DE4031731A1 - PROTEIN FROM HIRUDO MEDICINALIS - Google Patents

Abstract

Described is a new protein, suitable for use in the treatment of illnesses, produced from Hirudo medicinalis. The protein extends the clotting time of blood, is relatively stable to heat and has a molecular weight of 25-34 KDa. Also described are the muteins of the new protein.

Description

The present invention relates to a new protein from the leech Hirudo medicinalis, its muteins as well as their production and use.

From the secretions of different leeches a number are already medical interesting factors have been described, which among other things in the Blood coagulation and fibrinolysis play a role. To this Factors include fibrinolytics that break down fibrin or fibrinogen directly, as well as plasminogen activators. To the fibrin or fibrinogen degrading Substrates include hementin (from Hementeria ghilianii) and destabilase (from Hirudo medicinalis; I.P. Baskova et al. Folia Hematol., Leipzig, 115, 1988, p. 166). A plasminogen activator was in secretions described by Hementeria depressa.

Inhibitors of blood coagulation, e.g. B. the thrombin inhibitor hirudin (from Hirudo medicinalis), a factor X _a inhibitor (from Hirudo medicinalis), trypsin and plasmin or chymotrypsin and elastase inhibitors such as. B. the Bdellin or Egline (from Hirudo medicinalis) is described (Leech Biology and Behavior, Vol. 11, Oxford, Science Publication / RT Sawyer, 1986.

A new factor has now been isolated from Hirudo medicinalis.

The invention relates to a new protein from Hirudo medicinalis, the the clotting time of the blood is prolonged, relatively heat-stable and a Has molecular weight of 25 to 34 KDa, as well as its muteins.

The new protein significantly increases the intrinsic clotting time (APTT) and the extrinsic clotting time (PT) of the blood. It differs in its anticoagulant properties from hirudin and the factor X _a inhibitor. In fractions in which the APTT prolonging activity is present, neither corresponding amounts of hirudin (by ELISA) nor factor X _a inhibitor activity (by color substrate) can be detected.

The muteins of the new protein are to be understood as those proteins which differ from the new protein by exchange, deletion and / or addition derived from amino acids or peptides without changing their properties differentiate strongly from the new protein.  

The new protein can be obtained from leeches (Hirudo medicinalis). For this, the leeches are placed in water. After stimulation with arginine the leeches release the factor into the aqueous solution from which the new one Protein in a known manner by ion exchange, affinity, chelation and Gel permeation chromatography or by hydrophobic chromatography is isolated.

You can also puncture the leeches after arginine stimulation and the new one Isolate protein from the punctate as described above.

It is also possible to get the new protein from the homogenate of heads decapitated leeches in an analogous manner.

To make the protein available in larger quantities for therapeutic purposes known genetic engineering methods (cf. Maniatis, T. et al; Molecular cloning; A Laboratory Manual, Cold Spring Harbor Press, N.Y., 1989) apply. For this, the protein is first produced in pure form. The protein is then cleaved and the resultant is obtained Peptides separated by reversed phase HPLC (rHPLC). From some of these The amino acid sequence is determined for peptides. With oligonucleotides coming from The determined sequences are then derived in a c-DNA bank through sequence-specific filter hybridization for the new protein coding gene identified. The genetic information thus obtained can one then in different host cells according to known methods of expression bring. On the one hand, the expression systems include prokaryotes like E. coli or Bacillus subtilis, but also eukaryotes like yeast and Mammalian cells (e.g. C127 mouse fibroblasts, hamster, insect cells).

The new protein and its muteins are suitable for the treatment of diseases, especially for the treatment and prophylaxis of thrombotic Conditions.

example a) Extraction of leech extract

Extracts from Hirudo medicinalis were made according to the following procedure receive:

1. leeches were treated with a 10 mM arginine solution for 75 min. In this The solution took a yellowish time due to the leech's secretion activity Color.  

After exchanging the liquid for water, the leeches secreted continue.

2. leeches that had been treated with arginine according to 1. were dotted and the stomach contents won.

b) Purification of the new protein

The liquids obtained according to a) were individually or together Heated to 90 ° C for 10 min and then immediately cooled on ice. After centrifugation (20 min, 10,000 rpm, Sorvall® SA 600 rotor) the supernatant was first diluted 1: 2 to 1: 5 with PBS. The pH was set to 7.4. After that, the solution was put on one with PBS Equilibrated Q-Sepharose® column (10 ml, Pharmacia, Sweden) applied. After washing away unbound material, the column became with a linear gradient from 0 to 100% buffer B (PBS + 1 M NaCl) eluted. Aliquots of the fractions were extended to APTT tested. The new factor eluted between these conditions 0.3 M and 0.5 M NaCl.

