NZ236620A - Labelled anticoagulant annexine family polypeptide agents for distinguishing between phosphatidyl serine and phosphatidyl choline for detecting activation of the haemostatic system and prothrombotic states - Google Patents
Labelled anticoagulant annexine family polypeptide agents for distinguishing between phosphatidyl serine and phosphatidyl choline for detecting activation of the haemostatic system and prothrombotic statesInfo
- Publication number
- NZ236620A NZ236620A NZ236620A NZ23662090A NZ236620A NZ 236620 A NZ236620 A NZ 236620A NZ 236620 A NZ236620 A NZ 236620A NZ 23662090 A NZ23662090 A NZ 23662090A NZ 236620 A NZ236620 A NZ 236620A
- Authority
- NZ
- New Zealand
- Prior art keywords
- anticoagulant
- radioisotope
- polypeptide
- vac
- agent
- Prior art date
Links
- 239000003146 anticoagulant agent Substances 0.000 title claims abstract description 39
- 229940127219 anticoagulant drug Drugs 0.000 title claims abstract description 39
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 38
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 38
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 38
- 239000003795 chemical substances by application Substances 0.000 title claims description 30
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 title claims description 23
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 title claims description 23
- 230000003331 prothrombotic effect Effects 0.000 title claims description 17
- 230000004913 activation Effects 0.000 title claims description 16
- 229940030225 antihemorrhagics Drugs 0.000 title claims description 13
- 230000000025 haemostatic effect Effects 0.000 title claims description 13
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 title claims description 12
- 239000003550 marker Substances 0.000 claims abstract description 23
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 230000005298 paramagnetic effect Effects 0.000 claims abstract description 9
- 239000002872 contrast media Substances 0.000 claims abstract description 8
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229960002897 heparin Drugs 0.000 claims abstract description 6
- 229920000669 heparin Polymers 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 35
- 230000008569 process Effects 0.000 claims description 20
- 210000004369 blood Anatomy 0.000 claims description 11
- 239000008280 blood Substances 0.000 claims description 11
- 208000007536 Thrombosis Diseases 0.000 claims description 10
- 238000002372 labelling Methods 0.000 claims description 10
- 229910052791 calcium Inorganic materials 0.000 claims description 9
- 239000011575 calcium Substances 0.000 claims description 9
- 238000005259 measurement Methods 0.000 claims description 9
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 7
- 239000000032 diagnostic agent Substances 0.000 claims description 7
- 229940039227 diagnostic agent Drugs 0.000 claims description 7
- 229910052736 halogen Inorganic materials 0.000 claims description 7
- 150000002367 halogens Chemical class 0.000 claims description 7
- 229910052738 indium Inorganic materials 0.000 claims description 7
- APFVFJFRJDLVQX-UHFFFAOYSA-N indium atom Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 claims description 7
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 claims description 7
- 229910052753 mercury Inorganic materials 0.000 claims description 7
- 229910052713 technetium Inorganic materials 0.000 claims description 7
- GKLVYJBZJHMRIY-UHFFFAOYSA-N technetium atom Chemical compound [Tc] GKLVYJBZJHMRIY-UHFFFAOYSA-N 0.000 claims description 7
- 229910052716 thallium Inorganic materials 0.000 claims description 7
- BKVIYDNLLOSFOA-UHFFFAOYSA-N thallium Chemical compound [Tl] BKVIYDNLLOSFOA-UHFFFAOYSA-N 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 5
- -1 "Tc Chemical compound 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 238000001361 intraarterial administration Methods 0.000 claims description 3
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- 230000005291 magnetic effect Effects 0.000 claims description 3
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical group O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 2
- 239000007850 fluorescent dye Substances 0.000 claims 9
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 claims 1
- 229960001231 choline Drugs 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 18
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
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- 239000004475 Arginine Substances 0.000 abstract description 3
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- 229910052740 iodine Inorganic materials 0.000 abstract description 3
- 239000011630 iodine Substances 0.000 abstract description 3
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- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 abstract description 3
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- 239000002506 anticoagulant protein Substances 0.000 abstract description 2
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- 239000002504 physiological saline solution Substances 0.000 abstract 1
- 150000003904 phospholipids Chemical class 0.000 description 40
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- WTBFLCSPLLEDEM-JIDRGYQWSA-N 1,2-dioleoyl-sn-glycero-3-phospho-L-serine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC WTBFLCSPLLEDEM-JIDRGYQWSA-N 0.000 description 26
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- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 description 6
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- DSNRWDQKZIEDDB-GCMPNPAFSA-N [(2r)-3-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-2-[(z)-octadec-9-enoyl]oxypropyl] (z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C/CCCCCCCC DSNRWDQKZIEDDB-GCMPNPAFSA-N 0.000 description 5
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
- A61K49/0043—Fluorescein, used in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
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-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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Abstract
Anticoagulant polypeptides (I) of the annexin family, labelled with a detectable marker are new. Also new are compsns. contg. (I) plus auxiliaries. (I) is esp. VAC (vascular anticoagulant protein) and suitable markers include fluorescein isothiocyanate, a radioisotope (esp. 131- or 125-iodine) or a paramagnetic contrast agent. (I), which can be administered intravenously or intraarterially, are formulated with physiological saline, 'Tween 80', arginine and/or phosphate buffer. The compsn. may also include an anticoagulant, esp. heparin, which has no effect on plasma Ca concn.
