Polypeptide with biological activity of inhibitor of collagen-induced adhesion of platelets, its preparing and using

FIELD: biochemistry, biotechnology, peptides.

SUBSTANCE: invention relates to protein of molecular mass 12000 Da isolated from medicinal leech saliva (Hirudo medicinalis) comprising 6 cysteine residues able to form bonds -S-S-, and pI value about 3.7. The novel protein possesses ability to block platelets adhesion induced by collagen. As result of screening cDNA library of H. medicinalis a novel gene of new inhibitor of adhesion is identified and its nucleotide sequence is determined. Invention describes a method for preparing recombinant form of protein involving transformation of suitable cells with vector comprising DNA sequence encoding inhibitor, culturing cells under condition providing its expression and isolation of expressed protein. Invention proposes using natural and recombinant forms of inhibitor in therapy states associated with vessels congestion and diseases of circulatory system, and for treatment of articles surface made of natural and artificial collagen.

EFFECT: valuable biological and medicinal properties of polypeptide.

17 cl, 10 dwg, 13 ex

 

In the present invention is described natural protein isolated from the saliva of the medicinal leech Hirudo medicinalis, which actively binds with collagen, acting thus as a natural inhibitor of the adhesion of platelets to collagen. The protein has a molecular weight of about 12,000, has an acidic isoelectric point and contains six cysteines. The protein sequence with the subsequent cloning of the gene from a cDNA library of N. medicinalis. In addition, the disclosed methods of obtaining such polypeptide by recombination. Recombinant and natural proteins are strong inhibitors induced collagen adhesion of platelets and, thus, can be used in the treatment of various conditions associated with blockage of blood vessels and circulatory disease. Finally, this protein is possible to cover the surface of natural or synthetic collagens, depriving them of the ability to stick with the cells and not letting the past be activated.

The technical field of the invention

When hemostasis or thrombosis platelets stick together with the extracellular matrix of the damaged vessel and cover the surface of the affected area. Impeding this process, which is the initial stage of the pathogenesis of thrombosis and occlusion of the arteries, should be a new step towards solving warnings trombotic the ski diseases.

Collagen is the most thrombogenic surface component. It is established that he is actively stimulates leukocyte adhesion and aggregation and release of platelet granules, which leads to attraction (Ruggeri, Z.M. and others; Seminars in Hematology, 1994, 31, 229-39) to the site where this process, additional platelets and, as a result, the formation of aggregates or thrombus. The primary interaction of platelets with the surface of the vessel occurs when the part associated with collagen factor von Willebrand's disease (FFV) and specific receptor FFV on the platelet glycoprotein complex Ib-V-IX. At the same time, ADP, epinephrine, and blood coagulation factors activate platelets, and increased thrombin activity formed by cross-linking fibrin clot. This process promotes platelet aggregation is mediated by fibrinogen, connecting the cells in the receptor glycoprotein IIb/IIIa (bridge connection).

This is a normal physiological reaction becomes very dangerous if it occurs in the course of the pathological process, when platelets stick together with generated in areas of sclerotic damage collagen (Van der Rest M. and others; FASEB Journal, 1991, 5, 2814-23) and start to be aggregated in occlusion. Depending on the location and size of the occlusion, it can lead to serious complications is the s, such as myocardial infarction, stroke, pneumonia or pulmonary embolism.

Today, the most well-known drug used to eliminate thrombotic diseases, is pamodesti antithrombin heparin, blocking the activity of thrombin and thus prevent the formation of fibrin clots. Heparin is widely used for diseases such as progressive angina and acute myocardial infarction. However, despite this, he also has several disadvantages. Here they are: it can be administered only intravenously, it has low affinity with the associated with clot thrombin, can inaktivirovanie some plasma proteins, sometimes causes thrombocytopenia, biologically heterogeneous and is not valid without cofactor, which is necessary to apply antithrombin III. Thus, the use of heparin in clinical conditions did not produce the expected results.

Was recently received low molecular weight heparin, which can be administered subcutaneously. However, such therapy was not much more powerful one with the use of standard heparin. Unfortunately, the same can be said about other pravdastaying antithrombin, such as hirudin, hirulog and warfarin. There is every reason to believe that one of the main problems is to thrombin production to the showing in the course of elimination of blood clots increases (Rao, AK and others, Circulation, 1996, 94, 389-2395).

Thus, in recent years special attention was paid to the inhibition of activation of prothrombin, managed by factor XA. The primary objective is to obtain inhibitors of this factor, however, the prospects of their application in therapy has not been studied.

