NO305276B1 - Use of an Anticoagulant Polypeptide as a Diagnostic Agent and Method for Detecting Prothrombic Condition - Google Patents
Use of an Anticoagulant Polypeptide as a Diagnostic Agent and Method for Detecting Prothrombic Condition Download PDFInfo
- Publication number
- NO305276B1 NO305276B1 NO922528A NO922528A NO305276B1 NO 305276 B1 NO305276 B1 NO 305276B1 NO 922528 A NO922528 A NO 922528A NO 922528 A NO922528 A NO 922528A NO 305276 B1 NO305276 B1 NO 305276B1
- Authority
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- Norway
- Prior art keywords
- vac
- polypeptide
- anticoagulant
- use according
- detectable marker
- Prior art date
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Abstract
Description
Foreliggende oppfinnelse angår anvendelse av et antikoagulerende polypeptid, spesielt et annexin, som er merket med en påvisbar substans, til diagnose, og en fremgangsmåte for konstatering av den protrombiske tilstand. The present invention relates to the use of an anticoagulant polypeptide, in particular an annexin, which is labeled with a detectable substance, for diagnosis, and a method for ascertaining the prothrombic state.
Blod består av spesielle ubundne celler som er dispergert i et plasma-medium. Celleinnholdet er skilt fra det omgivende plasma gjennom såkalte plasma-membraner. Disse membranene er bygget opp av fosfolipider i form av et dobbelt skikt og av assosierte proteiner som delvis trenger gjennom dobbeltskiktet eller stikker ut fra dette. Blood consists of special unbound cells that are dispersed in a plasma medium. The cell contents are separated from the surrounding plasma through so-called plasma membranes. These membranes are made up of phospholipids in the form of a double layer and of associated proteins that partially penetrate the double layer or protrude from it.
De forskjellige fosfolipidene er ikke fordelt vilkårlig over den utvendige og innvendige omhylling av dobbeltskiktet, men holdes i en asymmetrisk konfigurasjon av cellen (7, 8). Mens fosfatidylkolin (PC) og sfingomyelin (SPH) utgjør de dominerende arter av den ytre omhylling, er fosfatidylserin (PS), fosfatidyletanolamin (PE) og fosfatidylinositol (PI) vesentlig lokalisert i den indre omhylling som vender mot cytosolet. Denne energiforbrukende asymmetri-tilstand er av eksepsjonell fysiologisk betydning. PC og SPH er de mest inerte representanter av fosfolipidfamilien og viser i sterk motsetning til de andre representanter, ytterst nøytrale forhold overfor plasmakomponentene. Denne reaksjonstreghet ved den ytre omhylling i forhold til plasmaproteinene er en absolutt forutsetning for å sikre blodets flytende tilstand. Spesielle plasmaproteiner som hører med til blodkoagulasjons-kaskaden, de såkalte koagulasjonsfaktorene, kan nemlig over-føre den flytende blodtilstand til en fast tilstand når de aktiveres (9). Disse koagulasjonsfaktorene kan aktiveres gjennom fosfolipider som fosfatidylserin. The different phospholipids are not distributed arbitrarily over the outer and inner envelope of the bilayer, but are held in an asymmetric configuration by the cell (7, 8). While phosphatidylcholine (PC) and sphingomyelin (SPH) constitute the dominant species of the outer envelope, phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) are substantially located in the inner envelope facing the cytosol. This energy-consuming asymmetry state is of exceptional physiological importance. PC and SPH are the most inert representatives of the phospholipid family and, in stark contrast to the other representatives, show extremely neutral conditions towards the plasma components. This reaction inertia of the outer covering in relation to the plasma proteins is an absolute prerequisite for ensuring the liquid state of the blood. Special plasma proteins that belong to the blood coagulation cascade, the so-called coagulation factors, can actually transfer the liquid blood state to a solid state when they are activated (9). These coagulation factors can be activated through phospholipids such as phosphatidylserine.
I mange tilfeller, f.eks. etter beskadigelse av et blod-kar, er det nødvendig, ja faktisk livsviktig, at koagulasjonsfaktorene aktiveres. I en slik situasjon kan en spesiell blodcelle, blodplaten, oppgi sin membran-asymmetri gjennom aktiveringsmekanismer som transporterer fosfatidylserin til den ytre omhylling, hvor den understøtter aktiveringen av koagulasjonsfaktorene (10). In many cases, e.g. after damage to a blood vessel, it is necessary, indeed vital, that the coagulation factors are activated. In such a situation, a special blood cell, the platelet, can express its membrane asymmetry through activation mechanisms that transport phosphatidylserine to the outer envelope, where it supports the activation of the coagulation factors (10).
Denne systematiske forandring av fosfolipidsammen setningen i den ytterste omhylling av blodplateplasma-membranen har stor fysiologisk betydning for hemostasen, som antydet for eksempel gjennom Scott-syndromet (11). This systematic change of the phospholipid composition in the outermost envelope of the platelet plasma membrane has great physiological significance for hemostasis, as suggested for example through the Scott syndrome (11).
Til fysiologien hører imidlertid også patologien. Blodets homeostase kan således enkelte ganger også gli over i en patologisk tilstand som ved arterielle, koronare og venøse tromboser. However, pathology also belongs to physiology. The homeostasis of the blood can thus sometimes also slip into a pathological state, such as in arterial, coronary and venous thromboses.
Disse hemostatiske forstyrrelsene er for det meste idiopatiske og gjør det umulig for legen å forutsi deres forekomst og utvikle profylaktiske terapier. These hemostatic disorders are mostly idiopathic and make it impossible for the physician to predict their occurrence and develop prophylactic therapies.
Formålet med foreliggende oppfinnelse har vært å tilveie-bringe en fremgangsmåte som kunne bidra til tidlig å fastslå hemostatiske forstyrrelser. The purpose of the present invention has been to provide a method which could contribute to the early determination of hemostatic disturbances.
I motsetning til symptomutviklingen, forløper utviklingen av forstyrrelsene oftest langsomt. Under denne fase med symptomløs progresjon, den såkalte protrombotiske tilstand, forløper det lokalt en kontinuerlig aktivering av koagulasjonssystemet. Denne lokale aktivering er perifert forbundet med forekomst av blodplater som befinner seg i en tidlig tilstand av aktiveringen. Disse blodplatene har allerede begynt å forandre fosfolipidsammensetningen av deres ytre plasmamembran-omhylling. Fosfatidylserin forekommer i den ytre omhylling. Hjelpemidler som diagnostiserer denne såkalte protrombotiske tilstand, ville være av største kliniske betydning. In contrast to the development of symptoms, the development of the disorders usually proceeds slowly. During this phase of symptomless progression, the so-called prothrombotic state, there is a continuous activation of the coagulation system locally. This local activation is peripherally associated with the occurrence of platelets that are in an early state of activation. These platelets have already begun to change the phospholipid composition of their outer plasma membrane envelope. Phosphatidylserine occurs in the outer envelope. Aids that diagnose this so-called prothrombotic condition would be of the greatest clinical importance.
Ved siden av forekomsten av svakt aktiverte blodplater i periferien, vil det klebe seg fullstendig aktiverte blodplater der hvor karveggen befinner seg i patologisk tilstand. Disse stedene er å anse som utløsere av aktiveringen av det hemostatiske system. Lokaliseringen av disse patologiske stedene og den trombe som her dannes, ville være av største terapeutiske interesse. Next to the presence of weakly activated platelets in the periphery, fully activated platelets will adhere where the vessel wall is in a pathological state. These places are to be regarded as triggers for the activation of the hemostatic system. The localization of these pathological sites and the thrombus that forms here would be of greatest therapeutic interest.