Description
<div class="application article clearfix" id="description">
<p class="printTableText" lang="en">236 <br><br>
Priority Date(s): <br><br>
Comptot# Specification Filed: <br><br>
ciaM-. (6) <br><br>
j6<PA&$A./.£§ <br><br>
Publican Da,.:ZZT.™' <br><br>
.0. Journal No: iHr.Lt.. <br><br>
Patents Form No. 5 <br><br>
NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION <br><br>
USE OF AN ANTICOAGULANT AS A DIAGNOSTIC AGENT <br><br>
WE, BOEHRINGER INGELHEIM INTERNATIONAL GMBH, a body corporate under the laws of FEDERAL REPUBLIC OF GERMANY of D-6507 Ingelheim am Rhein, FEDERAL REPUBLIC OF GERMANY <br><br>
hereby declare the Invention, for which we pray that a patent may be granted to us, and the method by which it is to be perfoamed, to be particularly described in and by the following statement: <br><br>
- 1 - <br><br>
(followed by page la) <br><br>
236 6 2 0 <br><br>
- i <*- <br><br>
56265.17 <br><br>
Use of an anticoagulant as a diagnostic agent <br><br>
The present invention relates to agents, <br><br>
particularly annexines, which are labelled with a detectable substance and the use thereof for diagnostic purposes. <br><br>
5 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 10 associated proteins which partially penetrate this double layer or protrude from it. <br><br>
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 15 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 2 0 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 25 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 30 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. <br><br>
In many cases, e.g. after injury to a blood vessel, <br><br>
(followed by page 2) <br><br>
236 6 2 0 <br><br>
- 2 - <br><br>
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 5 transport phosphatidyl serine to the outer coat, where it aids the activation of the coagulation factors (10). <br><br>
This systematic change in the phospholipid composition of the outer coat of the platelet plasma membrane is of major physiological importance in 10 haemostasis, as indicated for example by Scott syndrome (11) . <br><br>
However, physiology also includes pathology; thus, the homoeostasis of the blood may in some cases slip into a pathological state, as occurs in arterial, 15 coronary and venous thrombosis. <br><br>
These haemostatic disorders are usually iodiopathic and make it impossible for doctors to predict their occurrence and develop preventive treatment. <br><br>
The aim of the present invention was to provide 20 agents which would help in the early recognition of haemostatic disorders. <br><br>
Unlike the development of the symptoms, the development of the disorders usually occurs slowly. During this phase of symptomless progression, the 25 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 30 change the phospholipid composition of their outer plasma membrane coat. Even at this stage, phosphatidyl serine is present in the outer coat. Consequently, agents which are able to recognise platelets having phosphatidyl serine in their outer membrane surface 35 would be able to diagnose the prothrombotic state; they would therefore be of extremely great clinical importance. <br><br>
- 3 - <br><br>
A further object of the invention was to provide adjuvants which are capable of specifically distinguishing phosphatidyl serine from phosphatidyl choline. <br><br>
5 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 10 haemostatic system. Therefore, at this position, sufficient phosphatidyl serine is exposed to the surrounding blood to enable it to locate the pathological position. However, not only the trigger location but also the thrombus forming at this point can 15 be located. <br><br>
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. <br><br>
20 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 25 factors Xa and Va are catalysed by phospholipid surfaces to which the clotting factors bind. <br><br>
The proteins which bind to phospholipids and interfere with processes dependent on phospholipid surfaces constitute a family which are Ca2+-dependent in 30 their binding to phospholipids. <br><br>
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, 35 endonexin II and lipocortin V. <br><br>
The structural features common to the annexines are presumably the basis for their similar Ca2+ and <br><br>
236 6 2 C <br><br>
- 4 - <br><br>
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. <br><br>
5 The physiological functions of the annexines relate to membrane-associated processes. The basic mechanism 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, 10 thereby preventing the formation of the coagulation-promoting complex on their surface. <br><br>
Studies of binding have shown that VAC associates reversibly with procoagulatory phospholipids in calcium-dependent manner. <br><br>
15 Other bivalent cations from the series Cd2+, Zn2+, <br><br>
Mn2+ and Co2+ also have a positive effect on association, but not to the same extent as Ca2+. <br><br>
Furthermore, it has surprisingly been found that VAC absorption on phospholipids is positively influenced 20 to an exceptional degree in the presence of Ca2+ and Zn2+ ions. <br><br>
Surprisingly, it has been found that, at plasma-calcium concentrations, VAC binds to phosphatidyl serine but not to phosphatidyl choline and sphingomyelin. VAC 25 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. <br><br>
30 This differentiation among phospholipids makes VAC, <br><br>
like the other annexines, an ideal reagent for achieving early recognition of the prothrombotic state as described above. <br><br>
Accordingly the invention provides, as one aspect 35 thereof an anticoagulant polypeptide selected from the family of annexines and provided with a detectable marker. <br><br>
236 6 2 0 <br><br>
- 5 - <br><br>