In addition, there are also thrombolytic methods. The main objective is to obtain plasminogen activator type staphylokinase, streptokinase, urokinase, plasminogen activator tissue type and activator complex Antilibanus plasmodesmata. These thrombolytic tools vary widely in the time they need for the resumption of perfusion, however, the total mortality rate does not depend on the use of one or another of them. In addition, often as a result of their introduction are complications such as re-occlusion or prolonged bleeding. This can be explained by a low specificity for fibrin and short half-life of these compounds. At the present time to correct the deficiencies that still make themselves felt when thrombolytic therapy, tested different ways of application and combination of a whole number of fibrinolytic effectors. However, these studies do not expect any results.

Ned is but there is a new group of patients with such problems, as an acute thrombotic occlusion and late restenosis at the angioplasty, arteriectomy, transplant artery or stent implantation in the vessel wall. Therapy can be performed by applying antithrombotics, antithrombotic and thrombolytic funds. The ability of a direct impact on the activity of platelets also has a number of other substances which are antagonists of ADP, such as ticlopidine or calcium ionophor A-23187 and, in particular, aspirin. These substances were either applied or used for the purpose of preventing or minimizing the process of platelet aggregation. The new substance of the present invention, the blocking adhesion of platelets, may also help to avoid aggregation of the latter in the course of surgical operations.

Another problem occurs when the interaction of artificial surfaces with blood. Artificial materials have a tendency to induce thrombotic processes, activating platelets and/or inducyruya coagulation. This can lead to disruption of the function of vascular grafts, heart valves, stents, catheters or any other devices or materials that interact with blood. Thus, the protein of the present invention, due to its ability to form nitropropene surface, can also be used, immobilization on the above materials and devices. Such treatment should be to ensure biocompatibility and resistance of such materials or devices to the formation of blood clots.

When applying available antithrombotic means we have to reckon with a number of limitations. Thus, there is a real need to develop new alternative methods and medicines.

Background of invention

The key to further advances in the treatment of cardiovascular diseases can be disclosed in the present invention methods directly aimed at blocking the adhesion of platelets induced by collagen and/or FFV.

Some new inhibitors that block the adhesion of platelets, are monoclonal antibodies against FPV. It was also suggested that inhibitors of glycoprotein IIb/IIIa with the same success it is possible to apply for inhibiting adhesion of platelets.

Some of these inhibitors, such as monoclonal Ab se, managed to go through clinical trials, while others, such as inhibitors KGD and RGDF, are still the subject of intense study. The specificity of most of these new inhibitors are not yet fully understood, thus, common side effects that may accompany their use, are not yet known.

Nova is a connection blocking induced by collagen adhesion of platelets, do not require a long search. They abound in nature, namely, the blood-sucking organisms. The literature describes several inhibitors isolated from natural material: a protein with a molecular mass of 65 KD called Calin isolated from Hirudo medicinalis (US 5537360, WO 92/07005) (Munro, R., and others, Blood Coagulation and Fibrinolysis, 1991, 2, 179-184), and a protein with a molecular weight of 16 KD (LAPP), isolated from the salivary glands of the leech Haementeria officinalis (US 5324715). Both protein described as inhibitors of aggregation to which they are ranked on the basis of the results of the research-induced collagen platelet aggregation in static conditions.

Despite the proven activity in vitro, LAPP was inactive for a number of proven first models in vivo (Schaffer L.W. and others; Arterioscler. Thromb., 1993, 13, 1593-1601) and Connolly T.M. and others; Thromb. Haemostas., 1993, 69, 589). In the soft tick Ornithodoros moubata also contains antithrombolytic protein (mountain)capable of block-stimulated collagen platelet aggregation (Waxman, L. and others; J. Biol. Chem., 1993, 268, 5445-49). Another inhibitor of platelet aggregation, selected from the bug described Noeske-Jungblut and With others in WO 9309137. Smith et al. were isolated from the snake venom protein with a molecular mass of 50 KD and from the saliva of the bug Triatoma pallidipennis one with a molecular weight of 19 KD. It was found that the protein contains a specific factor inhibiting platelet aggregation induced by collagen. Protein with a molecular weight of 19 KD, called pallidipennis, inhibits mediated collagen platelet aggregation in plasma. Inhibition of aggregation induced by other effectors (ADP, thrombin, simulator thromboxane A2 U46619, complex turbolover ether), not detected. Pallidipes not affect the adhesion of platelets with collagen, however, inhibited the release of platelet ATP. It reversibly interacts with platelets without interfering with collagen. The exact mechanism of action and therapeutic value of this protein are being studied. The Gan and others described echistatin, which, according to the authors, is an inhibitor binding to the fibrinogen receptor GP IIa/IIIb (J. Biol. Chem, 1988, 263, 19827-32).

