for labellinct The starting material used was a solution of 50 mg VAC-a in 4 ml of 20 mmol/1 sodium phosphate buffer + 150 mmol/1 NaCl, pH 7.2, diluted with 1 ml of twice distilled water. Molecular mass VAC-a:
34000 g/mol.
1.1.6. I-125 used for labelling Na~~SI made by Dupont, NEN Products, with 67.3 MBq (=1.82 mCi) total radioactivity on the date of calibration. The specific activity was 15.9 Ci/mg of iodine = 1.98 kCi/mmol 1/2 I2, corresponding to 0.115 ~.g of iodine (9.2 x 10-~ mmol 1/2 IZ) . The active NaI was dissolved in 5.5 ~,1 of 0.1 mol/1 NaOH.
1.2. Iodisation Al.l the work was carried out with removal of the isotope behind leaded glass screens. 20 ~,1 (= 200 ~cg) of the solution of VAC-a described in 1.1.5. were transferred into the reaction vessel pretreated with IODO-GEN. This vessel was sealed and shaken for 20 minutes at ambient temperature.
Then the reaction solution was applied by means of a pipette to the prepared PD-10 column (see 1.1.3.). The reaction vessel was rinsed again with 500 ~.l of the elution buffer (see 1.1.2.) and this solution was also applied to the PD-10 column. The eluate flowing out was discarded.
1.3. Purification By the application of 0.5 ml aliquots of elution buffer (see 1.1.2) at 2 minute intervals, the VAC-a [ ~25I ] was separated from the free ~ZSI/Na ~ZSI . After 12 fractions, this purification step was complete.

2fl'~0~4'~

The relative activity content of the fraction was measured using a laboratory monitor (GMZ) (Fig. 7).
Fractions 6 and 7 were combined, made up to precisely 2.0 ml with elution buffer, divided into 100 ul portions and frozen at -20°C. The substance was kept available in these portions for analysis and developmental studies.
2. Analytical part 2.1. Measurement of content In the chromogenic substrate assay a VAC-a content of 71.8 ~,g/2.0 ml of solution was measured.
2.2. Measurement of radioactivity 2.2.1. Total radioactivity After a 100 ~cl portion had been thawed, 50 ~,1 of this [~zSI] VAC-a solution was added by pipette to 950 ~,1 of the inactive VAC-a solution (see 1.1.5.), thoroughly mixed and 50 ~,1 thereof was placed in an LSC-Counter (Beckman) for measurement. 24.5 MBq (= 0.663 mCi)/2.0 ml of total solution.
2.2.2. Specific activity Measurement of the content and total radioactivity yielded a specific activity of 341.5 MBq/mg = 11.61 TBq/mmol (9.23 mCi/mg = 313.8 Ci(mmol) 2.2.3. Measurement of the protein-bound radioactivity by TCA precipitation 100 ~.1 of the VAC-a solution were combined with 50 ~.1 of 3% BSA solution and 150 ~,1 of 40% aqueous trichloroacetic acid, shaken thoroughly and left to stand for 60 minutes in a refrigerator. The ~o~~s~~