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 5 substances, of agents which are capable of recognising the prothrombotic state of the platelets, which is different from the normal state. Singe the prothrombotic state differs from the normal state of the platelets in that the outer coat of only the 10 prothrombotic platelet shows phosphatidyl serine, this 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 15 characterised by their specificity for phosphatidyl serine, which can be determined by the binding tests described in the specification. <br><br>
By making use of this specificity of the agents according to tha invention it is also possible to locate 20 the starting point for the activation of the haemostatic system and/or the thrombus. <br><br>
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 25 health-threatening condition, to adopt suitable therapeutic measures. <br><br>
Preferred agents according to the invention are anticoagulant polypeptides provided that they have the necessary specificity for the phospholipid phosphatidyl 30 serine. <br><br>
The family of the annexines, particularly VAC, is particularly preferred. <br><br>
In order to be able to use the agents according to the invention, particularly VAC or the other annexines 35 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 <br><br>
236620 <br><br>
- 6 - <br><br>
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, 5 particularly those of iodine, for example 1311 or 125I or lead, mercury, thallium, technetium or indium (203Pb, 198Hg, Z01T1, To, 111In) . <br><br>
Fluorescein isothiocyanate (Serva) may be used for labelling VAC in a manner known per se. Labelling is 10 also possible by means of a paramagnetic contrast 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 15 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 <br><br>
2 0 recorded. <br><br>
In a further aspect of the invention there is provided a process for detecting the starting point of activation of the haemostatic system comprising the steps of <br><br>
25 a) introducing into the system an anticoagulant polypeptide, selected from the family of annexines and provided with a detectable marker; and <br><br>
3 0 b) observing the distribution of said polypeptide after an incubation period. <br><br>
The anticoagulant polypeptide thus provided with a detectable marker may then administered by the 35 intraarterial or intravenous route. The quantity applied has to be such that it suffices for the subsequent measurement after a sufficient incubation <br><br>
236 6 20 <br><br>
- 7 - <br><br>
period. <br><br>
Another aspect of the invention provides a process for detecting a prothrombotic state comprising the steps 5 of: - <br><br>
a) mixing blood to be examined extracorporally with an anticoagulant polypeptide selected from the family of annexines and which carries a detectable marker; and <br><br>
10 <br><br>
b) analysing the labelling associated with specific types of cells. <br><br>
Thus for example, in order to diagnose a 15 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, 20 heparin, and then the labelling associated with specific types of cells is analysed. <br><br>
The polypeptide, labelled according to the invention, may be used for the purpose of the invention in blood-isotonic aqueous solution or with adjuvants. 25 Adjuvants may, for example, include TWEEN 80, arginine, phosphate buffers and physiologically compatible preservatives. Other substances are well known to the skilled man and may also be used. <br><br>
The radioactive labelling is carried out using, for 3 0 example, the known iodogen method (12) or the conventional chloramine-T method. In view of its half-life of 8 days, 131I is recommended for in vivo diagnosis. The radioactively labelled agent is taken up in blood-isotonic aqueous solution. After sterile 35 filtration it is injected. The whole body scintigraphs are taken with a gamma camera, e.g. for 1311, l, 2, 4 and 7 days after the injection. <br><br>
236 6 2 0 <br><br>
- 8 - <br><br>
As well as the genuine forms of the annexines it is also possible to use altered forms for the purposes of the invention. <br><br>
Reference should be made in particular to the 5 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 10 prothrombotic state of the platelets involved. <br><br>
The present invention relates specifically to inter alia: <br><br>
An anticoagulant polypeptide selected from the 15 family of the annexines, preferably VAC and has a detectable label. <br><br>
An anticoagulant polypeptide which, comprises a detectable marker, selected from fluorescent <br><br>
20 labels, preferably fluorescein isothiocyanate, <br><br>
radioisotopes, e.g. of a halogen, technetium, lead, mercury, thallium or indium, particularly preferably 131I or 125I or a paramagnetic contrast agent. <br><br>
25 <br><br>
An anticoagulant polypeptide as described above for distinguishing phosphatidyl serine from phosphatidyl choline. <br><br>
30 - An anticoagulant polypeptide as described above for use as a diagnostic agent. <br><br>
A process for locating the starting point for the <br><br>
35 <br><br>
activation of the haemostatic system, comprising the steps of <br><br>
236 6 2 0 <br><br>
- 9 - <br><br>
a) administering to the system, preferably by intraarterial or intravenous route an anticoagulant polypeptide or agent, provided with a detectable label, from the family of <br><br>
5 annexines, preferably VAC; and b) after an incubation period, observing the distribution of said polypeptide, <br><br>
extracorporally with for example either a gamma-10 scintillation camera or by magnetic resonance measurement. <br><br>
- A process for diagnosing the prothrombotic state, in which <br><br>
15 <br><br>
a) 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 <br><br>
20 <br><br>
b) the labelling associated with specific cell types is analysed. <br><br>
An agent comprising an anticoagulant polypeptide 25 according to the invention, preferably VAC which has a detectable marker, and additionally containing an adjuvant such as, for example, a physiological solution of sodium chloride, TWEEN 80, arginine and/or phosphate buffer, an 3 0 anticoagulant, preferably heparin, which does not reduce the plasma calcium concentration. <br><br>