However, despite all these apparent successes, there is one problem still to be solved. It consists in obtaining new anticoagulants and anti-thrombin, which would have increased the ability of inhibiting the formation of clots induced FFV activated platelets and activated endothelial cells, could be used in the pharmaceutical industry and is produced in quantities that allow to speak about their commercial value.

Because none of the above is still protein is not ideal from the point of view of therapeutic the ski applications, the creators of the present invention decided to resort to new methods of screening, in order to find compounds that would approach the solution of this problem.

Description of the invention

In the present invention are described isolated from N. medicinalis inhibitor, impacting directly on the interaction of collagen with platelet count and, thus, inhibiting the activation of platelets and their interaction at the initial stage.

In the literature there is not a single example of a new release mechanism or compounds identified by the screening method involving an exception from a natural source aggregation inhibitors and lytic proteins. However, this technique is used in the creation of the present invention. At first, in the identification of new inhibitors of adhesion was difficult to believe, because it is known that at least six platelet surface glycoproteins involved in adhesion to collagen and, in addition, several compounds of platelet origin, such as the factor a background of Villebranda, fibronectin and thrombospondin are indirect mediators of adhesion of collagen with platelet count.

However, recognizing the impossibility exception, all described or unknown inhibitors FFV and connections, operating right near St is the only receptors on platelets, screening saliva Hirudo medicinalis was carried out using that method. The result exceeded all expectations - got a new protein, called saracino. This protein is able to block adhesion of platelets, can be isolated from tissues and secretions well studied leeches of the species Hirudo medicinalis

The present invention relates to an active polypeptide to saracino isolated from the leech Hirudo medicinalis This protein was extracted from saliva using the last method, which is a combination of dialysis with high blood pressure, at least one phase chromatography, for example, anion exchange, and at least one stage of high-resolution chromatography with reversed phase (IHVR with PF). Dialysis under pressure in this case was simply necessary - increased concentration of saliva helped to avoid substantial losses of biologically active saracina. Selected saratin actively associated with several collagens and, depending on the dose, in varying degrees, blocks the adhesion of platelets with collagen.

For the optimization of cascade screening available techniques have been modified with a clear delimitation of adhesiveness of platelets and their tendency to aggregation. Such modifications include an assessment of the ability of platelets to slow down or stop the flow passing through the fiber, the degree is regarding platelets in clot formation in vitro, evaluation of adhesiveness with a glass fraction or filtration of whole blood and the determination of the degree of clumping of platelets treated with anticoagulant plasma with high concentration of platelets with filters consisting of glass fibers or collagen, in the regulated pressure gradient.

Protein (called saratin) is characterized by the amino acid sequences shown in sequence (SEQ. ID. NO. 2), and consists of 103 amino acids with a theoretical relative molecular mass of approximately 12068 daltons ± 1 KD. The protein has a unique primary structure, no significant similarity with other sequences described previously. The protein contains large amounts of aspartic and glutamic acids, so that the molecule has a low isoelectric point (pH 3,7±0,5, determined by the method of polyacrylamide gel electrophoresis with isoelectric focusing).

After electrophoresis in polyacrylamide gel with sodium dodecyl sulfate pre recovered protein, found a dramatic change in its mobility, indicating that post-translational modifications. In the sequencing of the polypeptide received six molecules of cysteine, which could be post-translational modification of the protein. what Ecodom electron-beam mass spectrometry has been defined, the actual molecular weight of saratin, equal 12061 that pointed to participate in the formation of the secondary structure of the native form of the protein from one to three disulfide bonds.

The adhesion inhibitor according to the present invention is new, as is different from the known isolated from leeches aggregation inhibitors, in particular, Kalina or LAPP, molecular weight, isoelectric point, amino acid sequence and biological activity.

In addition, this invention relates to a selected DNA comprising polynucleotide encoding obtained from leeches inhibitor of adhesion of platelets with the amino acid sequence similar to that described for protein. The nucleotide sequence representing the cDNA clone shown in SEQ. ID. NO. 1. Position 1-63 this nucleotide sequence is the putative leader sequence of 21 amino acids, and the position 64-372 contains an open reading frame encoding a polypeptide consisting of 103 amino acid residues, and the amino acid sequence shown for the Mature protein of SEQ. ID. NO. 2.