I henhold til oppfinnelsen anvendes et antikoagulerende polypeptid innen annexin-familien som er forsynt med en påvisbar markør, som middel for å skjelne mellom fosfatidylkolin og fosfatidylserin. Videre angår oppfinnelsen anvendelse av et slikt polypeptid som middel for diagnose av protrombiske tilstander, utgangspunktet for forstyrrelsen eller aktiveringen av det hemostatiske system og/eller tromben. According to the invention, an anticoagulant polypeptide within the annexin family which is provided with a detectable marker is used as a means of distinguishing between phosphatidylcholine and phosphatidylserine. Furthermore, the invention relates to the use of such a polypeptide as a means of diagnosing prothrombic conditions, the starting point for the disturbance or activation of the hemostatic system and/or the thrombus.
Oppfinnelsen angår også anvendelse av et antikoagulerende polypeptid innen annexin-familien, som er forsynt med en påvisbar markør, for fremstilling av et preparat til å fastslå utgangspunktet for aktiveringen av det hemostatiske system. The invention also relates to the use of an anticoagulant polypeptide within the annexin family, which is provided with a detectable marker, for the production of a preparation to determine the starting point for the activation of the hemostatic system.
Videre angår oppfinnelsen en fremgangsmåte for konstatering av den protrombiske tilstand, og denne fremgangsmåte karakteriseres ved at a) blodet som skal undersøkes, blandes ekstrakorporealt med et antikoagulerende polypeptid innen annexin-familien, som bærer en påvisbar markør og b) markeringen assosiert med spesifikke celletyper, analyseres . Furthermore, the invention relates to a method for ascertaining the prothrombic state, and this method is characterized by a) the blood to be examined is mixed extracorporeally with an anticoagulant polypeptide within the annexin family, which carries a detectable marker and b) the marking associated with specific cell types, are analyzed.
Blodkoaguleringsmekanismen utgjør en kaskade av enzymatiske reaksjoner, hvor dannelsen av trombin som tilslutt omdanner fibrinogen til fibrin, befinner seg på slutten av denne. Forskjellige prokoagulante reaksjoner, som for eksempel aktiveringen av protrombin ved hjelp av faktorene Xa og Va, katalyseres av fosfolid-overflater som koagulasjonsfaktorene binder seg til. 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 the activation of prothrombin by means of factors Xa and Va, are catalyzed by phospholide surfaces to which the coagulation factors bind.
Blant de proteiner som binder seg til fosfolipder og som interfererer med prosesser som er avhengige av fosfolipid-overflater, finnes en familie som for sin binding til fosfolipider, er avhengig av Ca<2+>. Among the proteins that bind to phospholipids and that interfere with processes that depend on phospholipid surfaces, there is a family that depends on Ca<2+> for its binding to phospholipids.
Til denne familie, som også kalles annexiner, hører foruten lipocortin I, calpactin I, protein II, lipocortin III, p67-calelectrin, også det vaskulære antikoagulantprotein (VAC) og IBC, PAP, PAPI, PP4, endonexin II og lipocortin V. 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 belong to this family, which is also called annexins.
De strukturelle felles karakteristika for annexiner, er sannsynligvis grunnlaget for deres lignende Ca<2+>og fosfolipid-bindingsegenskaper. Selv om denne generelle egenskap gjelder for alle annexiner, består det en klar individualitet med hensyn til deres affinitet til Ca<2+>og til de forskjellige fosfolipid-arter. The structural common features of annexins are likely the basis for their similar Ca<2+> and phospholipid binding properties. Although this general property applies to all annexins, there remains a clear individuality with respect to their affinity for Ca<2+> and for the different phospholipid species.
De fysiologiske funksjoner til annexinene angår membran-assosierte prosesser. Den grunnleggende mekanisme for den koagulasjonshemmende virkning av VAC er erkjent som en hemming av fosfolipidenes katalytiske kapasitet til å binde VAC til deres overflate, hvorved dannelsen av det koagulasjons-befordrende kompleks på deres overflate forhindres. The physiological functions of the annexins concern membrane-associated processes. The basic mechanism for the anticoagulation effect of VAC is recognized as an inhibition of the catalytic capacity of the phospholipids to bind VAC to their surface, thereby preventing the formation of the coagulation-promoting complex on their surface.
Bindingsstudier har vist at VAC kalsiumavhengig reversibelt assosierer seg med prokoagulatoriske fosfolipider. Binding studies have shown that VAC reversibly associates with procoagulant phospholipids in a calcium-dependent manner.
Også andre bivalente kationer fra rekken Cd<2+>, Zn<2+>, Mn<2+>og Co<2+>påvirker assosiasjonen positivt, riktignok ikke i samme utstrekning som Ca<2+>. Other bivalent cations from the series Cd<2+>, Zn<2+>, Mn<2+>and Co<2+> also affect the association positively, although not to the same extent as Ca<2+>.
Ut over dette er det nå overraskende oppdaget at VAC-adsorpsjonen til fosfolipider påvirkes meget positivt i nærvær In addition to this, it has now surprisingly been discovered that VAC adsorption to phospholipids is very positively affected in its presence
„2+ „ 2+ . „2+ „ 2+ .
av Ca - og Zn -ioner. of Ca and Zn ions.
Overraskende er det også fastslått at VAC ved plasma-konsentrasjoner binder seg til fosfatidylserin men ikke til fosfatidylkolin og sfingomyelin. VAC erkjenner og binder således spesifikt perifere blodplater med PS i deres ytre membran-omhylling. Dessuten erkjenner VAC også spesifikt slike steder i det vaskulære systemer som PS utsetter blodet for. Surprisingly, it has also been established that VAC at plasma concentrations binds to phosphatidylserine but not to phosphatidylcholine and sphingomyelin. VAC thus specifically recognizes and binds peripheral platelets with PS in their outer membrane envelope. Moreover, VAC also specifically recognizes such sites in the vascular system to which PS exposes the blood.
Denne differensiering ved fosfolipidene gjør VAC så vel som de andre annexinene, til et ideelt reagens for tidlig å fastslå den såkalte protrombotiske tilstand som gir seg de ovenfor angitte utslag. This differentiation at the phospholipids makes VAC, as well as the other annexins, an ideal reagent for early determination of the so-called prothrombotic condition that produces the results indicated above.
Gjennom foreliggende oppfinnelse er det for første gang mulig å erkjenne den protrombotiske tilstand i det vaskulære system. Denne diagnose muliggjøres gjennom spesifisiteten av substanser, av midler som er i stand til å erkjenne den, i forhold til normaltilstanden, endrede protrombotiske tilstand til blodplatene. Da denne tilstand adskiller seg fra blod-platenes normaltilstand ved at den ytre omhylling bare oppviser fosfatidylserin i den protrombotiske blodplate, er i prinsippet ethvert middel som kan skjelne fosfatidylserin spesifikt fra fosfatidylkolin, egnet til å utnytte dette oppfinnelsesmessige prinsipp. Midler som kan benyttes kjenne-tegnes ved sin spesifisitet overfor fosfatidylserin, hvilket kan fastslås gjennom de bindingsforsøk som er angitt i foreliggende beskrivelse. Through the present invention, it is possible for the first time to recognize the prothrombotic state in the vascular system. This diagnosis is made possible through the specificity of substances, of means which are able to recognize the, in relation to the normal state, changed prothrombotic state of the platelets. As this condition differs from the normal condition of blood platelets in that the outer covering only exhibits phosphatidylserine in the prothrombotic platelet, in principle any agent that can distinguish phosphatidylserine specifically from phosphatidylcholine is suitable for utilizing this inventive principle. Agents that can be used are characterized by their specificity towards phosphatidylserine, which can be determined through the binding tests indicated in the present description.