- A kit for the diagnostic detection of the prothrombotic state or the starting point of <br><br>
35 activation of the haemostatic system and/or a thrombus containing an agent or an anticoagulant polypeptide of the invention which is capable of distinguishing phosphatidyl serine from <br><br>
236 6 2 <br><br>
- 10 -phosphatidyl choline. <br><br>
Use of an anticoagulant polypeptide or agent of the invention for distinguishing phosphatidyl serine 5 from phosphatidyl choline. <br><br>
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 10 haemostatic system or the thrombus. <br><br>
The invention vill now be described by way of non-limiting Examples with reference to the drawings in which:- <br><br>
15 <br><br>
Fig. 1 shows graphically alternating adsorption and desorption of VAC on a phospholipid surface, induced by increasing and lowering the Ca2+ concentration. The adsorption of VAC (1 jug/ml) on a 20% DOPS/80% DOPC 20 phospholipid double layer. Addition of Ca2+ (3,4,6 mM) is indicated by t or v; <br><br>
Fig. 2 shows graphically the influence of the phospholipid composition and Ca2+ concentration on the 25 adsorption of VAC on a phospholipid surface. <br><br>
O 100% DOPS; • 20% DOPS; A 5% DOPS; □ 1% DOPS; 100% DOPC; all the mixtures were supplemented with DOPC. [VAC] = 1 /xg/mi; <br><br>
30 Fig. 3 shows graphically the 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; <br><br>
35 Fig. 4 shows graphically the synergistic effect of Zn2+ on the Ca2+-dependent adsorption of VAC on the phospholipid surface. The effect of Ca2+ on the VAC <br><br>
2366 20 <br><br>
- n - <br><br>
adsorption on 1% DOPS and 99% DOPC in the presence of 50 /tM Zn2+ was measured. [VAC] = l /xg/ml; <br><br>
Fia. 5 shows graphically adsorption of VAC on phospholipid double layers of varying composition. VAC adsorption on dioleoyl phosphatidyl serine (DOPS), <br><br>
•^rdiolipin (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 /xg/ml; [Ca2+] = 3 mM; <br><br>
Fig. 6 shows the structure of 1,3,4,6-Tetrachloro-3a-6a-diphenyl-glycoluril (IODO-GEN); <br><br>
Fig. 7 shows the distribution of radioactivity in fractions from a column; and <br><br>
Fig. 8: shows the distribution of radioactivity in a sample under a linear analyser. <br><br>
Materials and Methods <br><br>
VAC is produced analogously to either EPA 0 181 465 or EPA 0 293 567. The following experiments were carried out with VACot, but the results can also be applied to the other annexines, particularly VAC/3. <br><br>
Lipids <br><br>
Dioleoyl-phosphatidyl choline (DOPC, No. P-1013) Dioleoyl-phosphatidyl ethanolamine (DOPE, No. P-0510), Cardiolipin (CL, No. C-5646), <br><br>
Dioleoyl-phosphatidyl glycerol (DOPG, No. P-9664), <br><br>
Phosphatidyl inositol (PIf No. P-0639), <br><br>
Dioleoyl-phosphatidic acid (DOPA, No. P-2767), <br><br>
Stearylamine (SA, S-6755) and egg yolk sphinogmyelin <br><br>
236 6 2 0 <br><br>
- 12 - <br><br>
(S—0756) were obtained from the firm Sigma Chemical Co. <br><br>
The purity of DOPC and DOPE was tested by thin layer chromatography. Dioleoyl-phosphatidyl serine 5 (DOPS) was produced by conversion of DOPC according to (1) . uC-labelled DOPS (specific activity 100,000 dpm//xg) was obtained from Amersham. <br><br>
Preparation of the phospholipid double layers on silicon 10 plates <br><br>
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 15 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 jliM CaCl2. 20 nl of a solution containing about 2 g/1 20 of phospholipid in chloroform were applied to this substrate. The DOPS fractions in the double layers were tested with 14C-labelled DOPS mixed with DOPC. The double layers filled up were removed from the silicon plates with the scintillation detergent (Du Pont Formula 25 989) and the total radioactivity was measured in a scintillation counter. <br><br>
Measurement of binding bv ellipsometrv <br><br>
The adsorption of VAC on the phospholipid double 3 0 layers was measured by means of an automatic ellipsometer as described (2,3). <br><br>
The binding tests were carried out in a hydrophilic cuvette containing 5 ml of a stirred buffer (0.05 M Tris/HCl; 0.1 M NaCl; pH=7.5; T=20°C). The divalent 35 cations were added stepwise as chlorides. <br><br>
At VAC-concentrations of <0.1 jitg/ml, the buffer which contained the specific VAC concentration was added <br><br>
2366 20 <br><br>
- 13 - <br><br>
continuously in order to create an adequate buffer capacity for VAC. <br><br>
The refractive index and the thickness d of the adsorbed film vere determined from the combined 5 polarising and analysing data (4). The quantity r of the adsorbed protein layer was determined from the refractive index and the thickness using a modified Lorentz-Lorenz equation [1] (3,5): <br><br>
10 [1] r=3d(n2-nb2)/[ (n2+2) (r(nb2+2) -v(nb2-l)) ] ; <br><br>
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). <br><br>
15 <br><br>
Results <br><br>
The effect of divalent cations on the binding of the VAC to phospholipids <br><br>
20 <br><br>
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 25 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 30 desorption are therefore improbable. The binding was also totally reversible when the cuvette was rinsed out with Ca2+-free buffer. <br><br>
The Ca2+ dependency of the VAC binding to phospholipids is shown in Fig. 2. The Ca2+-dosage-35 activity curve shows quite clearly a Ca2+ concentration at which half the maximum VAC adsorption is achieved: [Caz+]1/2. The [Ca2+]1/2 value depends on the composition <br><br>
236 6 2 0 <br><br>
- 14 - <br><br>
of the phospholipid surface. With phospholipid surfaces containing 100%, 20%, 5% and 1% DOPS, [Ca2+]1/2 values of 36 [M, 220 (Mil, 1.5 mM and 8.6 mM were measured, respectively (Table 1). These results accord 5 particularly well with the [Ca2+]1/2 value of 53 /uM, which was measured for the endonexin II (=VAC) binding to equimolar mixtures of PS/PC vesicles (6). The maximum quantity of protein adsorbed (TmaX) was independent of the DOPS fraction of the membrane and amounted to 10 approximately 0.217 jzg/cm2. No adsorption was detected with pure DOPC double layers up to a Ca2+ concentration of 3 mM. <br><br>