The present invention also relates to recombinant vectors, including synthetic gene encoding obtained from leeches inhibitor of adhesion of platelets according to this invention, and the cell host containing the recombinant vectors based methods of selection and allocation of newly expressed proteins were used methods of tagging or adapt any of the purification of natural saracina. How is the selection of the recombinant protein from the supernatant liquid or sludge, is determined by the pattern of extracellular or intracellular expression in cells, for example, yeast cells, insect cells, cells of the kidney hamster, such as E. coli, transformed corresponding vectors. How to link these processes must be known to the person skilled in the art. Beautiful expression occurred when master roles were E. coli. In this case periplasmatic expression was initiated by the insertion of the leading sequence pelB. The product (about 5 mg/l) from Escherichia coli (E. coli) was preceded by osmosis and centrifugation. In parallel, an experiment was conducted on Saccharomyces cerevisiae (S. cerevisiae) (>10 mg/l of culture fluid) with the vector adapted to the yeast. The substance of the glands were isolated by centrifugation. Purification was carried out by the method crossflow filtration and ion exchange chromatography. When using COS cells or Cho expression product was about 750 ng/ml. the Results of electrophoretic and chromatographic analyses testified to the purity and homogeneity of purified material, Sequeira same amino acid and determining the molecular weight, was able to verify his identity derived from the saliva of sarati is at. In addition, the present invention relates to a method of purification of active protein from the saliva of leeches and assessment of its level of antitromboticos activity using static and dynamic analysis, and the application of these methods for the selection of recombinant protein.

Methods of obtaining saracina described in Examples 6, 7, 8, and 13. Here it should be noted that the methods of expression not confined to these examples. For example, to Express saratin well as in transgenic mice, or other organisms, including mammals.

The proteins of the present invention also includes variations that preserve the activity of the disclosed sequences here, including fragments or subunits, natural variation allelic variation, random artificial mutants and directional variations of the sequences, such as, for example, result from the extension, keeping the specified activity. Fragment or subunit can be any sequence containing fewer amino acids than the full protein, for example an incomplete sequence that does not contain N - and/or C-terminal parts of the protein.

In addition, the present invention relates to hybrid proteins, such as fused proteins or protein, resulting from expression of several genes within vecto the and expression, which can include a polypeptide having the specific activity of the protein according to this invention, linked through peptide bonds with the second polypeptide. In addition, the present invention applies to other variations of proteins according to this invention, mainly, to any variations that differ from the selected protein only conservative substitutions of amino acids. The last described by Taylor and others, J. Mol. Biol., 1986, 188, 233.

The present invention also relates to methods of applying proteins to block or delay the activation of platelets by inhibiting the interaction of collagen with platelet. The protein can be used to prevent, prevention, therapy and treatment of thrombotic diseases. The mechanism of action of the protein according to this invention differs from all previously described proteins, which affect different platelet surface proteins. It is closely associated with the surface of collagen and, in one embodiment, covers its specific areas, thus depriving them of the possibility of interaction with platelets and binding of the latter. The advantage of this option, a new mechanism of action consists in the complete preservation of functional activity of platelets, which allows almost or even completely avoid cromatica the Oia after the introduction of the protein.

This protein can also be used, which is highly significant for the treatment of various surfaces, thereby giving them the ability to repel the platelets, which suggests the creation of compatible blood units.

As mentioned above, the polypeptides of the present invention can be used as a pharmaceutically effective compounds in the form of pharmaceutical compositions and combined preparations.

The pharmaceutical compositions according to this invention may contain additional active ingredients, such as aspirin, anticoagulants such as hirudin or heparin or thrombolytic agents such as plasminogen activators or streptokinase.

With any non-toxic organic or inorganic acid, a new polypeptide of the present invention can form pharmaceutically acceptable salts. As examples of inorganic acids can lead hydrochloric, Hydrobromic, sulfuric or phosphoric acid and acid metal salts such as secondary acid phosphate and sodium acid sulfate potassium. Examples of organic acids with mono-, di - and tricarboxylic acids such as acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, ximalayasha benzoic, oxybenzone, phenylacetic, cinnamic, salicylic acid and sulfonic acids, such as methanesulfonate acid. Salts carboxykinase amino acid residues are non-toxic salt of carboxylic acid, obtained using any suitable inorganic or organic bases. These salts include, for example, salts of alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, light metals of Group street 111A, including aluminum, as well as salts of organic primary, secondary and tertiary amines, such as trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethylenimine, N,N'-dibenziletilendiaminom, dehydroabietylamine and N-alkylpiperidines.