precipitate formed was removed by centrifuging.
Aliquots of the supernatant were measured in an LSC-counter.
Result: 99.3% of the radioactivity was precipitable.
2.2.4. Radioactivity yield Of the 67.3 MBq put in, 24.5 MBq were found in the VAC-a. The activity yield was therefore 36.4%.
2.3 Degree of modification From the specific activity and the quantity of inactive VAC-a used, it was calculated that statistically every 6th VAC-a molecule was labelled with a ~zSI-atom.
3.4 Identity and purity Using the SDS-PAGE (gradient gel 7 to 17%, non-reducing conditions) and subsequent evaluation of the gel by silver staining (Oakley method), autoradiography and detection under the linear analyser (made by Berthold LB 282, probe LB 2820) (Fig. 8) the substance was investigated by comparison with the VAC-a used. The substances were identical, the proportion of dimeric product was significantly below the limit of 8o tolerated for inactive charges.
c Legend accompan~ing the drawings Fiq. 1: Alternating adsorption and desorption of VAC on a phospholipid surface, induced by increasing and lowering the Ca2' concentration. The adsorption of VAC
(1 ~g/ml) on a 20% DOPS/80% DOPC phospholipid double layer. Addition of Caz+ (3,4,6 mM) is indicated by t or v.
Fig. 2: Influence of the phospholipid composition and CaZ+ concentration on the adsorption of VAC on a phospholipid surface.
0 100% DOPS; ~ 20% DOPS; 0 5% DOPS; D to DOPS% 100%
DOPC% all the mixtures were supplemented with DOPC.
[VAC] - 1 ~g/ml.
Fig. 3: Effect of bivalent ions on the adsorption of VAC. VAC adsorption on double layers of 20% DOPS and 80% DOPC in the presence of the ions specified (1 or 3 mM). [VAC] - 1 ~,g/ml.
Fist. 4: Synergistic effect of ZnZ' on the Caz'-dependent adsorption of VAC on the phospholipid surface. The effect of Ca2' on the VAC adsorption on 1% DOPS and 99%
DOPC in the presence of 50 ~,M Znz+ was measured.
[VAC] - 1 ~.g/ml.
Fig'. 5: Adsorption of VAC on phospholipid double layers of varying composition.
VAC adsorption on dioleoyl phosphatidyl serine (DOPS), cardiolipin (CL) and dioleoyl phosphatidyl ethanolamine (DOPE), either pure or mixed with 80% dioleoyl phosphatidyl choline (DOPC), on dioleoyl phosphatidyl glycerol (DOPG), phosphatidyl inositol (PI) and stearylamine mixed with 80% DOPC or on pure DOPC. [VAC]
- 1 ug/ml % [ Caz+ ] - 3 mM .

Fig. 6: 1,3,4,6-Tetrachloro-3a-6a-diphenyl-glycoluril (IODO-GEN) Fiq_ 7: Distribution of radioactivity in the fractions 1-12.
Fig. 8: Distribution of radioactivity under the linear analyser.
Table 1 Evaluation of the phospholipid double layers applied to silicon plates ~4C-DOPS Quantity of Activity DOPS
on film phospholipids fraction balance (by ellipsometry) measured ug/cm2 DPM %
2 0.396 453 1.9 5 0.409 1133 4.5 20 0.401 5006 20 100 0.442 27174 99 The calculated mixture was placed on the film balance. The quantity of double layer was measured by ellipsometry and the activity of the '4C-labelled DOPS
was measured using a Beckmann 6S 3801 scintillation counter (s. d. <2%) and corrected by the background radiation (60 DPM). The DOPS fraction in the double layer was calculated using equation 2:
Quantity(~g/cm2) x spec. activity (DPM.ug'~) Fraction =
Activity (DPM) x area (cm2) The specific activity of DOPS was 100,000 DPM.~,g'~, the area occupied by phospholipids was 0.62 cm2.
Trade-mark _ 20 _ 2U7~~~'~
Table 2 Half the maximum VAC-binding to various phospholipid surfaces.
Lipid (mol%/molo) rmax ~ S.D. [Ca2']»2~S.D.
( I~g/ cm2 ) mM
DOPS(100) 0.195 0.025 0.036 0.013 DOPS/ DOPC (20/80) 0.222 0.014 0.22 0.06 DOPS/ DOPC (5/95) 0.229 0.004 1.5 0.5 DOPS/ DOPC (1/99) 0.234 0.007 8.6 2.5 Cardiolipin / DOPC (20/80) 0.209 0.011 0.039 0.022 DOPG/ DOPC (20/80) 0.212 0.003 0.155 0.027 PI / DOPC (20/80) 0.221 0.005 0.47 0.05 DOPA/ DOPC (20/80) 0.207 0.006 0.75 0.26 DOPE/ DOPC (20/80) 0.213 0.003 0.86 0.21 Sphingomyelin 0.225 0.014 7 3 /
DOPC
(20/80) DOPC(100) n.d. >30 mM

The maximum VAC-adsorption (rmax) on the phospholipid surfaces specified together with the calcium concentration resulting in half the maximum VAC binding [Ca2+]»2 are given as the averages of at least three different experiments with the corresponding standard deviations.
n.d. - not determined.
z:

.~ ~o~o~~~

Biblioqraphy 1. Confurius, P & Zwaal, R. F. A. (1977) Biochim.
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Claims

CLAIMS:

1. Use of an anticoagulant polypeptide from the family of the annexines as an agent for diagnosing the prothrombotic state, the starting point of the disruption or activation of the haemostatic system and/or of a thrombus.