The adsorption of VAC to the phospholipid double layers of different concentrations is shown in Figure 5. 15 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. <br><br>
In experiments with cations other than Ca2+, it was found that the binding of VAC to the phospholipids is 20 strongly Ca2+-specific (Fig. 3). Cd2+, Zn2+, Mn2+ and Co2+ showed little promotion of binding; Ba2+ and Mg2+ had no influence. This property of the cations can to some extent be correlated with the ion radii thereof. <br><br>
25 Zinc synergism <br><br>
High concentrations of zinc ions (1 mM) promote VAC-adsorption culy to a small extent (Fig. 3) ; 50 juM have no effect whatsoever on adsorption. Surprisingly, this concentration does influence binding in the 30 presence of Ca2+; there is some synergism. The [Ca2+]1/2 value fell from 8.6 to 2.7 mM for double layers having only 1% DOPS ([Zn2+]=50 juM) (Fig. 4). 50 jzM [Zn2+] is within the normal range of the plasma zinc concentrations. <br><br>
35 <br><br>
2366 20 <br><br>
- 15 - <br><br>
Diagnostic methods 1• In vitro diagnosis <br><br>
5 a) VAC is labelled by methods known per se with the fluorescein group, e.g. with fluorescein isothiocyanate; in this way yAC-FITC is obtained. <br><br>
10 b) A patient's blood is placed in a plastic test tube containing an anticoagulant which does not reduce the plasma-calcium concentration (e.g. heparin) and VAC-FITC. <br><br>
15 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. <br><br>
20 <br><br>
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-25 threatening condition can be recognised early by this method and consequently treated at an early stage. <br><br>
2. In vivo diagnosis <br><br>
30 <br><br>
a) VAC is labelled with a short-lived isotope, for example 131I using methods known per se; 131I-VAC is obtained. <br><br>
35 <br><br>
b) <br><br>
131I-VAC is administered intravenously to a patient. <br><br>
2366 20 <br><br>
35 <br><br>
- 16 - <br><br>
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- <br><br>
5 field-of-view gamma camera. <br><br>
d) Intravascular sites with 131I-VAC accumulation mark the point where the thrombosis is progressing. Suitable thrombosis-preventing or <br><br>
10 thrombosis-alleviating measures can be taken early. <br><br>
Table 1 <br><br>
15 Evaluation of the phospholipid double layers applied to silicon plates uC-D0PS Quantity of Activity DOPS <br><br>
on film phospholipids fraction <br><br>
20 balance (by ellipsometry) measured <br><br>
% Mg/cm2 DPM % <br><br>
2 0.396 453 1.9 <br><br>
5 0.409 1133 4.5 <br><br>
25 20 0.401 5006 20 <br><br>
100 0.442 27174 99 <br><br>
The calculated mixtuie as placed on the film balance. The quantity of double layer was measured by 30 ellipsometry and the activity of the 14C-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: <br><br>
Quantity (jig/cm2) x spec, activity (DPM.^g"1) <br><br>
Fraction = <br><br>
Activity (DPM) x area (cm2) <br><br>
2 3 6 6 2 C <br><br>
- 17 - <br><br>
The specific activity of DOPS was 100,000 DPM./ig"1, the area occupied by phospholipids was 0.62 cm2. <br><br>
10 <br><br>
Taftlq 3 <br><br>
Half the maximum VAC-binding to various phospholipid surfaces. <br><br>
Lipid (mol%/raol%) rmax ± S.D. [Ca2+]1/2±S.D. <br><br>
(/ug/cm2) mM <br><br>
DOPS (100) <br><br>
0. <br><br>
195 <br><br>
+ <br><br>
0. <br><br>
025 <br><br>
0. <br><br>
036 <br><br>
+ <br><br>
0. <br><br>
013 <br><br>
DOPS / DOPC (20/80) <br><br>
0. <br><br>
222 <br><br>
± <br><br>
0. <br><br>
014 <br><br>
0. <br><br>
22 <br><br>
± <br><br>
0. <br><br>
06 <br><br>
DOPS / DOPC (5/95) <br><br>
0. <br><br>
229 <br><br>
± <br><br>
0. <br><br>
004 <br><br>
1. <br><br>
5 <br><br>
± <br><br>
0. <br><br>
5 <br><br>
DOPS / DOPC (1/99) <br><br>
0. <br><br>
234 <br><br>
± <br><br>
0. <br><br>
007 <br><br>
8. <br><br>
6 <br><br>
± <br><br>
2. <br><br>
5 <br><br>
Cardiolipin / DOPC (20/80) <br><br>
0. <br><br>
209 <br><br>
+ <br><br>
0. <br><br>
Oil <br><br>
0. <br><br>
039 <br><br>
+ <br><br>
0. <br><br>
022 <br><br>
DOPG / DOPC (20/80) <br><br>
0. <br><br>
212 <br><br>
± <br><br>
0. <br><br>
003 <br><br>
0. <br><br>
155 <br><br>
± <br><br>
0. <br><br>
027 <br><br>
PI / DOPC (20/80) <br><br>
0. <br><br>
221 <br><br>
± <br><br>
0. <br><br>
005 <br><br>
0. <br><br>
47 <br><br>
+ <br><br>
0. <br><br>
05 <br><br>
DOPA / DOPC (20/80) <br><br>
0. <br><br>
207 <br><br>
+ <br><br>
0. <br><br>
006 <br><br>
0. <br><br>
75 <br><br>
± <br><br>
0. <br><br>
26 <br><br>
DOPE / DOPC (20/80) <br><br>
0. <br><br>
213 <br><br>
+ <br><br>
0. <br><br>
003 <br><br>
0. <br><br>
86 <br><br>
+ <br><br>
0. <br><br>
21 <br><br>
Sphingomyelin / DOPC (20/80) <br><br>
0. <br><br>
225 <br><br>
+ <br><br>
0. <br><br>
014 <br><br>
7 <br><br>
+ <br><br>
3 <br><br>
DOPC (100) <br><br>
n. <br><br>
d. <br><br>
>30 mM <br><br>
The maximum VAC-adsorption (Tmax) on the phospholipid surfaces specified together with the calcium concentration resulting in half the maximum VAC binding [Ca2+]1/Z are given as the averages of at least three different 30 experiments with the corresponding standard deviations, n.d. = not determined. <br><br>
1. Radioactive labelling <br><br>
35 <br><br>
1.1 Preparations <br><br>
1.1.1. Preparation of a 500 mmol/1 sodium phosphate buffer <br><br>
236 6 <br><br>
- 18 - <br><br>
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 5 water until a pH of 7.5 was achieved. <br><br>
1.1.2. Preparation of a 20 mmol/1 sodium phosphate buffer + 150 mmol NaCl (elution buffer) <br><br>
2.76 g of sodium dihydrogen phosphate monohydrate 10 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) 15 to 1 litre of the buffer. <br><br>
1.1.3. Equilibration of the purification column <br><br>
A PD-10 column (Sephadex G25, Messrs. Pharmacia) was equilibrated with about 30 ml of the elution buffer. <br><br>
20 <br><br>
1.1.4. Preparation of the reaction vessel <br><br>