Fractions containing activity were pooled 1: 2 at 20 mM Phosphate buffer, 250 mM NaCl pH 7.0 diluted and placed on a Cu chelate Column (1 ml) applied. After rinsing with 10 to 15 column volumes Equilibration buffer (20 mM Na phosphate, 250 mM NaCl pH 7.0) was carried out elution by applying a linear gradient from 0 to 100% Buffer B (20 mM succinic acid, 1 M NaCl pH 4.0). The pillar was then either with 20 mM phosphate, 2 M NaCl, 10 mM EDTA, pH 7 or post-eluted with 20 mM succinic acid 2 M NaCl, 100 mM histidine pH 4. Aliquots of individual fractions were extended to APTT tested, activity-containing fractions pooled and over Centricon® 10 (Amicon, Order No. 4206) concentrated on PBS buffered and on an FPLC gel filtration column (Superose® 12, Pharmacia) at a flow rate of 0.5 ml / min cleaned up.

The fractions containing activity were pooled and made up to one Mono Q Sepharose® equilibrated with PBS (Pharmacia, Sweden) applied. From the Mono Q column, the elution was carried out either with a pH gradient (0 to 100% B, buffer B = 20 mM succinic acid 1 M NaCl pH 4) or with a salt gradient (0 to 100% B; Buffer B = PBS + 1 M NaCl). Further purification of the new protein was then carried out via rHPLC or ion exchange and affinity chromatography. Fractions containing activity were pooled and opened SDS gels analyzed.  

c) Characterization of the new protein 1. Temperature stability

An aliquot of the new factor was heated to 95 ° C for 10 minutes and then immediately cooled on ice. Activity was measured by measuring inhibition the APTT determines. Due to the heat treatment, the activity decreased by about 10%.

2. Solvent stability

An aliquot of the activity-containing samples was taken at room temperature with 50% acetonitrile in 20 mM ammonium acetate pH 6.0 and Incubated for 30 min. After removing the solvent, the Influence of the samples on the APTT analyzed. It turned out that the new factor in its activity is not affected by acetonitrile becomes.

3. Protein nature

An aliquot of the new factor was mixed with 10 µg trypsin for 16 h at 37 ° C incubated. The reaction was started by boiling for 5 min and immediately Cooling stopped with ice and then the influence of the sample on the Inhibition of APTT determined. Likewise, with a control sample procedures that have not been treated with trypsin but otherwise the same was. The results show that after incubation with trypsin the Activity of the factor disappears, which proves its protein nature.

4. Molecular weight

a) The molecular weight of the new protein was determined using membranes defined pore size. To this end, fractions that contained new protein with buffer (PBS pH 7.2 or 20 mM succinic acid pH 4) diluted in a Centricon® micro concentration (10 or 30 kDa exclusion limit) and centrifuged at 5000 × g for 60 min. The runs of the membranes were in a Speed Vac concentrator evaporated to dryness, resuspended in PBS and just like that Retentate of the membranes measured in the APTT.

At both pH values, the new protein was found in Centricon® 10, but not concentrate with Centricon® 30. That means that new protein must have a molecular weight between 10 and 30 kDA.  

b) The molecular weight was further measured on a calibrated gel filtration column (Superose® 12). PBS was used as the eluent at a flow of 0.3 ml / min. There were fractions of 150 ul fractionated and tested for APTT extension. The maximum activity was in a range from 25 kDa to 34 kDa under these conditions certainly.

5. Point of attack of the new protein in the coagulation cascade

The new factor has been tested in various assay systems determine how and what factors in the coagulation cascade he inhibited.

a) APTT (= activated partial thromboplastin time) for the detection of a anticoagulant activity in the intrinsic coagulation cascade

Samples to be tested were diluted in 50 μl human plasma (eg Preciclot®, Boehringer / Ma, no. 654 370). For this purpose, 50 μl of PTT _a reagent (Boehringer / Ma, No. 886 050) were pipetted in and incubated at 37 ° C. for 5 min. The coagulation cascade was started by adding 50 μl of 25 mM CaCl₂. The time until the occurrence of plasma clots was measured.