Utnyttelsen av denne spesifisitet gjør det dessuten mulig å lokalisere utgangspunktet for aktiveringen av det hemo- The utilization of this specificity also makes it possible to locate the starting point for the activation of the hemo-
statiske system og/eller tromben. static system and/or the thrombus.
Gjennom foreliggende oppfinnelse er det således for første gang mulig å gripe til egnede terapeutiske forholdsregler ved en tidlig diagnose av en truende tilstand som muligens er under utvikling. Through the present invention, it is thus for the first time possible to resort to suitable therapeutic measures in the event of an early diagnosis of a threatening condition which is possibly developing.
De midler som anvendes i henhold til oppfinnelsen, er antikoagulerende polypeptider i den utstrekning de oppviser den nødvendige spesifisitet for fosfolipidet fosfatidylserin. The agents used according to the invention are anticoagulant polypeptides to the extent that they exhibit the necessary specificity for the phospholipid phosphatidylserine.
Særlig foretrukket er annexin-familien, spesielt VAC. Particularly preferred is the annexin family, especially VAC.
For å kunne gjøre bruk av midlene som anvendes i henhold til oppfinnelsen, særlig VAC eller de andre annexinene, som diagnostikum, merkes de på i og for seg kjent måte. Som egnet markør kan for eksempel benyttes markering med fluorescerende grupper, eller radioaktiv markering. Som fordelaktig anvendelig fluorescens-markør kan nevnes fluoresceinisotiocyanat, som fordelaktig anvendelig radioaktiv markør, radio-131 isotopene av halogenene, spesielt av jod, eksempelvis I eller 12 5I eller også bly, kvikksølv, tallium, technetium eller indium (<2>03Pb, 198Hg, 201T1,<99m>Tc, 111In) . In order to be able to use the agents used according to the invention, especially VAC or the other annexins, as diagnostics, they are labeled in a manner known per se. For example, marking with fluorescent groups or radioactive marking can be used as a suitable marker. Fluorescein isothiocyanate can be advantageously used as a fluorescent marker, and fluorescein isothiocyanate can be mentioned as an advantageously applicable radioactive marker, the radio-131 isotopes of the halogens, especially of iodine, for example I or 12 5I or also lead, mercury, thallium, technetium or indium (<2>03Pb, 198Hg , 201T1,<99m>Tc, 111In) .
Fluoresceinisotiocyanat (Serva) kan på kjent måte (13) benyttes til merking av VAC. Fluorescein isothiocyanate (Serva) can be used in a known manner (13) for labeling VAC.
Merking med et paramagnetisk kontrast-agens som er påvisbart i et MRI-system (magnetic resonance imaging) er også mulig. Det kan benyttes gadolinium, kobolt, nikkel, mangan eller jernkomplekser som det kan fremstilles konjugater med, som diagnostiske agens som er påvisbare i et MRI-system. I slike systemer benyttes et sterkt magnetfelt for i organismen å rette ut atomenes nukleære spinnvektorer. Deretter oppheves feltet, hvorved kjernene vender tilbake til sin utgangs-tilstand. Denne prosess observeres og registreres. Det antikoagulerende polypeptid som således er forsynt med en påvisbar markør, administreres herunder intraarterielt eller intravenøst. Den tilførte mengde avmåles slik at den etter tilstrekkelig inkubasjonstid, holder til den etterfølgende måling. Labeling with a paramagnetic contrast agent that is detectable in an MRI (magnetic resonance imaging) system is also possible. Gadolinium, cobalt, nickel, manganese or iron complexes with which conjugates can be prepared can be used as diagnostic agents that are detectable in an MRI system. In such systems, a strong magnetic field is used to align the nuclear spin vectors of the atoms in the organism. The field is then removed, whereby the nuclei return to their initial state. This process is observed and recorded. The anticoagulant polypeptide, which is thus provided with a detectable marker, is administered intra-arterially or intravenously. The added quantity is measured so that, after sufficient incubation time, it lasts for the subsequent measurement.
Til påvisningen av en protrombotisk tilstand tilsettes testblodet ekstrakorporealt polypeptidet som anvendes i hen hold til oppfinnelsen, eventuelt i nærvær av et ytterligere antikoagulant som ikke nedsetter plasma-kalsiumkonsentrasjonen, eksempelvis heparin, hvoretter den markør som er assosiert med den spesifikke celletype analyseres. For the detection of a prothrombotic condition, the extracorporeal polypeptide used in accordance with the invention is added to the test blood, possibly in the presence of an additional anticoagulant that does not reduce the plasma calcium concentration, for example heparin, after which the marker associated with the specific cell type is analysed.
Det merkede polypeptid kan anvendes så vel i blod-isotonisk vandig oppløsning, som med hjelpestoffer, for å oppnå formålet i henhold til oppfinnelsen. Hjelpestoffer kan eksempelvis være TWEEN 80, arginin, fosfatbuffer og fysiologisk akseptable konserveringsmidler. Ytterligere stoffer er velkjent for fagmannen og kan likeledes benyttes. The labeled polypeptide can be used both in blood-isotonic aqueous solution, as well as with excipients, to achieve the purpose according to the invention. Excipients can be, for example, TWEEN 80, arginine, phosphate buffer and physiologically acceptable preservatives. Additional substances are well known to the person skilled in the art and can likewise be used.
For den radioaktive merking benyttes f.eks. den kjente jodogenmetodé (12) eller den vanlige kloramin-T-metode. På grunn av dens halveringstid på 8 dager, er<131>lå anbefale for in vivo-diagnosen. Det radioaktivt merkede middel tas opp i blod-isoton vandig oppløsning og injiseres etter steril-filtrering. Helkropps-scintigrafier utføres med et gamma-kamera, f.eks. for<131>II, 2, 4 og 7 dager etter injeksjonen. For the radioactive marking, e.g. the known iodogen method (12) or the usual chloramine-T method. Due to its half-life of 8 days, <131> is not recommended for the in vivo diagnosis. The radioactively labeled agent is taken up in blood-isotonic aqueous solution and injected after sterile filtration. Whole-body scintigraphs are performed with a gamma camera, e.g. for<131>II, 2, 4 and 7 days after the injection.
Ved siden av de genuine annexinformer, kan også endrede former benyttes ved anvendelsen i henhold til oppfinnelsen. Alongside the genuine annex forms, modified forms can also be used in the application according to the invention.
Det skal særlig henvises til mutanter beskrevet i Particular reference should be made to mutants described in
EPA 0 293 567. I tillegg er også de fragmenter eller kjemisk modifiserte derivater av annexiner som oppviser spesifisiteten overfor fosfolipidene, fosfatidylserin/fosfatidylkolin og som dermed kan erkjenne den protrombotiske tilstand av de med-virkende blodplater, anvendelige. EPA 0 293 567. In addition, the fragments or chemically modified derivatives of annexins which show specificity towards the phospholipids, phosphatidylserine/phosphatidylcholine and which can thus recognize the prothrombotic state of the contributing platelets, are also applicable.
Foreliggende oppfinnelse angår spesifikt: The present invention relates specifically to:
Anvendelse av et antikoagulerende polypeptid fra annexin- familien, fortrinnsvis VAC, som er forsynt med en påvisbar markør. Use of an anticoagulant polypeptide from annexin- the family, preferably VAC, which is provided with a detectable marker.
Anvendelse av et antikoagulerende polypeptid som, som påvisbar markør har fluorescensmerking, fortrinnsvis fluoresceinisotiocyanat, en radioisotop av et halogen, av technetium, bly, kvikksølv, tallium eller indium, særlig Use of an anticoagulant polypeptide which, as a detectable marker, has fluorescence labelling, preferably fluorescein isothiocyanate, a radioisotope of a halogen, of technetium, lead, mercury, thallium or indium, in particular
131 I eller<125>I eller et paramagnetisk kontrastmiddel. Anvendelse av et antikoagulerende polypeptid som ovenfor beskrevet for å skjelne mellom fosfatidylserin og fosfatidylkolin. 131 I or<125>I or a paramagnetic contrast agent. Use of an anticoagulant polypeptide as described above to distinguish between phosphatidylserine and phosphatidylcholine.
