EP0975975A1

EP0975975A1 - Method for determining activated coagulation factors in plasma and plasma derivatives

Info

Publication number: EP0975975A1
Application number: EP98913654A
Authority: EP
Inventors: Günter WÖBER; Beate Gesien
Original assignee: Blutspendedienst Der Drk-Landesverbande Nordrhein und Westfalen-Lippe GmbH
Current assignee: Blutspendedienst Der Drk-Landesverbande Nordrhein und Westfalen-Lippe GmbH
Priority date: 1997-04-09
Filing date: 1998-03-06
Publication date: 2000-02-02
Also published as: NO994736D0; WO1998045713A1; US6124110A; NO994736L; CA2286164A1

Abstract

The invention relates to a method for determining activated coagulation factors in plasma or plasma derivatives. A sample of the plasma or plasma derivative to be examined is incubated with an activated prothrombin complex and procoagulative phospholipid vesicles, the latter containing integrated tissue factor, and thrombin formation is initiated by addition of an appropriate amount of Ca ions. After a determined incubation time, thrombin formation is terminated and the amount of thrombin formed is determined by known methods.

Description

They are used to determine activated blood coagulation factors in plasma and plasma derivatives

The invention relates to a method for determining activated blood coagulation factors in plasma and plasma derivatives.

When plasma is stored at low temperatures or during the course of cleaning processes, activated blood coagulation factors can occur in plasma or plasma derivatives. Blood coagulation factors are normally enzymatically inactive proenzymes, which are converted into their biologically active form by limited proteolytic cleavage. For example, at the end of the coagulation cascade from prothrombin (factor II) thrombin (activated factor II = factor Ha) is formed, which cleaves fibrinogen. Thrombin also activates factor XIII, which crosslinks fibrin into a stable clot. This physiological activation is desirable in most cases.

On the other hand, an artificial activation of blood coagulation factors in stored plasma or in the production of a purified prothrombin complex from plasma containing factors II, VII, IX, X as therapeutically active components is undesirable. In the treatment of patients with umfas ^¬ Send coagulation disorders with prothrombin complex concentrates, eg by blood loss during major surgery, thrombo-hemorrhagic complications may occur as an undesirable side effect whose cause is unknown. What is certain is that the risk of such complications from tissue thromboplasia in that after severe surgery 1 pathologically high concentration circulates in the patient's circulation, is increased. It is also suspected that activated factors IX or X in prothrombin complex concentrates may contribute to an increased risk of thrombosis.

5

Laboratory tests are therefore carried out on the plasmas and plasma derivatives mentioned in order to demonstrate the absence of activated factors. For plasma is a method for the selective measurement of factor VIIa in the presence of factor

I _Q VII known (U.S. Patent 5,472,850). The patent describes an assay for a factor VIIa which uses a truncated (shortened) tissue factor tTF. The tissue factor is an integral membrane protein that binds both factor VII and factor VIII with high affinity

15 and thereby triggers the coagulation cascade. The complex of factor VIIa and tissue factor also catalyzes the activation of factor VII to factor VIIa. In the patent 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

25 of such a soluble mutant tissue factor in a coagulation test selectively determine factor VIII in the presence of factor VII in plasma.

However, the test is relatively complicated to do and

30 also brings only one result for the presence of the factor Vlla.

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).

In this test, the clotting time of plasma is measured in the presence of the sample to be examined in the absence of substances, such as kaolin, which initiate coagulation in vitro. In the absence of activated factors, this clotting time is approximately 150 s; the clotting time is reduced in the presence of activated factors. However, this test is relatively insensitive and does not always show activated factors. A criticism of this test can be found in the article by C.V. PRO SE and A.E. WILLIAMS "A Comparison of the in vitro and in vivo thrombogenic activity ..."; Thromb. Haemost. 1980, pp. 81-86.

It is therefore the task of specifying a more precise and sensitive test for determining the presence of activated factors, which specifies a broad spectrum of activated factors.

This problem is solved by a process with the following process steps:

a) 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,

b) after a fixed incubation time of, for example, 5 min and 10 min, the thrombin formation is ended and the amount of thrombin formed is determined by known methods.

A test method is known (Y. SULTAN, F. LOYER, In Vitro Evaluation of Factor VHI-bypassing Activity ...; j. Lab.

