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

New protein that lengthens blood coagulation time, derived from the land leech haemadipsa sylvestris Download PDF

Info

Publication number
WO1994009039A1
WO1994009039A1 PCT/EP1993/002793 EP9302793W WO9409039A1 WO 1994009039 A1 WO1994009039 A1 WO 1994009039A1 EP 9302793 W EP9302793 W EP 9302793W WO 9409039 A1 WO9409039 A1 WO 9409039A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
factor
land
leu
new protein
Prior art date
Application number
PCT/EP1993/002793
Other languages
German (de)
French (fr)
Inventor
Karl-Hermann Strube
Thomas Meyer
Siegfried Bialojan
Burkhard Kroeger
Original Assignee
Basf Aktiengesellschaft
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
Priority to DEP4234921.4 priority Critical
Priority to DE19924234921 priority patent/DE4234921A1/en
Application filed by Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Publication of WO1994009039A1 publication Critical patent/WO1994009039A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81Protease inhibitors
    • C07K14/815Protease inhibitors from leeches, e.g. hirudin, eglin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides

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:

Figure imgf000009_0001

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

Claims
1. " Protein from land blood gels of the genus Haemadipsa with the 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.
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.
PCT/EP1993/002793 1992-10-16 1993-10-12 New protein that lengthens blood coagulation time, derived from the land leech haemadipsa sylvestris WO1994009039A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
DEP4234921.4 1992-10-16
DE19924234921 DE4234921A1 (en) 1992-10-16 1992-10-16 New blood coagulation protein from Haemadipsa sylestris

Publications (1)

Publication Number Publication Date
WO1994009039A1 true WO1994009039A1 (en) 1994-04-28

Family

ID=6470630

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1993/002793 WO1994009039A1 (en) 1992-10-16 1993-10-12 New protein that lengthens blood coagulation time, derived from the land leech haemadipsa sylvestris

Country Status (2)

Country Link
DE (1) DE4234921A1 (en)
WO (1) WO1994009039A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8501241B1 (en) 2012-09-17 2013-08-06 Biopep Solutions, Inc. Treating cancer with a whole, leech saliva extract
CN108101975A (en) * 2017-12-14 2018-06-01 中国科学院昆明动物研究所 A kind of forest mountain leech antithrombotic polypeptide Sylvestin and its vivoexpression preparation method and application

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0346894A2 (en) * 1988-06-16 1989-12-20 Merrell Dow Pharmaceuticals Inc. Extraction and purification of an anticoagulant substance from the south american leech, haementeria ghilianii
EP0352903A2 (en) * 1988-06-24 1990-01-31 Yissum Research Development Company Of The Hebrew University Of Jerusalem Bovine factor Xa inhibiting factor and pharmaceutical compositions containing the same
WO1993011239A1 (en) * 1991-11-26 1993-06-10 Basf Aktiengesellschaft New thrombin-inhibiting proteins from terrestrial leeches

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0346894A2 (en) * 1988-06-16 1989-12-20 Merrell Dow Pharmaceuticals Inc. Extraction and purification of an anticoagulant substance from the south american leech, haementeria ghilianii
EP0352903A2 (en) * 1988-06-24 1990-01-31 Yissum Research Development Company Of The Hebrew University Of Jerusalem Bovine factor Xa inhibiting factor and pharmaceutical compositions containing the same
WO1993011239A1 (en) * 1991-11-26 1993-06-10 Basf Aktiengesellschaft New thrombin-inhibiting proteins from terrestrial leeches