Anvendelse av et antikoagulerende polypeptid som ovenfor Use of an anticoagulant polypeptide as above
beskrevet for anvendelse som diagnostikum. described for use as a diagnostic.
Fremgangsmåte for konstatering av den protrombotiske tilstand, hvorunder a) blodprøven ekstrakorporealt blandes med et antikoagulerende polypeptid eller middel innen annexin-familien, fortrinnsvis VAC, som bærer en påvisbar markør, og b) markeringen som er assosiert med spesifikke celletyper, analyseres. Method for ascertaining the prothrombotic state, during which a) the blood sample is mixed extracorporeally with an anticoagulant polypeptide or agent within the annexin family, preferably VAC, which carries a detectable marker, and b) the marker associated with specific cell types is analyzed.
Et middel som ved siden av et antikoagulerende polypeptid innen annexin-familien, fortrinnsvis VAC, som er forsynt med en påvisbar markør, dessuten inneholder et hjelpestoff som for eksempel fysiologisk natriumklorid oppløsning, TWEEN 80, arginin og/eller fosfatbuffer, hvorunder eventuelt et antikoagulant som ikke reduserer plasma-kalsiumkonsentrasjonen, fortrinnsvis heparin, kan benyttes. An agent which, in addition to an anticoagulant polypeptide within the annexin family, preferably VAC, which is provided with a detectable marker, also contains an auxiliary substance such as, for example, physiological sodium chloride solution, TWEEN 80, arginine and/or phosphate buffer, including possibly an anticoagulant which does not reduce the plasma calcium concentration, preferably heparin, can be used.
Et analysesett til diagnostisk påvisning av den protrombotiske tilstand eller utgangspunktet for aktiveringen av det hemostatiske system og/eller en trombe, kan inneholde et antikoagulerende polypeptid som beskrevet ovenfor, som er i stand til å skjelne mellom fosfatidylserin og fosfatidylkolin. An analysis kit for diagnostic detection of the prothrombotic condition or the starting point for the activation of the hemostatic system and/or a thrombus can contain an anticoagulant polypeptide as described above, which is capable of distinguishing between phosphatidylserine and phosphatidylcholine.
I henhold til oppfinnelsen anvendes et antikoagulerende polypeptid som beskrevet ovenfor for å skjelne mellom fosfatidylserin og fosfatidylkolin. According to the invention, an anticoagulant polypeptide as described above is used to distinguish between phosphatidylserine and phosphatidylcholine.
Et antikoagulerende polypeptid som beskrevet ovenfor kan anvendes i henhold til oppfinnelsen, til diagnose av den protrombotiske tilstand eller av utgangspunktet for forstyrrelse av det hemostatiske system eller for tromben. An anticoagulant polypeptide as described above can be used according to the invention, for diagnosis of the prothrombotic condition or of the starting point for disturbance of the hemostatic system or for the thrombus.
Materialer og metoder Materials and methods
VAC ble fremstillet analogt med EPA 0 181 465 eller VAC was produced analogously to EPA 0 181 465 or
EPA 0 293 567. De etterfølgende eksperimenter ble utført med VACa, men resultatene er overførbare til de andre annexinene, spesielt også til VACS. EPA 0 293 567. The subsequent experiments were carried out with VACa, but the results are transferable to the other annexins, especially also to VACS.
Lipider Lipids
Dioleoyl-fosfatidylkolin (DOPC, Nr. P-1013) Dioleoyl-fosfatidyletanolamin (DOPE, Nr. P-0510) Cardiolipin (CL, Nr. C-5646) Dioleoyl-phosphatidylcholine (DOPC, No. P-1013) Dioleoyl-phosphatidylethanolamine (DOPE, No. P-0510) Cardiolipin (CL, No. C-5646)
Dioleoyl-fosfatidylglycerol (DOPG, Nr. P-9664) Fosfatidylinositol (PI, Nr. P-0639) Dioleoyl-phosphatidylglycerol (DOPG, No. P-9664) Phosphatidylinositol (PI, No. P-0639)
Dioleoyl-fosfatidsyre (DOPA, Nr. P-2767) Dioleoyl phosphatidic acid (DOPA, No. P-2767)
Sterarylamin (SA, S-6755) og Stearylamine (SA, S-6755) and
eggeplomme-sfingomyelin (S-0756) ble anskaffet fra firma Sigma Chemical Co. Egg yolk sphingomyelin (S-0756) was purchased from Sigma Chemical Co.
Renheten av DOPC og DOPE ble kontrollert ved tynnskikt-kromatografi. Dioleoyl-fosfatidylserin (DOPS) ble fremstillet ved omdannelse av DOPC ifølge (1).<14>C merket DOPS (spesifikk aktivitet 100.000 dpm//xg) ble anskaffet fra Amersham. The purity of DOPC and DOPE was checked by thin-layer chromatography. Dioleoyl-phosphatidylserine (DOPS) was prepared by conversion of DOPC according to (1).<14>C labeled DOPS (specific activity 100,000 dpm//xg) was purchased from Amersham.
Fremstilling av fosfolipid- dobbeltskiktene på silisiumplater Production of the phospholipid bilayers on silicon wafers
Fosfolipid-dobbeltskikt ble påført med en "Langmuir-film balance" (Lauda type FW-1) som beskrevet av Corsel et al., (2). Hydrofile silisiumplater ble behandlet i 30% kromsvovel-syre og vann i 24 timer og oppbevart i 50% etanol/vann. Før anvendelse ble de grundig renset med et detergent og vann. "Film-balance"-anordningen ble fylt med demineralisert vann og 50/iM CaCl2. På denne nedre fase ble det påsatt 20 fil av en oppløsning som inneholdt 2 g/l fosfolipid i kloroform. DOPS-fraksjonene i dobbeltskiktet ble kontrollert med<14>C merket DOPS i blanding med DOPC. De dannede dobbeltskiktene ble fjernet fra silisiumplatene med scintillasjons-detergentet (Du Pont Formel-989) og den totale radioaktivitet målt i en seintiliasjonsteller. Phospholipid bilayers were applied with a "Langmuir film balance" (Lauda type FW-1) as described by Corsel et al., (2). Hydrophilic silicon wafers were treated in 30% chromosulfuric acid and water for 24 hours and stored in 50% ethanol/water. Before use, they were thoroughly cleaned with a detergent and water. The "film-balance" device was filled with demineralized water and 50 µM CaCl 2 . 20 μl of a solution containing 2 g/l phospholipid in chloroform was applied to this lower phase. The DOPS fractions in the bilayer were controlled with <14>C labeled DOPS mixed with DOPC. The formed bilayers were removed from the silicon plates with the scintillation detergent (Du Pont Formula-989) and the total radioactivity measured in a late scintillation counter.
Måling av bindingen ved ellipsometri Measurement of the binding by ellipsometry
Adsorpsjonen av VAC til fosfolipid-dobbeltskiktene ble målt ved hjelp av et automatisk ellipsometer (2,3) The adsorption of VAC to the phospholipid bilayers was measured using an automatic ellipsometer (2,3)
Bindingsforsøkene ble utført i en hydrofil kyvette som inneholdt 5 ml av en omrørt buffer (0,05M Tris/HCl; 0,1M NaCl; pH=7,5; T=20°C) . De divalente kationene ble tilsatt trinnvis som klorider. The binding experiments 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 cations were added stepwise as chlorides.