2 mg of IODO-GEN (molecular mass: 432.09 g/mol), available from Pierce Chemical Company and shown in Fig. 6, were dissolved in 50 ml of highly pure 25 dichloromethane. 200 Ml 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 fxg (1.85 x 10"2 mmol) of IODO-GEN were finely distributed over the wall of the 30 reaction vessel. IODO-GEN facilitates the iodination of proteins. <br><br>
1.1.5. VAC-a used for labelling <br><br>
The starting material used was a solution of 50 mg 35 VAC-a in 4 ml of 20 mmol/1 sodium phosphate buffer + <br><br>
150 mmol/1 NaCl, pH 7.2, diluted with 1 ml of twice distilled water. Molecular mass VAC-a: 34000 g/mol. <br><br>
10 <br><br>
25 <br><br>
236 6 <br><br>
- 19 - <br><br>
1.1.6. 1-125 used for labelling <br><br>
Na125I 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/mrool 1/2 I2, corresponding to 0.115 fig of iodine (9.2 x 10'7 mmol 1/2 I2) . The active Nal was dissolved in 5.5 pi of 0.1 mol/1 NaOH. <br><br>
1.2. Iodisation <br><br>
All the work was carried out with removal of the isotope behind leaded glass screens. 20 m 1 (= 200 /xg) of the solution of VAC-a described in 1.1.5. were transferred into the reaction vessel 15 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 /il of 20 the elution buffer (see 1.1.2.) and this solution was also applied to the PD-10 column. The eluate flowing out was discarded. <br><br>
1.3. Purification <br><br>
By the application of 0.5 ml aliquots of elution buffer (see 1.1.2) at 2 minute intervals, the VAC-a [125I] was separated from the free 125l/Na 12SI. After 12 fractions, this purification step tfas complete. 30 The relative activity content of the fraction was measured using a laboratory monitor (GHZ) (Fig. 7). <br><br>
Fractions 6 and 7 were combined, made up to precisely 2.0 ml with elution buffer, divided iirto loo Ml 35 portions and frozen at -20°C. The substance was kept available in these portions for analysis and developmental studies. <br><br>
236 6 2 0 <br><br>
- 20 - <br><br>
2. Analytical part <br><br>
2.1. Measurement of content <br><br>
In the chromogenic substrate assay a VAC-a content of 5 71.8 /ig/2.0 ml of solution was measured. <br><br>
2.2. Measurement of radioactivity <br><br>
2.2.1. Total radioactivity <br><br>
10 After a 100 /il portion had been thawed, 50 /il of this <br><br>
[125I] VAC-a solution was added by pipette to 950 /ul of the inactive VAC-a solution (see 1.1.5.), thoroughly mixed and 50 /il thereof was placed in an LSC-Counter (Beckman) for measurement. 24.5 MBq 15 (= 0.663 mCi)/2.0 ml of total solution. <br><br>
2.2.2. Specific activity <br><br>
Measurement of the content and total radioactivity yielded a specific activity of 20 341.5 MBq/mg = 11.61 TBq/mmol <br><br>
(9.23 mCi/mg = 313.8 Ci(mmol) <br><br>
2.2.3. Measurement of the protein-bound radioactivity bv TCA precipitation <br><br>
25 100 /il of the VAC-a solution were combined with 50 /xl of 3% BSA solution and 150 /xl of 40% aqueous trichloroacetic acid, shaken thoroughly and left to stand for 60 minutes in a refrigerator. The precipitate formed was removed by centrifuging. <br><br>
30 Aliquots of the supernatant were measured in an LSC- <br><br>
counter. <br><br>
Result: 99.3% of the radioactivity was precipitable. <br><br>
35 <br><br>
2.2.4. Radioactivity yield <br><br>
Of the 67.3 MBq put in, 24.5 MBq were found in the VAC-a. The activity yield was therefore 36.4%. <br><br>
236 6 2 0 <br><br>
- 21 - <br><br>
2.3 Degree of modification <br><br>
From the specific activity and the quantity of inactive VAC-a used, it was calculated that statistically every 6th VAC-a molecule was labelled 5 with a 125I-atom. <br><br>
3.4 Identity and purity <br><br>
Using the SDS-PAGE (gradient gel 7 to 17%, non-reducing conditions) and subsequent evaluation of the 10 gel by silver staining (Oakley method), <br><br>
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, 15 the proportion of dimeric product was significantly below the limit of 8% tolerated for inactive charges. <br><br>
Bibliography <br><br>
20 l. Confurius, P & Zwaal, R. F. A. (1977) Biochim. Biophys. Acta 488, -42. <br><br>
2. Corsel, J. W., Willems, G. M., Kop, J. M. M., <br><br>
Cuypers, P. A. & Hermens, W. Th. (1986) J. Colloid 25 Interface Sci. Ill, 544-554. <br><br>
3. Cuypers, P. A., Corsel, J. W., Janssen, M. P., Kop, J. M. M., Hermens, W. TH. & Hemker, H. C. (1983) J. Biol. Chem. 258, 2426-2431. <br><br>
30 <br><br>
4. McCrackin, F. L., Passaglia, E., Stromberg, R. R. & Steinberg, H. L. (1963) J.Res.Nat.Bur.Stand.Sect.A 67, 3-377. <br><br>
35 <br><br>
5. <br><br>
Kop, J. M. M., Cuypers, P. A., Lindhout, Th., Hemker, H. C. & Hermens, W. Th. (1984) J. Biol. Chem. 259, 13993-13998. <br><br>
236 6 2 <br><br>
- 22 - <br><br>
6. Schlaepfer, D. D., Mehlman, T., Burgess, W. H. & <br><br>
Haigler. H. T. (1987) Proc. Natl. Acad. Scl. USA 84, 6078-6082. <br><br>
5 7. Op den Kamp, J.A. F. Ann. Rev. Biochem. 1979, 48: 47-71. <br><br>
8. Zwaal, R.F.A. Biochim. Biophys. Acta 1978, 515: 163-205. <br><br>
10 <br><br>
9. Jackson, C.M. and Nemerson, Y. Ann. Rev. Biochem. 1980, 49: 765-811. <br><br>
10. Bevers, E.M., Comfurius, P. and Zwaal, R.F.A. 15 Biochim. Biophys. Acta 1983, 736: 57-66. <br><br>
11. Rosing, J., Bevers, E.M., Comfurius, P., Hemker, H.C. can Dieijen, G., Weiss, H.J. and Zwaal, R.F.A. Blood 1985, 65: 1557-1561. <br><br>
20 <br><br>
12. Fraker P.J., Speck, J.C., Biochem. Biophys. Res. <br><br>
Comm. 80, 849-857, 1978. <br><br>
13. Reisher, J.I. & Orr, H.C., Anal. Biochem. 1968, 26, 25 178-179. <br><br></p>
</div>
Claims (51)
1. Use of an anticoagulant polypeptide selected from 5 the family of annexines and provided with a detectable marker as an agent for distinguishing between phosphatidyl serine and phosphatidyl choline.<br><br>
2. Use according to claim 1, wherein the anticoagulant 10 polypeptide is VAC.<br><br>
3. Use according to claim 1 or to claim 2, wherein the detectable marker is selected from a fluorescent label, a radioisotope and a paramagnetic contrast agent.<br><br> 15<br><br>
4. Use according to claim 3 wherein the fluorescent label is fluorescein isothiocyanate.<br><br>