The samples showed a significant delay in plasma clotting time (intrinsically).

b) PT (= prothrombin time, quick test) for the detection of an anticoagulant Effect on the extrinsic coagulation cascade

Samples were taken in 100 µl human plasma (e.g. Preciclot®, Boehringer / Ma, No. 654 370). For this purpose, 100 ul thromboplastin (Diluted 1: 1 with NaCl 0.9%). With 50 µl 25 mM CaCl₂ the coagulation cascade was started. The time was measured until the appearance of plasma clots. The samples showed one significant delay in plasma clotting time (extrinsic).

c) FX _a inhibition test to differentiate FX _a inhibitor

Samples (diluted in PBS) were incubated with factor X _a (FX _a ) and a specific chromogenic substrate for FX _{a for} 60 min at room temperature:

25 µl sample
50 µl FX _a (Boehringer / Ma, No. 602 388, 0.025 U / ml)
100 µl S-2222 color substrate (Kabi-Vitrum, 16.9 ml H₂O / vial)

After 5 minutes, the optical density was measured at 405 mm for the first time. After 60 min the reaction was stopped with 50 μl glacial acetic acid and the optical density measured again at 405 mm.

Batches without FX _a and samples without inhibitor serve as controls. A Δ OD _405.40 ′ was calculated for the evaluation using the following formula:

Δ OD = [OD _60min -OD _5min ] _{+ FXa} - [OD _60min -OD _5min ] _-FXa

(Difference in absorbance change with and without FX _a ).

Appropriate approaches with and without inhibitor determine the degree of inhibition of FX _a (in%) by

d) thrombin time

A sample of the new protein was diluted in 100 ul plasma. By Addition of 50 ul thrombin reagent (Boehringer / Ma, No. 126 594) coagulation started. The time until plasma clots occur was determined turbidometrically.

The evaluation was carried out using a calibration curve with known thrombin Inhibitors (e.g. hirudin). The results obtained show that the new protein is a thrombin inhibitor.

6. Attempts to differentiate the new protein from hirudin a) Fractions of the new protein corresponding to an APTT extension 2500 ATU showed were introduced into a hirudin ELISA

Material:

• - Microtiter plates (Flow 77-173-05)
• - streptavidin-peroxidase complex (Boehringer / Ma 1089153)  
• - tetramethylbenzidine (Serva 35926)
• Peroxidase substrate: 0.1 ml TMB solution (42 mM TBM in dMSO) and Mix 10 ml substrate buffer (0.1 M Na acetate pH 4.9) slowly; then add 14.7 ml of 3% H₂O₂

Action:

• - Coating microtiter plates with 0.2 ml / well of hirudin (1 µg / ml, in 0.1 M NaHCO₃, pH 8.3)
• - Saturate with 1% BSA / PBS; 0.3 ml / well
• - Wash 3 times with 0.05% Tween 20 / PBS
• - 12 standard dilutions of hirudin, starting with 500 mg / ml 0.1% BSA / PBS in steps of two, each 0.1 ml / well; then addition of 0.1 ml / well poly-anti-hirudin (1 µg / ml 0.1% BSA / PBS) and Competition overnight / 4 ° C
• - Wash like above
• - 0.2 ml / well biotinylated anti-rabbit IgG antibody (1: 5000) 2 h at room temperature
• - Wash like above
• - 0.2 ml / well streptavidin-peroxidase complex (1: 10,000); 30 min at Room temperature
• - Wash like above
• - 0.1 ml / well peroxidase substrate
• - Stop reaction with 0.1 ml / well 2 M H₂SO₄
• - Measurement of the absorption at 450 nm

Evaluation:

The OD values obtained were against the logarithm of the standard dilutions applied and resulted in the range of about 60 to 2 ng / l a linear dependency.

Samples with unknown hirudin concentration were placed in parallel Calibration line in different dilutions in the test. In the An amount of hirudin could be detected in the measured samples corresponds to an activity of maximum 5 ATU / ml.

b) Thrombin time (= TZ)

When analyzing the new protein, measurement of TZ revealed an antithrombin Activity of 200 ATU / ml. This was a factor of 40 above the value determined for hirudin according to ELISA. That proves that new protein is not hirudin.  

c) Concentration

Attempts were made to use hirudin using Centricon® membranes at pH 4 and 7 focus. Hirudin was through the Centricon® 10 membrane not held back. The new protein will be under these conditions retained by the membrane (cf. 4a).

Claims (1)

Protein from hirudin medicinalis, which increases the clotting time of the blood, is relatively heat stable and has a molecular weight of 25 to 34 KDa owns, as well as its muteins.