Ved VAC-konsentrasjoner <0,1/xg/ml ble bufferen som inneholdt den spesifikke VAC-konsentrasjon, tilsatt kontinuerlig for å skaffe en tilstrekkelig bufferkapasitet for At VAC concentrations <0.1/xg/ml, the buffer containing the specific VAC concentration was added continuously to provide a sufficient buffer capacity for
VAC. VAC.
Ut fra de kombinerte polariserings- og analyseringsdata ble brytningsindeksen og tykkelsen av den adsorberte film bestemt (4). Mengden å av det adsorberte proteinskikt ble bestemt ut fra brytningsindeksen og tykkelsen ved hjelp av en modifisert Lorentz-Lorenz-ligning [1] (3, 5): From the combined polarization and analysis data, the refractive index and thickness of the adsorbed film were determined (4). The amount of the adsorbed protein layer was determined from the refractive index and thickness using a modified Lorentz-Lorenz equation [1] (3, 5):
[1] å = 3d(n2-nb2)/[(n2+2) (r(nb2+2)-v(nb2-l) ) ] ; [1] to = 3d(n2-nb2)/[(n2+2) (r(nb2+2)-v(nb2-l) ) ] ;
nb er bufferens brytningsindeks. Verdiene r=0,254 og v=0,71 ble innsatt for den spesifikke molare refraktivitet og det partielle spesifikke volum (3). nb is the buffer's refractive index. The values r=0.254 and v=0.71 were inserted for the specific molar refractivity and the partial specific volume (3).
Resultater Results
Effekten av divalente kationer på bindingen av VAC til fosfolipider The effect of divalent cations on the binding of VAC to phospholipids
VAC bindes til fosfolipid-membraner som består av 20% DOPS/80%DOPC, i avhengighet av kalsiumkonsentrasjonen. Påfølgende tilsetning av EDTA førte til omgående og fullstendig desorpsjon (Fig. 1). Ved forandringen av de frie Ca +-konsentrasjonene kunne adsorpsjonen utløses igjen flere ganger uten at den adsorberte mengde eller adsorpsjonsgraden endret seg merkbart. Irreversible forandringer av VAC-molekylet eller av fosfolipid-dobbeltskiktene ved adsorpsjonen eller desorpsjonen er derfor ikke sannsynlig. Bindingen var likeledes fullstendig reversibel når kyvetten ble spylt med Ca<2+->fri buffer. VAC binds to phospholipid membranes consisting of 20% DOPS/80% DOPC, depending on the calcium concentration. Subsequent addition of EDTA led to immediate and complete desorption (Fig. 1). When the free Ca + concentrations changed, the adsorption could be triggered again several times without the adsorbed amount or the degree of adsorption changing noticeably. Irreversible changes of the VAC molecule or of the phospholipid bilayers during the adsorption or desorption are therefore not likely. The binding was also completely reversible when the cuvette was flushed with Ca<2+->free buffer.
Ca<2+->avhengigheten av VAC-bindingen til fosfolipider er fremstillet i Fig. 2. Ca<2+->dose/virkningskurven viser helt entydig en Ca<2+->konsentrasjon hvor halvdelen av den maksimale VAC-adsorpsjon er nådd: [Ca<2+>]1^2. [Ca<2+>]-verdien avhenger av sammensetningen av fosfolipid-overflaten. Ved fosfolipid-overflater som inneholder 100%, 20%, 5% og 1% DOPS, ble [Ca +] 1y2-verdier på henholdsvis 36 itM, 220/iM, 1,5 mM og 8,6 mM målt (Tab. 1). Disse resultatene stemmer ganske godt overens med [Ca2 + ] 1/,2-verdien på 53fiM som ble målt for endonexin II (=VAC)-binding til ekvimolare blandinger av PS/PC vesikler (6). Den maksimale mengde av det adsorberte protein ^maks^ var uavnen<?i9 av membranens DOPS-fraksjon og utgjorde ca. 0,217 xig/cm2. Ingen adsorps jon kunne fastslås ved rene DOPC-dobbeltskikt inntil en Ca<2+->konsentrasjon på 3 mM. The Ca<2+->dependence of the VAC binding to phospholipids is shown in Fig. 2. The Ca<2+->dose/effect curve clearly shows a Ca<2+->concentration where half of the maximum VAC adsorption is reached : [Ca<2+>]1^2. The [Ca<2+>] value depends on the composition of the phospholipid surface. At phospholipid surfaces containing 100%, 20%, 5% and 1% DOPS, [Ca +] 1y2 values of 36 itM, 220/iM, 1.5 mM and 8.6 mM were measured respectively (Tab. 1 ). These results are in good agreement with the [Ca 2 + ] 1/.2 value of 53 fiM measured for endonexin II (=VAC) binding to equimolar mixtures of PS/PC vesicles (6). The maximum amount of the adsorbed protein ^max^ was equal to the DOPS fraction of the membrane and amounted to approx. 0.217 xig/cm2. No adsorption could be determined by pure DOPC bilayers up to a Ca<2+->concentration of 3 mM.
Adsorpsjonen av VAC til fosfolipid-dobbeltskikt av for-skjellig sammensetning, er vist i Fig. 5. Også her er det tydelig at praktisk talt ingen adsorpsjon av VAC kan iakttas ved rene DOPC-dobbeltskikt. Adsorpsjonen av VAC til stearylamid (SA) er likeledes svak. The adsorption of VAC to phospholipid bilayers of different composition is shown in Fig. 5. Here, too, it is clear that practically no adsorption of VAC can be observed with pure DOPC bilayers. The adsorption of VAC to stearyl amide (SA) is likewise weak.
Ved forsøk med andre kationer enn Ca<2+>ble det fastslått at bindingen av VAC til fosfolipidene i stor grad er Ca<2+->spesifikk (Fig. 3). Cd<2+>, Zn<2+>, Mn<2+>og Co<2+>viste svak befordring av bindingen; Ba 2 + og Mg 2 + hadde ingen innflytelse. Denne egenskap ved kationene kan i en viss utstrekning korreleres med deres ioneradier. In experiments with cations other than Ca<2+>, it was established that the binding of VAC to the phospholipids is largely Ca<2+->specific (Fig. 3). Cd<2+>, Zn<2+>, Mn<2+>and Co<2+>showed weak transport of the bond; Ba 2 + and Mg 2 + had no influence. This property of the cations can to a certain extent be correlated with their ionic radii.
Sink- synergisme Zinc synergism
Høye konsentrasjoner av sinkioner (1 mM) fremmer VAC-adsorpsjon (Fig. 3) bare i liten grad; 50 xxM har overhodet ingen innflytelse på adsorpsjonen. Merkelig nok påvirker denne konsentrasjon bindingen i nærvær av Ca<2+>;det kan således iakttas en synergisme. [Ca<2+>]1^2~verdien falt fra 8,6 til 2,7 mM for dobbeltskikt med kun 1% DOPS ( [Zn2 + ] =50/xM) (Fig. 4) . 50 xtM [Zn2 + ] ligger i det normale området for sink-konsentrasjoner i plasma. High concentrations of zinc ions (1 mM) promote VAC adsorption (Fig. 3) only to a small extent; 50 xxM has no influence whatsoever on the adsorption. Strangely enough, this concentration affects the binding in the presence of Ca<2+>; a synergism can thus be observed. The [Ca<2+>]1^2~ value dropped from 8.6 to 2.7 mM for bilayers with only 1% DOPS ( [Zn2 + ] =50/xM) (Fig. 4) . 50 xtM [Zn2 + ] is in the normal range for zinc concentrations in plasma.