2. Use of a polypeptide according to claim 1, characterised in that it is VAC.

3. Use of a polypeptide according to claim 1 or 2, characterised in that it is provided with a detectable marker.

4. Use of a polypeptide according to claim 3, characterised in that a fluorescent marker is used for fluorescent marking or a radioisotope of a halogen, technetium, lead, mercury, thallium or indium, is used for radio-active marking, or a paramagnetic contrast agent is used.

5. Use of a polypeptide according to claim 4, characterised in that the fluorescent marker is fluorescein isothiocyanate.

6. Use of a polypeptide according to claim 4, characterised in that the radioisotope of a halogen is 131I or 125I.

7. Use of a polypeptide according to any one of claims 1 to 6, characterised in that the agent additionally contains an adjuvant.

8. Use of a polypeptide according to claim 7, characterised in that physiological saline solution, TWEEN 80*, arginine and/or phosphate buffer is used as the adjuvant.

9. Use of a polypeptide according to any one of claims 1 to 8, characterised in that the agent additionally contains an anticoagulant which does not reduce the plasma-calcium concentration.

10. Use of a polypeptide according to claim 9, characterised in that the anticoagulant is heparin.

11. Use of a polypeptide according to any one of claims 1 to 10 as an agent for distinguishing between phosphatidyl serine and phosphatidyl choline.

12. Process for determining the starting point for the activation of the haemostatic system, characterised in that a) an anticoagulant polypeptide provided with a detectable marker from the family of the annexines is introduced into the system and b) after an incubation period the distribution of said polypeptide is observed.

13. Process according to claim 12, characterised in that the polypeptide is VAC.

14. Process according to claim 12, characterised in that a radioisotope, or a paramagnetic contrast element is used as the detectable marker.

15. Process according to claim 14, characterised in that the radioisotope is chosen from the group consisting of 125I, 123I, 131I, 111In, 99m TC, 203Pb, 198Hg and 201T1.

16. Process according to any one of claims 12 to 15 characterised in that the polypeptide is administered by intraarterial or intravenous route.

17. Process according to any one of claims 12 to 16, characterised in that the distribution of said polypeptide is observed extracorporally, using a gamma scintillation camera or magnetic resonance measurement.

18. Process for detecting the prothrombotic state, characterised in that a) blood to be examined is mixed extracorporally with an anticoagulant polypeptide from the family of annexines which carriers a detectable marker and b) the labelling associated with specific types of cells is analysed.

19. Process according to claim 18, characterised in that the polypeptide is VAC.

20. Process according to claim 18 or 19, characterised in that a fluorescein group or a radioisotope is used as the detectable marker.

21. Process according to any one of claims 18 to 20, characterised in that the detectable marker is selected from the group consisting of 125I, 123I, 131I, 111In, 99m TC, 203Pb, 198Hg and 201T1.

22. Process according to any one of claims 12 to 21, characterised in that additionally an adjuvant is added.

23. Process according to claim 22, characterised in that the adjuvant used is physiological saline solution, TWEEN 80*, arginine and/or phosphate buffer.

24. Process according to claim 22 or 23, characterised in that additionally an anticoagulant which does not reduce the plasma-calcium concentration is used.

25. Process according to claim 24, characterised in that the anticoagulant is heparin.

26. Process according to any one of claims 12 to 25 for distinguishing between phosphatidyl choline and phosphatidyl serine.

27. Kit comprising a fluorescent marker, a vascular anti-coagulant protein (VAC), an anti-coagulant which does not reduce the plasma-calcium concentration and a fluorescence activated cell sorter for the diagnostic detection of the prothrombotic state, the starting point of activation of the haemostatic system and/or the thrombus.