5. Use according to claim 3 wherein the radioisotope 20 is selected from halogen, technetium, lead, mercury,<br><br> thallium or indium.<br><br>
6. Use according to claim 5 wherein the radioisotope is 131I or 125I.<br><br> 25<br><br>
7. Use according to any one of the preceding claims as a diagnostic agent.<br><br>
8. A process for detecting the starting point of 30 activation of the haemostatic system comprising the steps of a) introducing into the system an anticoagulant polypeptide, selected from the family of 35 annexines and provided with a detectable marker;<br><br> and v.:,- • t \--fh r;;-««rn ?<br><br> -■8jUL1093 ;<br><br> 236620<br><br> - 24 -<br><br> b) observing the distribution of said polypeptide after an incubation period.<br><br>
9. A process according to claim 8, wherein said 5 polypeptide is VAC.<br><br>
10. A process according to claim 8 or claim 9 wherein the detectable marker is a radioisotope or a paramagnetic contrast element.<br><br> 10<br><br>
11. A process according to any one of claims 8 to 10 wherein the radioisotope is selected from the radioisotopes 125I, 123I, 131I, 111In, "Tc, 203Pb, 198Hg or 2°1Tl.<br><br> 15<br><br>
12. A process according to any one of claims 8 to 11, wherein the polypeptide is administered by intraarterial or intravenous route.<br><br> 20
13. A process according to any one of claims 8 to 12,<br><br> wherein the distribution of said polypeptide is observed extracorporally, using a gamma scintillation camera or magnetic resonance measurement.<br><br> 25
14. A process for detecting a prothrombotic state comprising the steps of:-<br><br> a) mixing blood to be examined extracorporally with an anticoagulant polypeptide selected from the 3 0 family of annexines and which carries a detectable marker; and b) analysing the labelling associated with specific types of cells.<br><br> 35<br><br>
15. A process according to claim 14, wherein the polypeptide is VAC.<br><br> • '} '■: * rv;;236620;- 25 -;
16. A process according to claim 14 or claim 15,;wherein the detectable marker is a fluorescein group, or a radioisotope.;5
17. A process according to claim 16 wherein the radioisotope is selected from 125I, 123I, 131I, 111In, ""To, 2°3pbf 198Hg and 201t1#;
18. An agent comprising an anticoagulant polypeptide lo selected from the family of annexines and provided with a detectable marker and an anticoagulant which does not reduce the plasma calcium concentration.;
19. An agent according to claim 18, wherein the 15 anticoagulant is heparin.;
20. An agent according to claim 18 or claim 19, wherein the anticoagulant polypeptide is VAC.;20
21. An agent according to any one of claims 18 to 20 wherein the detectable marker is selected from a fluorescent label, a radioisotope and a paramagnetic contrast agent.;25
22. An agent according to claim 21 wherein the fluorescent label is fluorescein isothiocyanate.;
23. An agent according to claim 21 wherein the radioisotope is selected from halogen, technetium, lead,;3 0 mercury, thallium or indium.;
24. An agent according to claim 23 wherein the radioisotope is 131I or 125I.;35
25. An agent according to any one of claims 18 to 24;for use as a diagnostic agent in a process according to any one of claims 8 -co |j;-8 JU1 1393 1;236620;- 26 -;
26. A kit for the diagnostic detection of the prothrombotic state or the starting point of activation of the haemostatic system and/or a thrombus comprising a first component which comprises an anticoagulant 5 polypeptide selected from the family of annexines and provided with a detectable marker, and a separate second component which comprises an anticoagulant which does not reduce the plasma calcium concentration.;10
27. A kit according to claim 26, wherein the anticoagulant is heparin.;
28. A kit according to claim 26 or claim 27, wherein the anticoagulant polypeptide is VAC.;15;
29. A kit according to any one of claims 26 to 28 wherein the detectable marker is selected from a fluorescent label, a radioisotope and a paramagnetic contrast agent.;20;
30. A kit according to claim 29 wherein the fluorescent label is fluorescein isothiocyanate.;
31. A kit according to claim 29 wherein the;25 radioisotope is selected from halogen, technetium, lead, mercury, thallium or indium.;
32. A kit according to claim 31 wherein the radioisotope is 131I or 125I.;30;
33. A method of distinguishing between phosphatidyl serine and phophatidyl choline which comprises a step of contacting a sample with an anticoagulant polypeptide selected from the family of annexines and provided with;35 a detectable marker.;
34. A method according to claim whp-rgin the;| patswt cits* j j -8 JUL 1993 |<br><br> 1 S<br><br> ?36620<br><br> - 27 -<br><br> anticoagulant polypeptide is VAC.<br><br>
35. A method according to claim 33 or claim 34, wherein the detectable marker is selected from a fluorescent<br><br> 5 label, a radioisotope and a paramagnetic contrast agent.<br><br>
36. A method according to claim 3 5 wherein the fluorescent label is fluorescein isothiocyanate.<br><br> 10
37. A method according to claim 35 wherein the radioisotope is selected from halogen, technetium, lead, mercury, thallium or indium.<br><br>
38. A method according to claim 37 wherein the 15 radioisotope is 131I or 125I.<br><br>
39. Use of an anticoagulant polypeptide selected from the family of annexines and provided with a detectable marker in the manufacture of an agent for detecting the<br><br> 20 starting point of activation of the haemostatic system and/or a thrombus.<br><br>
40. Use according to claim 39 wherein the anticoagulant polypeptide is VAC.<br><br> 25<br><br>
41. Use according to claim 39 or claim 40 wherein the detectable marker is selected from a fluorescent label, a radioisotope and a paramagnetic contrast agent.<br><br> 30
42. Use according to claim 41 wherein the fluorescent label is fluorescein isothiocyanate.<br><br>
43. Use according to claim 41 wherein the radioisotope is selected from halogen, technetium, lead, mercury,<br><br> 3 5 thallium or indium.<br><br>