Diagnostiske metoder Diagnostic methods
1. In vitro- diagnose 1. In vitro diagnosis
a) VAC merkes etter i og for seg kjente fremgangsmåter a) VAC is labeled according to procedures known per se
(13) med fluoresceingruppen, eksempelvis ved hjelp (13) with the fluorescein group, for example by means of
av fluoresceinisotiocyanat; det oppnås på denne måte of fluorescein isothiocyanate; it is achieved in this way
VAC-FITZ. VAC-FITZ.
b) Blod fra en pasient oppsamles i et lite plastikkrør som inneholder et antikoagulant (f.eks. heparin) som b) Blood from a patient is collected in a small plastic tube containing an anticoagulant (e.g. heparin) which
ikke reduserer plasma-kalsiumkonsentrasjonen, og does not reduce the plasma calcium concentration, and
VAC-FITC. VAC-FITC.
c) Etter blanding analyseres blodcellene under bruk av en FACS (fluorescence activated cell sorter). Ved c) After mixing, the blood cells are analyzed using a FACS (fluorescence activated cell sorter). By
denne analyse bestemmes den fluorescensintensitet som er assosiert med spesifikke celletyper. this analysis determines the fluorescence intensity associated with specific cell types.
d) Analyse-profilene viser mengden av blodplater med bundet VAC-FITC, dvs. plater med eksponert PS og d) The analysis profiles show the amount of platelets with bound VAC-FITC, i.e. platelets with exposed PS and
dermed forekomst av en protrombotisk tilstand. Denne helsetruende tilstand kan på denne måte erkjennes tidlig og følgelig behandles tidlig. thus occurrence of a prothrombotic condition. This health-threatening condition can thus be recognized early and consequently treated early.
2. In vivo- diagnose 2. In vivo diagnosis
a) VAC merkes med en kortlivet isotop, eksempelvis<131>I, etter i og for seg kjente fremgangsmåter; det a) VAC is labeled with a short-lived isotope, for example <131>I, according to procedures known per se; the
oppnås<131>I-VAC. is obtained<131>I-VAC.
b)<131>I-VAC tilføres pasienten intravenøst. b)<131>I-VAC is administered intravenously to the patient.
c) Etter en viss inkubasjonstid underkastes pasienten en hel- eller del-kropps scintigrafi. Fordelingen av radioaktiviteten kan iakttas med et large-field-off - view gamma-kamera. d) Intravaskulære steder med<131>I-VAC-akkumulasjon kjennetegner de steder hvor trombosen utvikler seg. c) After a certain incubation time, the patient is subjected to a whole or partial body scintigraphy. The distribution of the radioactivity can be observed with a large-field-off-view gamma camera. d) Intravascular sites of<131>I-VAC accumulation characterize the sites where the thrombosis develops.
Egnede trombose-hindrende eller trombose-helbredende forholdsregler kan tidlig innsettes. Appropriate thrombosis-preventing or thrombosis-healing measures can be implemented early.
Radioaktiv markering Radioactive marking
1.1 Forberedelser 1.1 Preparations
1.1.1 Fremstilling av en 500 mmol/ 1 natriumfosfatbuffer 24,5 g natrium-di-hydrogenfosfat-monohydrat ble oppløst i 1 liter bidestillert vann og tilsatt til en oppløsning av 1.1.1 Preparation of a 500 mmol/l sodium phosphate buffer 24.5 g of sodium dihydrogen phosphate monohydrate was dissolved in 1 liter of bidistilled water and added to a solution of
35,3 g di-natrium-hydrogenfosfat i 1 liter bidestillert vann til pH 7,5. 35.3 g of disodium hydrogen phosphate in 1 liter of bidistilled water to pH 7.5.
1.1.2. Fremstilling av en 20 mmol/ 1 natriumfosfatbuffer + 1.1.2. Preparation of a 20 mmol/l sodium phosphate buffer +
150 mmol NaCl ( elueringsbuffer) 150 mmol NaCl (elution buffer)
2,76 g natrium-di-hydrogenfosfat-monohydrat ble oppløst i 1 liter bidestillert vann og tilsatt til en oppløsning av 2.76 g of sodium dihydrogen phosphate monohydrate was dissolved in 1 liter of bidistilled water and added to a solution of
2,84 g dinatriumhydrogenfosfat i 1 liter bidestillert vann til pH 7,2. Ved tilsetning av 8,77 g NaCl (150 mmol) 2.84 g of disodium hydrogen phosphate in 1 liter of bidistilled water to pH 7.2. By adding 8.77 g of NaCl (150 mmol)
til 1 liter av bufferen, ble elueringsbufferen fremstillet. to 1 liter of the buffer, the elution buffer was prepared.
1.1.3. Likevektsinnstilling av rensesøylen En PD-10-søyle (Sephadex G25, Firma Pharmacia) ble like-vektsinnstillet med ca. 3 0 ml av elueringsbufferen. 1.1.4. Preparering av reaksionsbeholderen 2 mg IODO-gen (molmasse: 432,09 g/mol); Fig. 6) ble 1.1.3. Equilibration of the purification column A PD-10 column (Sephadex G25, Firma Pharmacia) was equilibrated with approx. 30 ml of the elution buffer. 1.1.4. Preparation of the reaction vessel 2 mg of IODO gene (molar mass: 432.09 g/mol); Fig. 6) became
oppløst i 50 ml diklormetan av høy renhet. 200/il av denne oppløsningen ble pipettert over i et 1,5 ml Eppendorf-glass og oppløsningsmidlet deretter avdampet ved 37°C (termostat-blokk). I reaksjonsglasset var dermed 8 tig (1,85 x 10 mmol) IODO-Gen finfordelt på veggen. dissolved in 50 ml of high purity dichloromethane. 200 µl of this solution was pipetted into a 1.5 ml Eppendorf tube and the solvent then evaporated at 37°C (thermostat block). In the reaction glass, 8 tig (1.85 x 10 mmol) of IODO-Gen was thus finely distributed on the wall.
1.1.5. VAC- a benyttet for merkingen 1.1.5. VAC-a used for the marking
Det ble gått ut fra en oppløsning av 50 mg VAC-a i 4 ml 20 mmol/1 natriumfosfatbuffer pluss 150 mmol/1 NaCl, pH It was assumed that a solution of 50 mg VAC-a in 4 ml of 20 mmol/1 sodium phosphate buffer plus 150 mmol/1 NaCl, pH
7,2, fortynnet med 1 ml bidestillert vann. Molmasse VAC-a: 34000 g/mol. 7.2, diluted with 1 ml bidistilled water. Molecular mass VAC-a: 34000 g/mol.
1.1.6. 1- 125 benyttet til merking 1.1.6. 1- 125 used for marking
1251 Na fra Fa. Dupont, NEN Products, med en total radioaktivitet på kalibreringsdagen på 67,3 MBq (=1,82 mCi). Den spesifikke aktivitet var 15,9 Ci/mg 1251 Na from Fa. Dupont, NEN Products, with a total radioactivity on the calibration day of 67.3 MBq (=1.82 mCi). The specific activity was 15.9 Ci/mg
jod = 1,98 kCi/mmol 1/2 J , hvilket tilsvarte 0,115 tig iodine = 1.98 kCi/mmol 1/2 J , which was equivalent to 0.115 tig
-7 -7
jod (9,2 x 10 mmol 1/2 J2). Det aktive Nal var oppløst 1 5,5/il 0,1 mol/l NaOH. iodine (9.2 x 10 mmol 1/2 J2). The active Nal was dissolved in 15.5/l 0.1 mol/l NaOH.
1.2. Jodering 1.2. Iodination
Alt arbeid ble foretatt i isotop-avtrekk bak blyglass-avskj erming. All work was carried out in isotope extraction behind lead glass shielding.