Clin. Med. 1993, pp. 444 ff.), In which thrombin formation is induced in the presence of a platelet suspension activated with collagen. Platelets must be freshly prepared from whole blood by repeated centrifugation before each test batch, washed and adjusted to a defined number of cells. Such platelet suspensions can only be kept for a few hours and cannot be preserved. This means a costly activity that can only be performed by trained personnel and limits the use of these known tests to highly qualified and well-equipped laboratories.

The new test method, on the other hand, 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.

In addition defibrinated plasma, a test batch contains a small amount of a ⁿ e and activated prothrombin prokoagu- latorische 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. Sets one _d ^d agegen urch adding a small fixed amount at a fixed _k ti _¬ fourth prothrombin a "background noise" is amplified by this activated factors. It can be in _th e _¬ sem case always according to the same incubation times, such as after 5 and 10 min, measured. It should be _emphasized, that a particular _SS in the _¬ make the use of the minimal "activated" prothrombin binkomplexes in this test and the possibility of activated factors XII, XI, and to determine iχ VII in a test _d s special value of the invention.

It has proven to be beneficial if the content of. EaJctαr— Xa in activated Pmthrmnhin nmp] PV f-'T-wa τ_% of., G-≥ _≤ official factor X content with non-measurable content of factor Ha, since this is the best way to reproduce the "background noise".

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.

Exemplary embodiments of the invention are explained as follows:

example 1

1. Production of Defibrinated Plasma

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 μl 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.

2. Production of the partial prothrombin complex (PPK1

Required reagents: normal plasma DEAE-Sephadex A 50 elution solution. 30 g NaCl per liter dist. water

Wash buffer. 9 g Na 2HP0-1.2 H20 + 7 g NaCl per liter dist.

Water, pH adjusted to 7.0;

Equilibration buffer. 6 g Na 3-citrate. 2 H20 + 7 g NaCl per

Liters of dist. Do not change water, pH.

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.

After filtering again, DEAE-Sephadex is suspended in 1 ml elution buffer and 15 min. protected at room temperature telt.

After removal of DEAE-Sephadex, 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.

3. Production of the activated prothrombin complex f PK ^

A suitable dilution of the PPK according to point 2 must be determined in the thrombin formation test (see point 5). For this purpose, a dilution series is prepared with HEPES buffer (eg 1:20, 1:40 and 1:80; HEPES = N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid)]. Each dilution is 5 minutes at 37 ° C incubated with CaCl at a final concentration of 10 mM.

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.

4. Production of procoagulatory phospholipid vesicles with integrated tissue factor

Reagents required:

Dialysis tubing, cellulose membrane from Sigma (Art. No. D 9402) or Slide-A-Lyzer ™ dialysis cassette from Fa.

Pierce;

Beef brain acetone dry powder (Brain Acetone Powder,

Sigma, Art. No. B 0508); n-octyl-β-D-glucopyranoside (short octylglucoside) from Alexis (Art. No. 500-001-G005); 100 mg / ml dist. Water (10% w / v

Solution);

Phosphatidylserine (PS), (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.

0.216 g of 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. While changing the dialysis buffer daily, dialysis is carried out for 3 days, followed by a further 2 days against 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.

For each new preparation, 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.

5. Thrombin Formation Test

Reagents required:

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;

Starting solution 375 mM CaCl, stop solution 40 M EDTA;

APK and vesicle (point 4) with integrated tissue factor in a suitable dilution

5.1. 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

131 740) and incubated for 4 h at 30 ° C. The two chemicals are then removed in a known manner by hydrophobic chromatography (Bonomo, R.J., EP-PS 366 946). To prepare for the test for activated coagulation factors, a plasma sample is defibrinated as described in Example 1.

Pipetting scheme

- for the blank value: 250 μl HEPES / NaCl buffer + 250 μl defined plasma (point 1);

- for the APK: 125 μl HEPES / NaCl buffer + 250 μl defibrinated plasma + 125 μl APK;

- For the plasma sample: 125 μl HEPES / NaCl buffer + 125 μl defibrinated plasma + 125 μl APK + 125 μl defibrinated plasma sample of the plasma to be examined for activated factors.

50 μl citrate buffer, 140 μl HEPES / NaCl buffer and 10 μl phospholipid suspension, prepared according to point 4, are added to each batch and incubated for 5 min at 37 ° C. The reaction is started with 10 μl of CaCl 2 solution, after 5 min and 10 min 50 μl of the mixture is added to 50 μl of an initially introduced EDTA solution (EDTA = ethylenediaminetetraacetic acid) in order to stop the formation of thrombin.