In the context of this description, the term "pharmaceutically acceptable carrier" refers to non-toxic solid or liquid filler, a solvent or a substance forming the shell of a capsule, which does not react with the active connection and cannot harm the patient. Appropriate, in the preferred embodiment, the liquid media is widely known in this field. Such carriers can serve as sterile water, saline, aqueous dextrose, sugar solutions, ethanol, glycols and oils derived from crude oil refining, as well as any animal or vegetable or synthetic, e.g. the, peanut oil, soybean oil and mineral oil.

Compounds of the present invention can be entered in single doses containing conventional non-toxic pharmaceutically acceptable carriers, solvents, adjuvants and binder, typical for parenteral administration.

The term "parenteral" here refers to subcutaneous, intravenous, intra-articular and intratracheal injection and infusion. In addition, other acceptable forms of application, such as oral and local. Compositions and combined preparations for parenteral use in the most preferred embodiment, is injected in the form of a bolus or as a permanent fuse, using known methods.

Tablets and capsules are intended for oral administration, contain conventional excipients such as binders, fillers, solvents, tabletting agents, lubricants, substances that contribute to the current release early, and surfactants. Using widely known in the field of methods of the tablets can be coated by a shell.

Liquid preparations for oral administration can be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or be produced as a dry product before applying SL is blowing to bring to the desired consistency, adding water or other suitable binder. Such liquid preparations may contain conventional additives such as suspendresume agents, emulsifying agents, non-aqueous binder and anticoagulants.

Preparations for topical application can be in the form of aqueous or oily suspensions, solutions, emulsions, gels or that is preferred emulsion ointments.

Single dose of the present invention can contain daily amount of protein according to this invention or its fraction of the units that make up the required dose. What dosage is optimal for each patient (mammal, including man), depends on several factors such as the activity of the applied active ingredient, age, body weight, General health, sex, diet, time and route of administration, rate of excretion, the purpose of the drug, i.e. is for the treatment or prophylactic treatment, the nature of thrombotic disorders, as well as the current activity beginning, which may act as antitromboticos funds or anticoagulant.

Thus, the pharmaceutically effective dose of the peptides of the present invention in the compositions and combined preparations are introduced into the patient's body (in vivo) as antithrombotic agents, composition is employed, about 0.01-100 mg/kg, preferably from 0.1 to 10 mg/kg of body weight. Depending on the form of application of a single dose may contain from 0.5 to 10 mg of inhibitor of thrombin. To achieve antivertigo action in vitro of blood, it should be 0.2 to 150 mg/l, preferably 1-20 mg/l peptides of the present invention.

The aim of the present invention is to develop an implantable or extracorporeal medical devices for use in areas where it will interact with circulating in the body fluids, the surface of which is by its cover immobilized polypeptide described above and in the claims, is largely resistant to thrombus formation. The polypeptide of the present invention immobilized on the medical device, thereby providing the biological compatibility of the surface of the latter and its resistance to the formation of blood clots. The surface of such devices sometimes have properties, usually inducing platelet aggregation, which leads to serious problems with their use in areas which involve interaction with blood or other present in the body fluids. As examples of such devices, usually made of plastics and synthetic fibers, can lead to dentures, iskusstvennye bodies, lenses, suture, artificial segments of blood vessels, catheters, dialyzers, pipes and vessels for the transport of blood.

Opacity of the posterior lens capsule (PSK) is a common complication after cataract extraction, to avoid which cannot even using modern surgical techniques and lenses used during this operation. PSCH arises from the proliferation and migration of epithelial cells of the lens in its posterior capsule that reduces visual acuity. To facilitate PSCH were asked to apply the methods of physiotherapy and chemically modified lenses. So, we used heparin coating for lenses or heparin eye drops, from which it can be concluded that PSK has thrombogenic nature.

It was found that saratin is much higher than the heparin from the point of view warnings and block thrombogenicity. Thus, another distinguishing feature of the present invention to provide a coating containing saratin, which would reduce thrombogenicity material used in refractive implants, surgically implanted in the front or rear camera. This new coating helps to avoid problems arising from stimulated cell growth. In combination with other drugs, e.g. the, causing cell death, sabatinovka floor gives you the ability to completely eliminate the opacity of the posterior lens capsule.

Brief description of figures

More detailed figures are explained in examples 1-10.

1

The separation of components of saliva. Elution of saratin marked *

a) the profile of the separation of saliva after anyoneeven with DEAE. Fraction of saratin collected in peak 3 (Example 2).

b) a second chromatography was carried out combined fractions on a Mono Q HR5/5. Samples were collected in the latter part of the main peak, as shown below (Example 2).

in the last stage prepreparation analytical GHUR with of saratin-positive fractions collected from Mono Q HR5/5 (Example 2). Active saratin selected in the main peak (peak 3).

