WO1994009039A1

WO1994009039A1 - New protein that lengthens blood coagulation time, derived from the land leech haemadipsa sylvestris

Info

Publication number: WO1994009039A1
Application number: PCT/EP1993/002793
Authority: WO
Inventors: Karl-Hermann Strube; Thomas Meyer; Siegfried Bialojan; Burkhard Kroeger
Original assignee: Basf Aktiengesellschaft
Priority date: 1992-10-16
Filing date: 1993-10-12
Publication date: 1994-04-28
Also published as: DE4234921A1

Abstract

Described is a new anticoagulant protein from land leeches of the genus Haemadipsa, having the N-terminal amino acid sequence: A-Val-Lys-Phe-Cys-Gly-His-Pro-Leu-Ser-Leu-Pro-Thr-Ala-Leu-Cys-Ala, where A represents an indefinite amino acid. This protein can be used in combatting diseases.

Description

New protein from the land blood gel Haemadipsa sylvestris that extends the clotting time of the blood

description

The present invention relates to a new protein from the land blood gel Haemadipsa sylvestris, which increases the clotting time of the blood, and a method for its production.

Anticoagulants are important therapeutic substances that are used, for example, for the prophylaxis or treatment of thromboses or arterial reocclusions. Coagulation can be inhibited by various mechanisms. There are substances that inhibit thrombin, for example hirudin, or substances that inhibit factor Xa, for example TAP (Waxman et al., Science 248, 593-596, 1990) or antistasin (Tuszynski et al., J. Biol. Chem., 262, 9718-9 * 723, 1989).

A new anticoagulant protein has now been isolated from the land blood gel Haemadipsa sylvestris.

The new protein (PT factor) has the following physicochemical properties.

In the Tris / Tricine-SDS polyacrylamide gel, the protein shows two bands, which are assigned a molecular mass of 8200 ± 2000 Da and 18600 ± 2000 Da (FIG. 2).

In the APTT test (Example 8a) and in the PT test (Example 8b), the protein in each case shows significant increases in the plasma coagulation time. Furthermore, it shows a specific factor Xa inhibition in a factor Xa inhibition test (example 8d). In an analog factor VII-He test (FIG. 3b) an inhibitory effect is also found; however, this can also occur indirectly through the inhibition of factor Xa.

The proteins determined the following N-terminal amino acid sequence:

A-Val-Lys-Phe-Cys-Gly-His-Pro-Leu-Ser-Leu-Pro-Thr-Ala-Leu-Cys-Ala, in which A represents an amino acid which cannot be clearly determined.

The new proteins can be obtained from the country's blood leeches

Isolate Haemadipsa. For this purpose, the leeches are expediently taken up in a buffer at pH 6 to 9, preferably pH 7 to 8 and homogenized with a homogenizer, preferably a mixer. The insoluble constituents are then separated off, preferably centrifuged off.

The proteins can be further purified from the solution obtained in this way by chromatographic methods, preferably ion exchange chromatography and / or reversed phase HPLC.

The purification of the proteins can be followed using the activity tests described in Example 8.

Genetic engineering processes are particularly suitable for producing the proteins according to the invention.

For this purpose, a cDNA library is created from the leech in a manner known per se. The gene coding for the protein according to the invention can be isolated from this gene bank by, for example, producing a DNA sample whose sequence is obtained from the N-terminal amino acid sequence described above by back-translation according to the genetic code. The corresponding gene can be found and isolated by hybridization with this DNA sample.

The polymerase chain reaction (PCR) technique can also be used to produce the corresponding gene.

For example, with the aid of a primer, the sequence of which was obtained by back-translation from the N-terminal amino acid sequence described above, and a second primer, the sequence of which is complementary to the 3 'end of the cDNA gene fragment, preferably with the sequence poly (dT) , produce the cDNA gene fragment for the protein according to the invention by PCR technique. The corresponding gene can also be isolated by creating an expression gene bank of leeches and screening them with an antibody which is directed against the protein according to the invention.

Further suitable DNA sequences are those which have a different nucleotide sequence but which, owing to the degeneration of the genetic code, code for the polypeptide chain according to the invention or parts thereof. DNA sequences which code for anticoagulant proteins and which hybridize with the above-mentioned nucleotide sequences under standard conditions are also suitable. The experimental conditions for DNA hybridization are described in textbooks in genetic engineering, for example in J. Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989. Standard conditions are understood to mean, for example, temperatures between 42 and 58 ° C. in an aqueous buffer solution with a concentration between 0.1 and 1 × SSC (1 × SSC: 0.15M NaCl, 15 mM sodium citrate pH 7.2).

