EP0975975A1 - Procede pour la determination des facteurs actives de la coagulation sanguine dans le plasma et les derives du plasma - Google Patents

Procede pour la determination des facteurs actives de la coagulation sanguine dans le plasma et les derives du plasma

Info

Publication number
EP0975975A1
EP0975975A1 EP98913654A EP98913654A EP0975975A1 EP 0975975 A1 EP0975975 A1 EP 0975975A1 EP 98913654 A EP98913654 A EP 98913654A EP 98913654 A EP98913654 A EP 98913654A EP 0975975 A1 EP0975975 A1 EP 0975975A1
Authority
EP
European Patent Office
Prior art keywords
plasma
factor
activated
thrombin
tissue factor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP98913654A
Other languages
German (de)
English (en)
Inventor
Günter WÖBER
Beate Gesien
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Blutspendedienst Der Drk-Landesverbande Nordrhein und Westfalen-Lippe GmbH
Original Assignee
Blutspendedienst Der Drk-Landesverbande Nordrhein und Westfalen-Lippe GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Blutspendedienst Der Drk-Landesverbande Nordrhein und Westfalen-Lippe GmbH filed Critical Blutspendedienst Der Drk-Landesverbande Nordrhein und Westfalen-Lippe GmbH
Publication of EP0975975A1 publication Critical patent/EP0975975A1/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors

Definitions

  • the invention relates to a method for determining activated blood coagulation factors in plasma and plasma derivatives.
  • Blood coagulation factors are normally enzymatically inactive proenzymes, which are converted into their biologically active form by limited proteolytic cleavage.
  • prothrombin factor II
  • thrombin factor Ha
  • Thrombin also activates factor XIII, which crosslinks fibrin into a stable clot. This physiological activation is desirable in most cases.
  • tissue factor is an integral membrane protein that binds both factor VII and factor VIII with high affinity
  • tissue factor also catalyzes the activation of factor VII to factor VIIa.
  • tissue factor a mutated form of the tissue factor is described, the amino acid sequence around that section of the
  • 2Q protein is shortened, which is responsible for the binding to the membrane.
  • the now soluble tissue factor tTF can bind factor VIII and is fully active in a coagulation test. However, he has lost the ability to auto-catalyze from factor VII to factor VIIa. Therefore you can with help
  • a coagulation test which is known and described under the name NAPPT 35 (non-activated partial prothrombine time), is prescribed as a standard test for prothrombin complex PPSB (see: HS KINGDON et al .; Potentially thrombogenic materials in factor IX concentrates; Thro b. Diath. Haemorrh. 1975; 617-631).
  • a sample amount of the plasma or plasma derivative to be examined is incubated with activated prothrombin complex and procoagulatory phospholipid vesicles, containing the latter integrated tissue factor, and the formation of thrombin is started by adding a suitable amount of Ca ions,
  • the thrombin formation is ended and the amount of thrombin formed is determined by known methods.
  • the new test method works with materials that can be produced in practically unlimited quantities and can be stored for a long time, so that tests can be carried out without delay because of the formation of a platelet suspension.
  • the new test measures thrombin formation in defibrinated plasma in the presence of activated factors.
  • the amount of thrombin formed is measured using known methods by cleaving a synthetic substrate, for example the known S 2238 (see HP Schwarz et al .; Kyoto Satellite Symposia of Xllth Congress of ISTH; Kyoto, Japan, 1989; p. 34 - abstract). The resulting color is measured photometrically.
  • a test batch contains a small amount of a n e and activated prothrombin prokoagu- latowitz phospholipid vesicles, the latter being integrated tissue factor.
  • the presence of the activated prothrombin complex is chosen because, because of the auto-catalytic nature of thrombin formation with very small amounts of activated factors, the start-up phase (lag phase) of thrombin formation would be of different lengths. It would not be precisely defined after which incubation times one would have to measure the thrombin formation.
  • the tissue factor to be used is produced from biogenic starting material, acetone dry powder in particular being extracted from the bovine brain by a detergent-containing solution.
  • the tissue factor is mixed with phosphatidylserine and phosphatidylcholine in a detergent-containing solution for incorporation into procoagulatory phospholipid vesicles and integrated into the procoagulatory phospholipid vesicles by dialysis.
  • Ancrod (venom of the snake species Ankistrodon r odostoma; Sig a Chemical Co .; Art. No. A 5042) is mixed with distilled water diluted to 10 U / ml. 1 ml of human plasma from normal donors is mixed with 10 ⁇ l of Ancrod solution, incubated for 1 hour at 37 ° C., rapidly cooled to -70 ° C., warmed again to 37 ° C. and centrifuged. The supernatant, defibrinated plasma, is stored at room temperature and must be used within 10 minutes The defibrinated plasma can also be frozen in portions and contains only negligible traces of thrombin.
  • cryogen supernatant 50 ml of normal plasma are frozen, thawed at 4 ° C. overnight and 15 min. centrifuged. The supernatant is "cryogen supernatant"; 0.5 units of heparin per ml of cryogen supernatant are added to the latter.
  • DEAE-Sephadex is allowed to swell in 1 M NaCl solution for 15 min at room temperature (21 - 24 ° C) or overnight at 4 ° C (20 mg dry matter to 2 ml solution), by repeated filtering through a nylon mesh and resuspending in an equilibration puff - he equilibrate. Heparin-binding proteins are adsorbed from the supernatant on the ion exchanger by incubation at 4 ° C. for one hour. 40 ml of cryogen supernatant are added to equilibrated DEAE-Sephadex corresponding to 20 mg dry matter. The loaded DEAE-Sephadex is filtered off and cleaned by resuspending in washing buffer.
  • DEAE-Sephadex is suspended in 1 ml elution buffer and 15 min. protected at room temperature telt.
  • the solution of the partial prothrombin complex is dialyzed overnight against distilled water that has been pre-cooled to 4 ° C. and frozen in portions.
  • a suitable dilution of the PPK according to point 2 must be determined in the thrombin formation test (see point 5).
  • HEPES buffer eg 1:20, 1:40 and 1:80
  • HEPES N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid
  • a suitable dilution is characterized by the linearity of the rate of cleavage of the chromogenic substrate S 2238.
  • the ready-to-use APK can be frozen in portions. With this procedure, an APK can be reproducibly produced with a factor Xa content of approximately 1% of the total amount of factor X in the preparation. Thrombin is not detectable after incubation of the APK with the chromogenic substrate S 2238.
  • Dialysis tubing cellulose membrane from Sigma (Art. No. D 9402) or Slide-A-Lyzer TM dialysis cassette from Fa.
  • Phosphatidylserine (Sigma Art. No. P 1185) Phosphatidylcholm (PC), (Sigma Art. No. P 4139).
  • Both phospholipids can contain unsaturated, but preferably saturated fatty acids;
  • Dialysis buffer in the form of 94.5 g sucrose and 0.27 g NaCl per liter dist. Water.
  • the dialysis buffer can also contain 0.5% NaN 3.
  • acetone dry powder is digested with 1 ml of the 10% octyl glucoside solution while shaking at about 30 ° C in an ultrasonic bath.
  • the insoluble material is removed by centrifugation, 20 mg of phospholipidic solution (molar ratio PC to PS 6 to 4) is dissolved per ml of supernatant and dialyzed against the dialysis buffer.
  • dialysis buffer which contains a gel for binding detergents (e.g. Biorad SM-2).
  • the detergent is removed by dialysis, and a suspension of vesicles is spontaneously formed, which contain the tissue factor integrated.
  • the vesicles obtained can be frozen in portions.
  • a suitable dilution is sought based on the blank value in the thrombin formation test (see point 5), the blank value being supposed to be a delta OD of 0.005 to 0.010.
  • HEPES / NaCl buffer 6 M HEPES, 139 M NaCl, 3.5 g albumin per 1 buffer, pH 7.35;
  • Citrate buffer 20 mM Na 3 citrate 2 H20, 125 mM NaCl, pH 7.35;
  • Plasma test for the presence of activated coagulation factors Pooled plasma, produced either from whole blood or by plasmapheresis, is treated with 1% Triton X-100 and 1% tributyl phosphate in a known manner to inactivate membrane-enveloped viruses (Neurath, AR and Horowitz, B., EP-PS
  • Chro ogenes substrate S 2238, Chro ogenix, Art. No. 41202 The content of a vial S 2238 (25 mg) is in 20 ml dist. Water dissolved; by dilution in the ratio l + i with dist. Water is made to a working dilution of 1 mM. Thrombin, 53 nkat, Chro Ogenix, Art. No. 41217, is in 1 ml of dist. Water dissolved. A buffer concentrate
  • a working buffer is made by mixing buffer concentrate (30 ml) with human serum albumin (HSA), 20% HSA (0.75 ml). This is used to mix substrate buffer containing 26 ml working buffer and 2.4 ml substrate solution.
  • HSA human serum albumin
  • thrombin formation from activated coagulation factors the values for APK and the blank value are subtracted from the measured extinction difference per minute. Using batch 9 as an example, this results in a value of 0.029.
  • a thrombin activity of 2 nkat is read off a calibration line with thrombin dilutions. If plasma samples contain heparin, this must be neutralized with protasulfate or Polybren TM (hexadimethrin bromide) before the test or preferably inactivated with heparinase.
  • Factor IXa, factor XIa and factor Xlla were obtained from Alexis, Art. No. 73510-1, 200-039-C025 and 200-042-UCO2. Defibrinated plasma was activated by incubation with thrombin (1.66 nkat, 10 min. At 37 ° C.), then the thrombin activity was increased using Pefabloc SC TM (4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride), Boehringer Mannheim , Art. No. 1 429 868, inhibited. The amount of Pefabloc SC TM required to inhibit the thrombin activity presented was determined in a preliminary test.
  • factor IXa The manufacturer of factor IXa, factor XIa and factor Xlla only states the activity in plasma units for factor IXa, where 1 plasma unit corresponds to the amount of the factor in 1 ml of normal plasma. Using the pipetting scheme and the test procedure, it can be calculated that 0.003 plasma units of factor IXa in the test trigger a clearly measurable thrombin formation after only 5 minutes of incubation time in the thrombin formation test.
  • Factor VIIIa was obtained from Alexis, Art. No. 73560-1 or HF086-1. Defibrinated plasma was not am activated. The pipetting scheme and test procedure were analogous to those given in Example 2. In a direct comparison, factor VIII phospholipid vesicles with and without integrated tissue factor were examined as cofactors in the thrombin formation test. Vesicles without tissue factor were used undiluted, while vesicles with integrated tissue factor had to be prediluted by a factor of 2000. The following results were obtained in such a comparison:

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Neurosurgery (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé pour la détermination des facteurs activés de la coagulation sanguine dans le plasma et les dérivés du plasma. Une quantité d'échantillon de plasma ou de dérivé de plasma à examiner est incubée avec un complexe activé de prothrombine et de vésicules phospholipidiques procoagulatrices, ces dernières renfermant un facteur de tissu intégré, et la formation de thrombine est démarrée par addition d'une quantité appropriée d'ions Ca. Après une durée d'incubation déterminée, la formation de thrombine est achevée et la quantité de thrombine formée est déterminée par des procédés connus.
EP98913654A 1997-04-09 1998-03-06 Procede pour la determination des facteurs actives de la coagulation sanguine dans le plasma et les derives du plasma Ceased EP0975975A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19714599 1997-04-09
DE19714599 1997-04-09
PCT/EP1998/001264 WO1998045713A1 (fr) 1997-04-09 1998-03-06 Procede pour la determination des facteurs actives de la coagulation sanguine dans le plasma et les derives du plasma

Publications (1)

Publication Number Publication Date
EP0975975A1 true EP0975975A1 (fr) 2000-02-02

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP98913654A Ceased EP0975975A1 (fr) 1997-04-09 1998-03-06 Procede pour la determination des facteurs actives de la coagulation sanguine dans le plasma et les derives du plasma

Country Status (5)

Country Link
US (1) US6124110A (fr)
EP (1) EP0975975A1 (fr)
CA (1) CA2286164A1 (fr)
NO (1) NO994736L (fr)
WO (1) WO1998045713A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6743596B1 (en) * 2000-10-27 2004-06-01 Biomerieux, Inc. Method of determining global coagulability hemostatic potential
US7247488B2 (en) * 2003-05-06 2007-07-24 Medtronic, Inc. Method and kit for testing a multi-channel blood assay cartridge
US20050221414A1 (en) * 2004-03-31 2005-10-06 Katalin Varadi Kit for measuring the thrombin generation in a sample of a patient's blood or plasma
AT501650A1 (de) * 2005-02-22 2006-10-15 Technoclone Ges M B H Verfahren zur bestimmung der gerinnungsaktivierung sowie gerät zur durchführung des verfahrens
US8071545B2 (en) * 2005-03-15 2011-12-06 Lewis S. Coleman, Md, Inc. Therapies and compositions for controlling the stress mechanism and for stabilizing hemostasis in an organism
SE1050124A1 (sv) * 2010-02-08 2011-08-09 Linkoping Biocontrols Ab Stabil lösning
WO2011148207A1 (fr) 2010-05-28 2011-12-01 Diagon Kft. Procédé pour la préparation biphasique de liposomes et son utilisation dans la fabrication de réactifs de diagnostic
CN107356768A (zh) * 2017-06-23 2017-11-17 宁波艾科生物科技有限公司 一种液体即用型凝血酶原时间检测试剂

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3516579A1 (de) * 1984-11-19 1986-05-22 Boehringer Mannheim Gmbh, 6800 Mannheim Gerinnungstest auf teststreifen
US5472850A (en) * 1991-04-10 1995-12-05 Oklahoma Medical Research Foundation Quantitative clotting assay for activated factor VII
ATE267250T1 (de) * 1995-03-03 2004-06-15 Eisai Co Ltd Prothrombin aktivierendes enzym, das kalzium benötigt
US5677162A (en) * 1995-05-01 1997-10-14 New York Blood Center, Inc. Method for activating prothrombin to thrombin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9845713A1 *

Also Published As

Publication number Publication date
NO994736D0 (no) 1999-09-29
WO1998045713A1 (fr) 1998-10-15
US6124110A (en) 2000-09-26
NO994736L (no) 1999-09-29
CA2286164A1 (fr) 1998-10-15

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