2

Electrophoresis in polyacrylamide gel with sodium dodecyl sulfate fractions collected at the stage GHUR with PF. Positive fraction of saratin marked with * (Examples 2 and 3).

3

The expression vector of ceratina in E. coli (Example 7).

4

Baculovirus donor plasmid of ceratina (Example 8).

5

Whole blood is introduced into the interaction with the surface of the artificial collagen visualization adhesion of platelets by colouring. Saratin was used as an inhibitor (protein #607) (Example 9).

6

Inhibition of adhesion of platelets on cover glasses coated to what lagena type III, under conditions of shear flow. Comparison of the inhibitory effect of saliva and ceratina (Example 9).

Fig.7.

Inhibition by saracino adhesion of platelets with cover glasses coated with collagen type III, under conditions of shear flow. The activity of saratin depends on the dosage (Example 9).

Fig

The expression vector of ceratina in yeast (Example 13).

Detailed description of the invention

Example 1

Screening for inhibitors of adhesion

Adhesion of platelets to collagen was adopted as the criterion by which carried out the screening of the components of saliva. In addition, to prevent the manifestation of the last functional properties that cannot be attributed to adhesion inhibitor, were four additional studies aimed at evaluating the effects of various antithrombotic funds: analysis of AZOCOLL, assessment activity amidase, test binding with the participation of the factor a background of Villebranda and analysis of platelet aggregation. All these tests are standard, however, the analysis of the adhesion of platelets, we had to modify. Briefly, the modification consists in the following: collagen Horm. (firm Nycomed) was coated 96-cellular tablets (firm Nunc) (used acidified collagen at a concentration of 20 μg/ml and the plates were incubated over night). Three times washing tablets SOP what limera of styrene and butadiene, collagen, remaining at the bottom of the cells, blocked with 1%serum albumin cattle. Then added fresh platelets isolated from citronellene human blood, and, in parallel with them, the fractions obtained in the individual stages of column chromatography. If necessary, before using platelets was re-activated, incubare in tertbutylamine in the presence of divalent ions of magnesium oxide. The source material of saliva total concentration of protein stabilized at the level of 200 ág/ml, were used as reference standards inhibitory effect. As it turned out, from the point of view of the activity of inhibitors of platelet highly sensitive to changes in the buffer and high salt concentrations. All samples were processed by the method of ion exchange chromatography, and therefore making direct study of fractions in the process of analysis of inhibition has proved very difficult and could not give accurate results. For this reason, all subject to study samples passed the stage of concentration (hardware Method), which allowed to reduce the ionic strength and at the same time to stabilize the concentration before measurements.

Example 2

Treatment of natural inhibitor

In the present invention was used saliva isolated from N. medicinalis. izvestno, it contains a number of biologically active proteins, such as hirudin, inhibitors of elastase, collagenase and inhibitor of platelet aggregation, such as Calin (Munro, R. and others; Blood Coagulation and Fibrinolysis, 1991, 2, 179-184) and LAPP (Schaffer, L.W. and others; Arterioscler. Thromb., 1993, 13, 1593-1601). These proteins are well studied, but mostly about eighty proteins that can be detected by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate, is still not known. This invention, by applying the procedure described in example 1 were fractionated screening of saliva on the subject of finding new proteins, directly blocking the interaction of platelets with collagen.