After the corresponding gene has been isolated, it can be genetically engineered in organisms, e.g. in bacteria, yeasts or higher eukaryotic cells can be expressed in a manner known per se with the aid of an expression vector.

The protein can be isolated from these recombinant host systems on the basis of the physicochemical properties described above.

The general procedure for the genetic engineering of a new protein with a known amino acid partial sequence is described in textbooks in genetic engineering, e.g. E.L. Winnacker, Gene and Clones, Verlag Chemie, Weinheim, 1984. The experimental conditions for the individual methods, such as for example the creation of a gene bank, hybridization, expression of a gene, are described in J. Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989.

The protein according to the invention is preferably used in the form of its pharmaceutically acceptable salts.

The new protein has anticoagulant properties. It can be used, for example, for the prophylaxis of thromboses or arterial reocclusions, for the treatment of thromboses, for the preservation of blood or for extracorporeal circulation.

The new protein is an effective anticoagulant. It can be used alone or together with known coagulation factors, for example thrombin inhibitors such as hirudin, as a medicament.

The invention is illustrated further by the following examples. example 1

Cleaning the PT factor from landing gels

Extraction of leech homogenates

150 g of live landing gel (Haemadipsa sylvestris) were homogenized in 400 ml of 20 mM sodium phosphate buffer (pH 7.4) with a mixer at 4 ° C. for 10 minutes. The homogenate was centrifuged for 15 minutes at 2000 rpm (Sorvall RC-5B, rotor GS-3) and then after removing the precipitate for 30 minutes at 8000 rpm. The precipitate was discarded and the supernatant was diluted to a volume of about 600 ml with 50 mM tris (hydroxymethyl) amino methane / HCl buffer pH 8.5 (Tris / HCl buffer).

The protein solution had a volume of 580 ml, the protein concentration was 4.49 mg / ml and the thrombin-inhibiting activity was 22.2 U / ml.

The coagulation time (APTT, see Example 8) of the blood was prolonged by about 200 sec in a 1:10 dilution and the partial thromboplastin time (PT) was prolonged by about 60 sec in a 1:10 dilution.

Alternatively, the new protein can be purified from homogenates of leech heads. For this purpose the leeches were first decapitated and the head preparations obtained in this way were homogenized in an analogous manner. Purification from leech heads has the advantage that the homogenate contains a smaller amount of protein with approximately the same amount of activity. The new protein is already here in a higher purity. Furthermore, a significantly reduced proteolytic activity was determined in the head homogenate. The disadvantage of isolation from leech heads is the limited amount of substance.

Example 2

Separation of landing gel homogenates by ion exchange chromatography

The protein solution obtained from the Egelhomogenaten was (50-100 ml, 2.5 cm diameter) on a 8.5 equilibrated with 50 mM Tris / HCl buffer pH Q-Sepharose ^® column applied. After washing away unbound material with the equilibration buffer, the bound proteins were washed with a

Gradients from 0-1 M NaCl in 20 mM Tris / HCl pH 8.5 eluted. Fractions of approx. 7 ml were collected and checked for protein content, thrombin inhibitory activity, APTT and PT prolonging activity examined. Fractions in which no or low antithrombin activity was measured, but high APTT and PT prolonging activity were measured (Fig. 1).

The new protein elutes from the column at a salt concentration of 300 to 500 mM NaCl.

Example 3

Purification of the PT factor by ion exchange chromatography on S-Sepharose FF

The value fraction of the Q-Sepharose chromatography was concentrated using a 3000 D membrane (Filtron Omega Alpha Cat. No. AM003062) and buffered to 20 mM succinic acid buffer pH 4.0 or simply with the succinic acid buffer pH 4.0 diluted and the pH adjusted if necessary. This fraction was applied to an S-Sepharose FF column (diameter 1.5-2.5 cm, volume 10-30 ml) equilibrated with 20 mM succinic acid at a flow of 40-80 ml / h. After washing away unbound material with equilibration buffer, the bound proteins were eluted from the column using a linear gradient from 0 to 1 M NaCl in 20 mM succinic acid pH 4.0. The elution was monitored at 280 nm in the UV and

Fractions of 3-7 ml volume collected. The fractions obtained were examined for PT-prolonging activity and the correspondingly active fractions were combined. The new factor eluted between 400 and 700 mM NaCl from the column under these conditions. To prepare for further purification of the PT factor, the value fraction was first concentrated over a 3000 D membrane (Filtron Omega Alpha Cat. No. AM003062) and finally concentrated to dryness in a vacuum concentrator.