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
M. SCHARF ET AL.: "Primary structures of new 'iso-hirudins'", FEBS LETTERS, vol. 255, no. 1, September 1989 (1989-09-01), AMSTERDAM NL, pages 105 - 110 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8501241B1 (en) 2012-09-17 2013-08-06 Biopep Solutions, Inc. Treating cancer with a whole, leech saliva extract
US8685462B1 (en) 2012-09-17 2014-04-01 Biopep Solutions, Inc. Whole, leech saliva product and applications thereof
US8784896B2 (en) 2012-09-17 2014-07-22 Biopep Solutions, Inc Antioxidant therapy with a whole, leech saliva extract
US8790711B2 (en) 2012-09-17 2014-07-29 Biopep Solutions, Inc. Treating diabetes with a whole, leech saliva extract
US8962034B2 (en) 2012-09-17 2015-02-24 Biopep Solutions, Inc. Antiviral therapy with a whole, leech saliva extract
US9017732B1 (en) 2012-09-17 2015-04-28 Biopep Solutions, Inc. Antibacterial therapy with a whole, leech saliva extract
US9149498B2 (en) 2012-09-17 2015-10-06 Biopep Solutions, Inc. Treating a parasitic disease with a whole, leech saliva extract
US9265803B2 (en) 2012-09-17 2016-02-23 Biopep Solutions, Inc. Treating a melanoma with a whole, leech saliva extract
US9265802B2 (en) 2012-09-17 2016-02-23 Biopep Solutions, Inc. Treating colorectal cancer with a whole, leech saliva extract
US9433649B2 (en) 2012-09-17 2016-09-06 Biopep Solutions, Inc. Treating a lymphoma with a whole, leech saliva extract
US9433648B2 (en) 2012-09-17 2016-09-06 Biopep Solutions, Inc. Treating renal cancer with a whole, leech saliva extract
US9480720B1 (en) 2012-09-17 2016-11-01 Biopep Solutions, Inc. Treating damaged dermal or mucosal tissue with a whole, leech saliva extract
US9597361B2 (en) 2012-09-17 2017-03-21 Biopep Solutions, Inc. Treating a bacterial skin infection with a whole, leech saliva extract
US10064897B2 (en) 2012-09-17 2018-09-04 Biopep Solutions, Inc. Treating a bacterial skin infection with a whole, leech saliva extract
CN108101975A (en) * 2017-12-14 2018-06-01 中国科学院昆明动物研究所 A kind of forest mountain leech antithrombotic polypeptide Sylvestin and its vivoexpression preparation method and application

Also Published As

Publication number Publication date
DE4234921A1 (en) 1994-04-21

Similar Documents

Publication Publication Date Title
Wesselschmidt et al. Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa
Tuszynski et al. Isolation and characterization of antistasin. An inhibitor of metastasis and coagulation.
Van Deerlin et al. The N-terminal acidic domain of heparin cofactor II mediates the inhibition of alpha-thrombin in the presence of glycosaminoglycans.
Baugh et al. Human leukocyte granule elastase: rapid isolation and characterization
AITKEN et al. The structure of the B subunit of calcineurin
Carrell et al. Mobile reactive centre of serpins and the control of thrombosis
US4791100A (en) Novel polypeptides with a blood coagulation-inhibiting action, processes for their preparation and isolation, their use and agents containing them
Ni et al. Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins
Müller-Esterl et al. Kininogens revisited
Salvesen et al. Human low-M r kininogen contains three copies of a cystatin sequence that are divergent in structure and in inhibitory activity for cysteine proteinases
FI120690B (en) Non-therapeutically used Thrombin inhibitors
US6096877A (en) Nematode-extracted serine protease inhibitors and anticoagulant proteins
McMullen et al. Location of the disulfide bonds in human coagulation factor XI: the presence of tandem apple domains
Travis et al. Human α-1-antichymotrypsin: Interaction with chymotrypsin-like proteinases
Markwardt Development of hirudin as an antithrombotic agent
Rydel et al. Refined structure of the hirudin-thrombin complex
JP2551551B2 (en) Desulfatohirudin, production thereof and pharmaceutical composition containing the same
Rzepecki et al. Characterization of a cystine-rich polyphenolic protein family from the blue mussel Mytilus edulis L
Santos et al. The A-superfamily of conotoxins structural and functional divergence
US5795954A (en) Factor VIIa inhibitors from Kunitz domain proteins
Waxman et al. Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa
Strube et al. Isolation, sequence analysis, and cloning of haemadin. An anticoagulant peptide from the Indian leech.
Van Damme et al. Granulocyte chemotactic protein‐2 and related CXC chemokines: from gene regulation to receptor usage
Maraganore et al. Anticoagulant activity of synthetic hirudin peptides.
Hon et al. Extraction and isolation of antifreeze proteins from winter rye (Secale cereale L.) leaves

Legal Events

Date Code Title Description
AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WA Withdrawal of international application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: CA