44. Use according to claim 4 3 wherein the radioisotope<br><br> O - irSt .<br><br> :J 1 !1 i 1 Q 0 X<br><br> ; \j s.. »J ^ 0<br><br> i<br><br> ! i<br><br> 236620<br><br> - 28 -<br><br> is 131I or 125I.<br><br>
45. Use according to claim 39 substantially as hereinbefore described.<br><br>
46. Use according to claim 1 substantially as hereinbefore described.<br><br>
47. A process according to claim 8 substantially as hereinbefore described.<br><br>
48. A process according to claim 14 substantially as hereinbefore described.<br><br>
49. An pgent according to claim 18 substantially as hereinbefore described.<br><br>
50. A kit according to claim 26 substantially as hereinbefore described.<br><br>
51. A method according to claim 33 substantially as hereinbefore described.<br><br> INGEIHEIM INTERNATIONAL GMBH<br><br> </p> </div>
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US5627036A (en) * | 1989-12-27 | 1997-05-06 | Boehringer Ingelheim International Gmbh | Use of an anticoagulant as a diagnostic agent |
ATE210464T1 (en) * | 1992-06-09 | 2001-12-15 | Neorx Corp | BIOTIN-DOTA CONJUGATES AND THEIR USE IN PRETARGETING PROCEDURES |
AU1186295A (en) * | 1993-11-24 | 1995-06-13 | University Of Washington | Blood coagulation retardants and devices |
CA2180555C (en) * | 1994-01-24 | 2004-12-14 | Sudhakar Kasina | Radiolabeled annexins |
US5968477A (en) * | 1994-01-24 | 1999-10-19 | Neorx Corporation | Radiolabeled annexin conjugates with hexose and a chelator |
US20030220233A1 (en) | 1994-01-24 | 2003-11-27 | Neorx Corporation | Radiolabeled annexins |
CA2185653C (en) * | 1994-03-16 | 2009-01-13 | Kathleen M. Miller | Stabilization of peptides and proteins for radiopharmaceutical use |
US5834196A (en) * | 1994-04-11 | 1998-11-10 | Nexins Research B.V. | Method for detecting and/or optionally quantifying and/or separating apoptotic cells in or from a sample |
DE69530841T2 (en) * | 1994-06-16 | 2004-05-06 | Neorx Corp., Seattle | RADIOACTIVELY MARKED ANNEXIN GALACTOSE CONJUGATES |
JPH10504534A (en) * | 1994-06-16 | 1998-05-06 | ネオルクス コーポレイション | Radiolabeled annexin-galactose conjugate |
US5886143A (en) * | 1994-12-07 | 1999-03-23 | Neorx Corporation | Hepatic-directed compounds and reagents for preparation thereof |
EP1486509A3 (en) * | 1994-12-07 | 2005-03-23 | Neorx Corporation | Radiolabeled annexin-galactose cluster conjugates |
WO1996017618A1 (en) * | 1994-12-07 | 1996-06-13 | Neorx Corporation | Radiolabeled annexin-galactose cluster conjugates |
US5955605A (en) * | 1995-02-21 | 1999-09-21 | Neorx Corporation | Biotinidase resistant biotin-DOTA conjugates |
FR2736197B1 (en) * | 1995-06-29 | 1997-09-12 | Univ Paris Curie | MAGNETIC NANOPARTICLES COUPLED WITH ANNEXIN AND THEIR USE |
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US4427646A (en) * | 1981-04-02 | 1984-01-24 | Research Corporation | Use of radiolabeled peptide derived from crosslinked fibrin to locate thrombi in vivo |
US4455290A (en) * | 1981-04-02 | 1984-06-19 | Research Corporation | Inhibition of fibrin polymerization by a peptide isolated from fibrin Fragment D1 |
US4820505A (en) * | 1985-04-04 | 1989-04-11 | Scripps Clinic And Research Foundation | Detection of activated platelets with antibodies to thrombospondin |
PT87083B (en) * | 1987-03-28 | 1992-07-31 | Boehringer Ingelheim Int | A PROCESS FOR THE PREPARATION OF A VASCULAR ANTICOAGULANT PROTEIN, OF DNA THAT CODIFIES FOR THIS PROTEIN AND OF PHARMACEUTICAL COMPOSITIONS CONTAINING IT |
DE3810331A1 (en) * | 1988-03-26 | 1989-10-05 | Boehringer Ingelheim Int | Monoclonal VAC antibodies |
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- 1990-12-19 EP EP97113801A patent/EP0806670B1/en not_active Expired - Lifetime
- 1990-12-19 KR KR1019920701497A patent/KR0181520B1/en not_active IP Right Cessation
- 1990-12-19 CA CA002070647A patent/CA2070647C/en not_active Expired - Lifetime
- 1990-12-19 EP EP91902118A patent/EP0509026B1/en not_active Expired - Lifetime
- 1990-12-19 ES ES91902118T patent/ES2113372T3/en not_active Expired - Lifetime
- 1990-12-19 JP JP03502409A patent/JP3135261B2/en not_active Expired - Lifetime
- 1990-12-19 AT AT97113801T patent/ATE429644T1/en not_active IP Right Cessation
- 1990-12-19 AU AU70711/91A patent/AU642202B2/en not_active Expired
- 1990-12-19 AT AT91902118T patent/ATE164083T1/en not_active IP Right Cessation
- 1990-12-19 HU HU9202149A patent/HU209650B/en unknown
- 1990-12-19 DE DE4040817A patent/DE4040817A1/en not_active Withdrawn
- 1990-12-19 DE DE59010946T patent/DE59010946D1/en not_active Expired - Lifetime
- 1990-12-19 DE DE59010815T patent/DE59010815D1/en not_active Expired - Lifetime
- 1990-12-19 WO PCT/EP1990/002257 patent/WO1991009628A1/en active IP Right Grant
- 1990-12-21 NZ NZ236620A patent/NZ236620A/en unknown
- 1990-12-21 ZA ZA9010319A patent/ZA9010319B/en unknown
- 1990-12-27 PT PT96385A patent/PT96385B/en not_active IP Right Cessation
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1992
- 1992-06-12 FI FI922719A patent/FI107586B/en active
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JP3135261B2 (en) | 2001-02-13 |
HU9202149D0 (en) | 1992-10-28 |
ATE164083T1 (en) | 1998-04-15 |
KR0181520B1 (en) | 1999-05-01 |
FI922719A0 (en) | 1992-06-12 |
AU7071191A (en) | 1991-07-24 |
ES2113372T3 (en) | 1998-05-01 |
DE59010815D1 (en) | 1998-04-23 |
EP0806670A3 (en) | 1997-12-10 |
WO1991009628A1 (en) | 1991-07-11 |
DE4040817A1 (en) | 1991-07-04 |
JPH05502877A (en) | 1993-05-20 |
HUT61491A (en) | 1993-01-28 |
EP0806670A2 (en) | 1997-11-12 |
NO922528D0 (en) | 1992-06-26 |
DE59010946D1 (en) | 2009-06-04 |
NO305276B1 (en) | 1999-05-03 |
EP0806670B1 (en) | 2009-04-22 |
ZA9010319B (en) | 1992-08-26 |
HU209650B (en) | 1994-09-28 |
FI107586B (en) | 2001-09-14 |
EP0509026A1 (en) | 1992-10-21 |
KR920703121A (en) | 1992-12-17 |
CA2070647A1 (en) | 1991-06-28 |
AU642202B2 (en) | 1993-10-14 |
EP0509026B1 (en) | 1998-03-18 |
PT96385B (en) | 1998-06-30 |
ATE429644T1 (en) | 2009-05-15 |
PT96385A (en) | 1991-10-31 |
CA2070647C (en) | 2001-04-10 |
NO922528L (en) | 1992-08-26 |
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