Example 4

Purification of PT-extending activity by reversed phase (r) HPLC '

The value fraction of S-Sepharose chromatography was dissolved in the smallest possible volume of 0.1% by weight trifluoroacetic acid and on a reversed phase (r) HPLC column (BioRad rp304) after 5 minutes of rinsing with 0.1% by weight. Trifluoroacetic acid using a linear gradient (0.1 wt .-% TFA in water / 0.1 wt .-% TFA in acetonitrile in 65 min to 0.1 wt .-% TFA in 50% acetonitrile) . The one from the HPLC column Eluting proteins were detected by UV detection at 210 nm and fractionated by hand. The PT-prolonging activity contained in the individual fractions was determined after removing the solvent and resuspending in water. The PT factor eluted between 20 and 28% acetonitrile from the column under these conditions.

Example 5

Amino terminal sequence of the PT factor

The amino-terminal sequence of the PT factor was determined using a peptide sequencer (Applied Biosystems, Model 477A). The following amino acid sequence 1 was determined in the eluates of the rp304 (r) HPLC column:

NH ₂ -XVKFVGHPLSLPTALXA ..., where X represents an amino acid which cannot be clearly determined. After reduction and reaction with vinyl pyridine (Huang et al., Biochemistry 1989, Vol. 28, 661 - 666), sequence 2 of this fraction could be determined:

NH ₂ -XVKFCGHPLSLPTALCA ..., where X represents an amino acid that cannot be clearly identified, possibly an aspartic acid residue. The valine identified in position 5 of sequence 1, like the amino acid which cannot be determined in position 16 of sequence 1, was clearly identified as pyridylethylated cysteine.

Example 6

Peptide mapping of the isolated PT-extending protein

To determine further amino acid partial sequences, the PT-extending fractions from Example 4 were subjected to reduction and pyridyl ethylation (Huang et al., Biochemistry, 1989, Vol. 28, 661-666). The reaction mixture was then rechromatographed on a C-18 (r) HPLC column (Nucleosil; Macherey and Nagel). After removal of the solvent, the reduced and vinyl pyridylated PT factor was subjected to cleavage by the protease trypsin (Boehringer Sequence Grade). The protein / protease ratio was 20 - 40 to 1.

The protease incubation was carried out for 16 hours at room temperature according to the manufacturer's instructions. The peptide fragments obtained were separated by (r) HPLC on a C-18 column. For this purpose, after 5 min with 0.1 wt.% TFA in A linear gradient of 0.1 wt% TFA in water in 120 min to 60% 0.1 wt% TFA in acetonitrile at a flow of 0.3 ml / min was used. The peptides detected at 210 nm were collected separately and analyzed after evaporation of the solvent in the gas phase sequencer (Applied Biosystems Model 477A).

The sequences of the individual fractions are shown in the table:

Example 7

Determination of the molecular mass by Tris / Tricine-SDS-polyacrylamide gel electrophoresis

Electrophoresis was carried out as described by Schägger and Jagow (Analytical Biochemistry, 166, 368-379 (1987)). An aliquot (20 μg) was separated on a 16% Tris / Tricine gel (BAI GmbH, Bensheim). Fig. 3 shows the result after staining the gel with Coomassie Brillant Blau (A) or Silber (B). The new protein (A: lane 2; B: lane 2) shows two bands with molecular masses of 8200 ± 1000 and 18600 ± 2000 Da.

Example 8

Target of the new protein in the coagulation cascade

The new factor was tested in various assay systems to determine how and which factors in the coagulation cascade are inhibited by it.

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

Samples to be tested ul human plasma (eg Preciclot ^®, Boehringer / Ma, no. 654370) was diluted in 50. For this purpose, 50 μl of PTT _a reagent (Boehringer / Mannheim, 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 CaCl ₂ . The time until the occurrence of plasma clots was measured. b) PT (= prothroid time, quick test) for the detection of an anticoagulant effect on the extrinsic coagulation cascade.

Samples were diluted in 100 μl human plasma (e.g. Preciclot®, Boehringer / Mannheim, No. 654 370). 100 μl thromboplastin (1: 1 diluted with NaCl 0.9%) were pipetted into this. The coagulation cascade was started with 50 μl of 25 M CaCl. The time until the occurrence of plasma clots was measured. The samples showed a significant increase in the plasma coagulation time (extrinsic).