The main problem was the separation of the adhesion inhibitor from the source material of saliva, which was caused mainly by the fact that in the first stage chromatographic separation inhibitor irreversibly lost almost all of its activity. The situation has not improved after supplementation (Munro, R., and others), such as 12%ethanol and divalent cations. At the same time, the high salt concentration in the saliva at low total concentrations of protein (190-250 mg/ml) required pre-concentration or replacement of the buffer. Thus, it was tested several methods of enrichment, concentration is whether the replacement buffer. Traditional dialysis resulted in complete loss of activity. Most of the other standard methods, such as ion-exchange or affinity chromatography and separation of molecules by size* (*size-Exclusion chromatography (approx. translat.)) (loss did not depend on the polymer contained in the column), did not yield any results, despite the use of different methods of separation and the use of a variety of buffers and additives. To the surprise of the authors of the present invention found that by dialysis with a high pressure of 500 ml of saliva can increase the concentration of protein in it (about 30-40 times) and at the same time get rid of the unwanted components of the buffer. Also it was found that the thus treated the source material of saliva was perfect for holding with him further purification. Thus, the selection of biologically active anti-adhesion components of saliva ceased to be a problem. Because cationogenic or columns for affinity chromatography was not possible to achieve the desired degree of purification, have switched to using weak anion-exchanger such as DEAE-Fastflow or EMD-DEAE-Fractogel. Has been tested with 12%ethanol and divalent cations, however, as with the use of these additives, and without them the results remained unchanged. As for optimal chromatographic sec the population settled on the following sequence: DEAE column, column Mono Q and at the end RP18 column for chromatography with reversed phase. Conditions chromatographic separation was optimized by applying chromatographic system BiaCore with analytical columns supplied by the company Pharmacia. The gradients used to work on columns of DEAE, Mono Q and RP18, shown in Figures 1A, b, C. the Division in the BiaCore system was scaled using the methods of liquid chromatography with the programming flow. Following the manufacturer's instructions, from the optimized operating conditions, except for the stage using a RP18 column, which, in order to minimize the loss of the pure material, continued to operate in the mode provided for methods Biacore passed directly to prepreparation division. The selection of purified protein obtained in the final phase chromatography with reversed phase, carried out by the method of high-speed vacuum centrifugation. Samples collected at the above-mentioned stages were combined and analyzed by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (Figure 2). Then the samples are re-suspended in the copolymer of styrene and butadiene, and then they were used for analytical and functional analyses. Typically, the output of saratin unprocessed saliva was about 750 μg/l

P the emer 3

Biochemical research

As described in Example 1, in the treatment of saracina was obtained mostly pure protein with an average molecular weight of about 21 KD (determined under the reduction conditions by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate) (Figure 2). Complete amino acid sequence obtained by direct sequencing of the first 48 amino acids of the purified protein. This sequence was completed by sequencing of several internal peptides generated by enzymatic cleavage. The full sequence of the protein shown in SEQ. ID. NO.2.

The protein consists of 103 amino acids with a calculated molecular mass of 12067,9 and such actual 12061,9 (defined by electron-beam mass spectrometry). From this difference between theoretical and measured molecular masses can be concluded that the formation of bridges S-S attended all six identified caratine cysteines. This is confirmed by the results of chromatographic analysis of protein by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate in reducing conditions and in the absence of these, when there is a sharp change in its mobility. In addition, the protein is rich in acidic amino acids such as Glu and As. The polyacrylamide gel electrophoresis with isoelectric focusing (firm Immobiline) allowed to determine the isoelectric point, which amounted to a pH of 3.7±0.5 in. In the comparative analysis as a benchmark, we used purified protein leeches, slica its physico-chemical properties with those of recombinant saracina obtained from expressing his baculovirus, yeast and E. coli. The properties of all three proteins were identical.

By electrophoresis in polyacrylamide gel with sodium dodecyl sulfate with visualization by staining method Kumasi (Figure 2) or silver plating and/or Western blotting revealed that the protein is homogeneous and is not glycosylated.

Example 4

Obtaining mRNA and synthesis of cDNA

RNA was obtained from the medical leech Hirudo medicinalis using the thiocyanate guanidine. mRNA from total RNA was isolated using a set of "Oligotex mRNA kit (firm QIAGEN).

cDNA was synthesized using a set of "Marathon cDNA Amplification kit (firm CLONTECH). Then the DNA sequence encoding saratin, following the supplier's instructions, amplified oligonucleotide primers PCR (polymerase chain reaction). Upon completion of the synthesis of cDNA from both her ends ligated universal adapter. The sequence of the universal adapter and universal primers AP1 or AR was chosen in accordance with the tvii with the instructions of the supplier of the mentioned set.

Example 5

Amplification and isolation of the gene of saratin PCR

Degenerate primers were synthesized on the basis of the nearest N-end back translated amino acid sequence of saratin.

These primers 01 and 02 are designed for specific hybridization to the 5'-end of cDNA saracina. The design of the primers was laid back translated amino acid sequence of the eight N-terminal amino acids of the purified protein of saratin, which were determined experimentally. Primers (01 and 02) were synthesized with the aim of stabilizing the degeneracy at the lower levels, and primer extension at critical 3'-end 8 of complementary base pairs, is perfectly consistent with the cDNA sequence of saratin that will provide an efficient and specific amplification matrix cDNA. In the degenerate alphabet DNA (IUPAC code) R=a or G; M=a or C; Y=C or T; and N=a or G or C or T.