2 0/il (= 200/xg) av oppløsningen av VAC-Q! beskrevet under 2 0/il (= 200/xg) of the solution of VAC-Q! described below
2.1.5., ble overført til den IODO-Gen forbehandlede reaksjonsbeholderen. Den ble lukket og ristet i 20 minutter ved romtemperatur. Deretter ble reaksjonsopp-løsningen påsatt med en pipette på den forberedte PD-10-søyle (s. 2.1.3.). Reaksjonsbeholderen ble etterspylt med 500/xl av elueringsbuf feren (s. 2.1.2.) og denne opp-løsningen likeledes anbragt på PD-10-søylen. Eluatet som derved rant gjennom ble kassert. 2.1.5., was transferred to the IODO-Gen pretreated reaction vessel. It was closed and shaken for 20 minutes at room temperature. The reaction solution was then applied with a pipette to the prepared PD-10 column (p. 2.1.3.). The reaction container was rinsed with 500 µl of the elution buffer (p. 2.1.2.) and this solution was also applied to the PD-10 column. The eluate that thereby flowed through was discarded.
1.3. Rensing 1.3. Cleansing
Ved påsetting av 0,5 ml porsjoner elueringsbuffer (s. 2.1.2. ) med 2 minutters mellomrom, ble det VAC-a [ 125I] adskilt fra det fremdeles frie<125>I/Na<125>I. Etter 12 fraksjoner var dette rensetrinn ferdig. Fraksjonenes relative aktivitetsinnhold ble målt med en laboratorie monitor (GMZ) (Fig. 7). By applying 0.5 ml portions of elution buffer (p. 2.1.2. ) at 2 minute intervals, the VAC-a [ 125I] was separated from the still free<125>I/Na<125>I. After 12 fractions, this purification step was finished. The relative activity content of the fractions was measured with a laboratory monitor (GMZ) (Fig. 7).
Fraksjonene 6 og 7 ble slått sammen, fylt opp til nøyaktig 2, 0 ml med elueringsbuf f er og oppdelt i 100/xl porsjoner og nedfrosset ved -20°C. Substansen ble holdt i beredskap i disse porsjoner for analyse- og Fractions 6 and 7 were pooled, made up to exactly 2.0 ml with elution buffer and divided into 100 µl aliquots and frozen at -20°C. The substance was kept in readiness in these portions for analysis and
utviklingsarbeid. development work.
2. Analytisk del 2. Analytical part
2.1 Innholdsbestemmelse 2.1 Determination of content
Ved den kromogene substrat-assay ble et VAC- a-innhold på In the chromogenic substrate assay, a VAC-a content of
71,8 fig/ 2, 0 ml oppløsning målt. 71.8 fig/ 2.0 ml solution measured.
2.2 Radioaktivitetsbestemmelse 2.2 Determination of radioactivity
2.2.1. Total radioaktivitet 2.2.1. Total radioactivity
Etter opptining av en 100/xl porsjon ble 50/xl av denne [ 12 5I] VAC-a-oppløsning tilsatt med en pipette til 950/xl av den inaktive VAC-a-oppløsning (s. 2.1.5), grundig blandet og 50/xl av denne bragt til måling i LSC-Counter (Beckman). 24,5 MBq (=0,663mCi)/2,0 ml totaloppløsning. After thawing a 100 µl aliquot, 50 µl of this [ 12 5I] VAC-a solution was added with a pipette to 950 µl of the inactive VAC-a solution (p. 2.1.5), thoroughly mixed and 50/xl of this brought to measurement in the LSC-Counter (Beckman). 24.5 MBq (=0.663mCi)/2.0 ml total solution.
2.2.2 Spesifikk aktivitet 2.2.2 Specific activity
Ut fra gehaltsbestemmelsen og total-radioaktiviteten ble det beregnet en spesifikk aktivitet på Based on the determination of the content and the total radioactivity, a specific activity was calculated
341,5 MBg/mg = 11,61 TBq/mmol 341.5 MBg/mg = 11.61 TBq/mmol
(9,23 mCi/mg = 313,8 Ci(mmol) (9.23 mCi/mg = 313.8 Ci(mmol)
2.2.3. Bestemmelse av proteinbundet radioaktivitet ved 2.2.3. Determination of protein-bound radioactivity by
TCA- felling TCA precipitation
100/xl av VAC-ot-oppløsningen ble tilsatt 50/xl 3% BSA-oppløsning og 150/xl 40% vandig trikloreddiksyre, ristet godt og plassert i kjøleskap i 60 minutter. Det dannede bunnfall ble frasentrifugert.Alikvoter av supernatanten ble overført for måling i LSC-Counter. 100 µl of the VAC-ot solution was added to 50 µl 3% BSA solution and 150 µl 40% aqueous trichloroacetic acid, shaken well and placed in a refrigerator for 60 minutes. The formed precipitate was centrifuged off. Aliquots of the supernatant were transferred for measurement in the LSC-Counter.
Resultat: 99,3% av radioaktiviteten var utfellbar. Result: 99.3% of the radioactivity was precipitable.
2.2.4. Radioaktivitetsutbytte 2.2.4. Radioactivity yield
Av de tilsatte 67,3 MBq ble 24,5 MBq funnet igjen i VAC-a. Aktivitetsutbyttet utgjorde dermed 36,4%. Of the added 67.3 MBq, 24.5 MBq was found again in VAC-a. The activity yield thus amounted to 36.4%.
2.3. Modifikasjonsgrad 2.3. Degree of modification
Av den spesifikke aktivitet og den anvendte mengde inaktiv VAC-a ble det beregnet at statistisk hvert 6. VAC-a-molekyl ble merket med<125>I-atom. From the specific activity and the amount of inactive VAC-a used, it was calculated that statistically every 6th VAC-a molecule was labeled with <125>I atom.
3.4. Identitet og renhet 3.4. Identity and purity
Under anvendelse av SDS-PAGE (gradient-gel 7 - 17%, ikke reduserende betingelser) og den påfølgende vurdering av gelen ved sølvfarving (Oakley-metoden), autoradiografi og påvisning under en Linearanalyzer (Fa. Berthold LB 282, Sonde LB 2 820) (Fig. 3) ble substansen undersøkt under sammenligning med det benyttede VAC-a. Substansene var identiske. Andelen av dimert produkt lå tydelig under grensen på 8% som tolereres for inaktive charger. Using SDS-PAGE (gradient gel 7 - 17%, non-reducing conditions) and the subsequent evaluation of the gel by silver staining (Oakley method), autoradiography and detection under a Linearanalyzer (Fa. Berthold LB 282, Probe LB 2 820) ) (Fig. 3) the substance was examined in comparison with the used VAC-a. The substances were identical. The proportion of dimerized product was clearly below the limit of 8% tolerated for inactive chargers.