Alternatively, the formation of individual extrinsic coagulation factors, e.g. Factor VIII or factor X, by measuring the conversion of chromogenic substrates.

For this purpose, the samples were diluted as described above, with the modification that the corresponding chromogenic substrate (25 μl) was pipetted in.

The extinction at 405 nm was then determined at different times. The extent of the inhibition can be determined from the difference between two experiments with and without inhibitor and the point of attack of the inhibitor in the coagulation cascade can be identified. S 2222 (for factor Xa) and CH ₃ S0 ₂ -D-CHA-But-Arg-pNA-AcOH (Pentapharm, Switzerland) (for factor Vlla) were used as chromogenic substrates.

As shown in FIG. 3, the new protein very effectively inhibits both factor Xa and the formation of factor VIIa.

c) Determination of the inhibition of thrombin

Thrombin (Boehringer / Mannheim) became a final concentration of (25 mU / ml) in phosphate buffered saline (PBS) (0.8 g / 1 NaCl; 0.2 g / 1 HCl; 0.144 g / 1 sodium phosphate; 0 , 2 g / 1 potassium phosphate, pH 7.5).

Chromozym TH (Boehringer / Mannheim) was dissolved in 20 ml H ₂ 0 / bottle.

50 μl of chromozyme as well as 25 μl of sample or buffer were added to the wells of a microtiter plate. Immediately afterwards, the absorption at 405 nm was measured at time 0 and after 30 min at 37 ° C. If the sample had a strong intrinsic color, a further control without thrombin was treated as above.

The activity of the thrombin releases a dye that absorbs at 405 nm from the chromogenic substrate. The inhibition of thrombin by a thrombin inhibitor can be recognized by a smaller increase in absorption at 405 nm and was quantified using a calibration curve.

FXa inhibition test

Samples (diluted in PBS) were incubated with factor Xa (FXa) and a specific chromogenic substrate for FXa for 60 min at room temperature:

25 ul sample

50 μl FXa (Boehringer / Mannheim, No. 602 388, 0.025 U / ml) 100 μl S-2222 color substrate (Kabi-Vitrum, 16.9 ml H ₂ 0 / vial) After 5 min the optical density at 405 nm measured for the first time. After 60 min, the reaction was stopped with 50 μl of glacial acetic acid and the optical density at 405 nm was measured again.

Batches without FXa and samples without inhibitor serve as controls. A Δ OD ₄ o ₅ , o- was calculated for the evaluation using the following formula:

Δ OD = [OD-OD ₆ 0min _5m i _n] + FXa "[OD o ₆ _m in OD _5m in] -FXA

(Difference in absorbance change with and without FXa) _

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

Δ OD _{+ In} hibitor% inhibition = x 100

Δ OD inhibitor

Legend to the figures

FIG. 1 Chromatography of leech homogenates on a Q-Sepharose column. After application, unbound material was removed by rinsing with the equilibration buffer (50 mM Tris-HCl, pH = 8.5). The bound protein was eluted by applying a salt gradient of 0-1 M NaCl. Individual fractions were examined for thrombin inhibition, extension of APTT and PT. Fractions in which APTT and PT were extended, but which showed little or no thrombin inhibition, were pooled.

FIG. 2 Tris / Tricine - SDS polyacrylamide gel electrophoresis

The molar mass of the value fraction of the (r) HPLC separation was determined on a 16% Tris / Tricine gel. (A): Coloring with Coomassie brilliant blue; (B): Silver color.

Lanes 1.3: Molar mass calibration substance, intact myoglobin 17.2 kDa, cyanogen bromide peptide myoglobin I + III 14.6 kDa, cyanogen bromide cyanide 8.2 kDa, cyanogen bromide cyanide 6.4 kDa, cyanogen bromide cyanide 2.6 kDa,

Myoglobin 1-14

Lanes 2.4: purified PT factor (20 μg) after (r) HPLC separation

FIG. 3: Measurement of the influence of PT factor on extrinsic coagulation.

a) Measurement of S 2222 turnover (factor Xa)

b) Measurement of the CH ₃ S0 -D-CHA-But-Arg-pNA-AcOH conversion (factor VIIa)

Claims

1. ^" Protein from land blood gels of the genus Haemadipsa with the N-terminal amino acid sequence:

2. DNA sequence coding for a protein according to claim 1.

3. DNA sequence which codes for an anticoagulant protein and hybridizes under standard conditions with the DNA sequence according to claim 2.

4. Use of a protein according to claim 1 for combating diseases.

5. Medicament containing a protein according to claim 1 and a further anticoagulant substance.

Sign.