6. Test for thrombin activity

The process is known per se and is supplied as a "kit". For the sake of completeness only

Reagents required:

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

500 mM Tris, 75 mM EDTA, 10% w / v PEG 6000, pH 8.4, is distilled with dist. Diluted water 1:10. 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.

20 μl of the sample quantities mentioned under point 5.1 "Pipetting scheme" are mixed with 500 μl substrate buffer, which is preheated to 37 "c. The extinction is measured in minute intervals. To check the linearity of the substrate cleavage, 5 extinction cycles of 1 min at 405 nm are used A calibration line is created using dilutions of thrombin with working buffer in the range from 5.3 to 0.53 nkat.

With virus-inactivated plasma samples, produced as in

Point 5, the following results were obtained:

1,2,3 ... different production batches of virus-inactivated plasma

B * APK 1 2 3 4

0.003 0.028 0.036 0.035 0.035 0.033

10

0.041 0.038 0.057 0.045 0.060 0.051

B * blank value The numbers given are absorbance changes per minute and were obtained after an incubation of 10 min in the thrombin formation test. The measuring point with 5 min incubation provided no or very little changes in extinction.

To evaluate 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 ™ (hexadimethrin bromide) before the test or preferably inactivated with heparinase.

Example 2 - Detection of activated factor IX. Factor XI and factor XII

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 ™ (4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride), Boehringer Mannheim , Art. No. 1 429 868, inhibited. The amount of Pefabloc SC ™ required to inhibit the thrombin activity presented was determined in a preliminary test.

Pipetting scheme:

- Blank value: 250 μl HEPES / NaCl buffer + 250 μl defined plasma;

- APK: 125 μl APK + 125 μl HEPES / NaCl buffer + 250 μl defibrinated plasma;

- Factor IXa, 10 μl of a solution with 0.2 plasma units or 10 μl factor XIa or 10 μl factor Xlla + 125 μl APK + 115 μl HEPES / NaCl buffer + 250 μl defibrinated plasma.

50 μl citrate buffer, 140 μl HEPES / NaCl buffer and 10 μl phospholipid suspension, prepared according to Example 1, point 4, are then added to each batch and incubated for 5 min at 37 ° C. The reaction is started with 10 μl CaCl 2 solution, after 5 min and 10 min 50 μl of the mixture is added to 50 μl of an EDTA solution, in order to thereby stop the formation of thrombin.

B * APK F IXa F XIa F Xlla incubation time in the thrombin + APK + ApK + ApK education test

0, 003 0, 006 0, 130 0, 034 0, 052 5 min.

0.022 0.039 0.112 0.083 0.088 10 min,

* Blank value

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.

Example 3 - Detection of activated factor VII

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:

B * APK F Vlla incubation time in the thrombin + ApK formation test

Vesicles without tissue factor 0.003 0.026 0.035 10 min,

Vesicle with tissue factor 0.015 0.039 0.061 10 min.

* Blank value

In both series of tests, 0.002 plasma units factor VIIa were tested. In a series of experiments with vesicles with integrated tissue factor, 0.001 plasma units factor VIII were still clearly detectable.

Claims

claims

1. Method for determining activated coagulation factors in plasma or plasma derivatives, with the following method steps:

10 a) a sample amount of plasma or plasma derivative to be examined is incubated with activated prothrombin complex and procoagulatory phospholipid vesicles, containing the latter integrated tissue factor, and thrombin formation is started by adding a suitable amount of Ca ions,

b) after a defined incubation time of, for example, 5 min and 10 min, the thrombin formation is stopped and

20 th ^e amount of thrombin formed by known methods determined.

2. The method according to claim 1, characterized in that the content of factor Xa in the activated

25 Prothrombin complex is about 1% of the total factor X content with an unmeasurable content of factor Ha.

3. The method according to claim 1 or 2, characterized in that the tissue factor is made of biogenic gg starting material.

4. The method according to claim 3, characterized in that the tissue factor is extracted from the bovine brain with a detergent-containing solution of dry acetone powder.

35

5. The method according to claim 1, characterized in that the tissue factor is integrated into procoagulatory phosp olipid vesicles.

6. The method according to claim 4 and 5, characterized in that the tissue factor is mixed in detergent-containing solution with phosphatidylserine and phosphatidylcholm and is integrated by dialysis in the procoagulant phospholipid vesicles.