Tekst til figurene Text for the figures
Fig, l: Alternerende adsorpsjon og desorpsjon av VAC på en fosfolipid-overflate, indusert ved økning hhv. senkning av Ca2 +-konsentrasjonen. Adsorpsjonen av VAC (1/xg/ml) på et 20% DOPS/80% DOPC fosfolipid-dobbeltskikt. Tilsetning av Ca<2+>(3, 4, 6 mM) er angitt med t hhv. E. Fig. 2: Innflytelse av fosfolipid-sammensetningen og Ca<2+->konsentrasjonen på adsorpsjonen av VAC på en fosfolipid-overf late . o 100% DOPS; o 20% DOPS; D 5% DOPS; □ 1 % DOPS; 100% DOPC; samtlige blandinger ble supplert med DOPC. [VAC] = 1/ig/ml. Fig. 3: Effekt av bivalente ioner på adsorpsjonen av VAC. VAC-adsorpsjon på dobbeltskikt av 20% DOPS og 80% DOPC i nærvær av de angitte ioner (1 eller 3 mM) . [VAC] = 1/ig/ml. Fig. 4: Synergistisk effekt av Zn2+ på den Ca<2>"""-avhengige adsorpsjon av VAC på fosfolipid-overflaten. Fig, l: Alternating adsorption and desorption of VAC on a phospholipid surface, induced by increasing or lowering the Ca2 + concentration. The adsorption of VAC (1/xg/ml) on a 20% DOPS/80% DOPC phospholipid bilayer. Addition of Ca<2+> (3, 4, 6 mM) is indicated by t or E. Fig. 2: Influence of the phospholipid composition and Ca<2+->concentration on the adsorption of VAC on a phospholipid surface. o 100% DOPS; o 20% VAT; D 5% DOPS; □ 1% DOPS; 100% DOPC; all mixtures were supplemented with DOPC. [VAC] = 1/ug/ml. Fig. 3: Effect of bivalent ions on the adsorption of VAC. VAC adsorption on bilayers of 20% DOPS and 80% DOPC in the presence of the indicated ions (1 or 3 mM). [VAC] = 1/ug/ml. Fig. 4: Synergistic effect of Zn2+ on the Ca<2>""-dependent adsorption of VAC on the phospholipid surface.
Effekten av Ca<2+>på VAC-adsorpsjonen på 1% DOPS og 99% DOPC i nærvær av 50/tM Zn<2+>ble målt. [VAC] = 1/xg/ml. The effect of Ca<2+> on the VAC adsorption of 1% DOPS and 99% DOPC in the presence of 50/tM Zn<2+> was measured. [VAC] = 1/xg/ml.
Fig. 5: Adsorpsjon av VAC på fosfolipid-dobbeltskikt av for-skjellig sammensetning. Fig. 5: Adsorption of VAC on phospholipid bilayers of different composition.
VAC-adsorpsjonen på dioleoylfosfatidylserin (DOPS), cardiolipin CL) og dioleoylfosfatidyletanolamin (DOPE), enten ren eller blandet med 80% dioleoylfosfatidylkolin (DOPC), på dioleoylfosfatidylglycerol (DOPG), fosfatidylinsositol (PI) og stearylamin blandet med 80% DOPC eller på ren DOPC. The VAC adsorption on dioleoylphosphatidylserine (DOPS), cardiolipin CL) and dioleoylphosphatidylethanolamine (DOPE), either pure or mixed with 80% dioleoylphosphatidylcholine (DOPC), on dioleoylphosphatidylglycerol (DOPG), phosphatidylinositol (PI) and stearylamine mixed with 80% DOPC or on pure DOPC.
[VAC] = 1/xg/ml; [Ca2 + ] = 3 mM. [VAC] = 1/xg/ml; [Ca 2 + ] = 3 mM.
Fig. 6: Fig. 6:
1,3,4,6-tetraklor-3a-6a-difenyl-glykoluril 1,3,4,6-tetrachloro-3a-6a-diphenyl-glycoluril
(IODO-GEN) (IODO GENE)
Fig. 7: Radioaktivitetsfordeling i fraksjonene 1-12. Fig. 7: Radioactivity distribution in fractions 1-12.
Fig. 8: Radioaktivitetsfordeling under "Linearanalyzer". Fig. 8: Radioactivity distribution under "Linearanalyzer".
Den beregnede blanding ble anbragt på "film balance"-anordningen. Mengden av dobbeltskikt ble målt ved ellipsometri og aktiviteten av<14>C-merket DOPS ble målt med en Beckman 6S 3801 scintillasjonsteller (s.d. <2%) og korrigert for bak-grunnsstrålingen (60 DPM). DOPS-fraksjonen i dobbeltskiktet ble beregnet ved bruk av ligning 2: The calculated mixture was placed on the "film balance" device. The amount of bilayer was measured by ellipsometry and the activity of <14>C-labeled DOPS was measured with a Beckman 6S 3801 scintillation counter (s.d. <2%) and corrected for the background radiation (60 DPM). The DOPS fraction in the bilayer was calculated using equation 2:
Den spesifikke aktivitet av DOPS utgjorde 100.000 -1 2 DPM.iig , overflaten besatt med fosfolipider 0,62 cm . The specific activity of DOPS amounted to 100,000 -1 2 DPM.iig, the surface covered with phospholipids 0.62 cm.
Den maksimale VAC-adsorpsjon (Tmaks) på de oppførte fosfolipid-overflater sammen med den kalsiumkonsentrasjon som fører til halvparten av den maksimale VAC-binding [Ca 2+^ 1/ 2' er angitt som middelverdi av minst tre forskjellige forsøk med angivelse av de tilsvarende standardavvik, The maximum VAC adsorption (Tmax) on the listed phospholipid surfaces together with the calcium concentration that leads to half of the maximum VAC binding [Ca 2+^ 1/ 2' is given as the mean value of at least three different experiments indicating the corresponding standard deviation,
n.d. = not determined = ikke bestemt n.d. = not determined = not determined
Litteraturfortegnelse Bibliography
1.. Confurius, P & Zwaal, R. F. A. (1977) Biochim. Biophys. Acta 488, -42. 2. Corsel, J. W., Willems, G. M., Kop, J. M. M. , Cuypers, P. A. & Hermens, W. Th. (1986) J. Colloid Interface Sei. 111, 544-554. 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. 4. McCrackin, F. L., Passaglia, E., Stromberg, R. R. & Steinberg, H. L. (1963) J.Res.Nat.Bur.Stand.Sect.A 67, 3-377. 5. Kop, J. M. M., Cuypers, P. A., Lindhout, Th., Hemker, H. C. & Hermens, W. Th. (1984) J. Biol. Chem. 259, 13993-13998. 6. Schlaepfer, D. D., Mehlman, T., Burgess, W. H. & Haigler. H. T. (1987) Proe. Nati. Acad. Sei. USA 84, 6078-6082. 7. Op den Kamp, J.A. F. Ann-. Rev. Biochem. 1979, 48: 47-71. 8. Zwaal, R.F.A. Biochim. Biophys. Acta 1978, 515: 163-205. 9. Jackson, CM. und Nemerson, Y. Ann. Rev. Biochem. 1980, 49: 765-811. 1.. Confurius, P & Zwaal, R.F.A. (1977) Biochim. Biophys. Acta 488, -42. 2. Corsel, J.W., Willems, G.M., Kop, J.M.M., Cuypers, P.A. & Hermens, W.Th. (1986) J. Colloid Interface Sei. 111, 544-554. 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. 4. McCrackin, F.L., Passaglia, E., Stromberg, R.R. & Steinberg, H.L. (1963) J.Res.Nat.Bur.Stand.Sect.A 67, 3-377. 5. Kop, J.M.M., Cuypers, P.A., Lindhout, Th., Hemker, H.C. & Hermens, W.Th. (1984) J. Biol. Chem. 259, 13993-13998. 6. Schlaepfer, D.D., Mehlman, T., Burgess, W.H. & Haigler. H.T. (1987) Proe. Nati. Acad. Pollock. USA 84, 6078-6082. 7. Op den Kamp, J.A. F. Ann-. Fox. Biochem. 1979, 48: 47-71. 8. Zwaal, R.F.A. Biochem. Biophys. Acta 1978, 515: 163-205. 9. Jackson, CM. und Nemerson, Y. Ann. Fox. Biochem. 1980, 49: 765-811.
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US5627036A (en) * | 1989-12-27 | 1997-05-06 | Boehringer Ingelheim International Gmbh | Use of an anticoagulant as a diagnostic agent |
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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 |
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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 |
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US5955605A (en) * | 1995-02-21 | 1999-09-21 | Neorx Corporation | Biotinidase resistant biotin-DOTA conjugates |
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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 |
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