PCR 3'-RACE was performed using a mixture of primers 01 and 02 with one universal primer, AP1 or AR. The PCR products were cloned into the cloning vector TA pCR2.1 or the vector pCR-Script SK(+) and sequenced. Sequeira several fragments PCR 3'-RACE, received the gene sequence of ceratina (except netransliruemoi region 5', the signal peptide sequence and a follower of the spine, encoding the first 8 amino acids of the N-Terminus of the Mature protein). To obtain information contained in these missing sequences of cDNA saratin, an experiment was conducted with 5'-RACE, during which used gene-specific primer from the middle of cDNA saracina and one of the universal primers AP1 or AR. Sequeira several resulting PCR fragments 5'-RACE, received the full sequence of the gene of saratin. As a result of amplification of the gene of saratin by PCR using gene-specific degenerate primers from both 3'-and 5'-end of the gene of saratin got the full gene of saratin.

In sequencing DNA was attended by more than 15 different clones of PCR products. When this was revealed only one significant change in the amino acid sequence in a single clone. It is highly likely that this change was caused by PCR. In addition, it was found five more silent changes, not Modestovich on the structure of the amino acid sequence. These changes were unlikely to be due to PCR, since the same modifications have been identified in different clones.

Gene ORF of saratin has a length, part 372 complementary base pairs, and contains the signal sequence of 21 amino acids and the sequence encoding the Mature the protein, which includes 103 amino acids. It was found that the amino acid sequence derived from clone PCR product identical to the sequence resulting from sequencing of natural protein isolated from saliva.

Example 6

Expression in COS cells and detection of expressed protein

For the purpose of gene expression of ceratina in mammalian cells such as COS or Cho, it using Xhol + Xbal cut out from the vector pCR-Script SK(+) and cloned into the expression vector pCl-neo (the company Promega) cells of the mammal. The latter was selected because it contains the promoters for T7 and T3, which allows expression in vitro and gene of resistance to neomycin, necessary for selection of G418, and also can be used as COS cells and Cho.

In addition to the signal sequence and the sequence of the Mature protein, the 5'-end of the insertion contains a sequence Koz., to allow efficient translation, and on the 3'-end (- end) - label his MRGS(H)6to detect the expression and tracking purification and concentration of protein. Plasmid construction called pNC-31.

DNA plasmids pNC-31 was used for transfection of COS cells. The COS cells were in the logarithmic growth phase, washed twice copolymer of styrene and butadiene was dissolved in the latest in a concentration of 1×107/ml. the ATEM 0.7-0.8 ml suspension COS was added 12 μg of plasmid DNA (less than 50 ál of N 2About or THE buffer) and mixed in a cuvette for electroporation. Electroporation was carried out at 1.9 kV, 25 microfarad for 10 minutes and Then the cells were transferred to 90 mm plate. Adding 8 ml of DMEM medium containing 10% FCS and antibiotics, and the cells were allowed to grow for three days. Supernatant and cells were used for further separation and detection of the protein. Expressed protein was detected using Western blotting using antibodies against MRGS(H)6. For cleaning used chelating agents, such as NTU or imiloxan acid immobilized on the matrix columns and modified metal ions such as Co, or Cu Mi.

Example 7

Construction of expression vector in E. coli and expression

Because in certain biological systems use different codons in E. coli, some of them are extremely rare. To Express the optimized version in E. coli using standard techniques, the gene was first necessary to cause compliance with the E. coli codon.

Expression in E. coli was performed using a modified version of the plasmid pASK75, carrier tet promotor region (Skerra, A. and others, Gene, 1994, 151, 131 -135). Modification, without going into details, was made by cloning a new linker between sites Xbal and Hind III (Figure 3). A new linker content is t leading the ompA sequence, another multiple cloning site and a tag 6 × His instead strep.

To construct the expression vector of saratin, PCR was necessary to introduce the restriction sites 5' Cla I and 3' Eco47III.

Used 5'-primer 03 and 3'-primer 04. The PCR product was first cloned in the vector PCR system II (company Invitrogen) and sequenced.

In the second stage of gene saracina, using the restriction sites 5'Clal and 3' HindIII, cloned into the modified vector pASK75.

At the end of the expression, having ascertained the existence of this recombinant of saratin activity during the second PCR was deleted His label and directly with leading sequence omp A poured the start codon of the gene of saratin. 5'-primer 05 and 3'-06 primer used in this PCR are listed in the sequence listing.

As an example, expression in E. coli can lead to the transformation of the expression vector pRG72 (Figure 3)containing the structural gene of saratin, merged with a leading sequence of ompA in competent cells W3110. Upon reaching the last mid-logarithmic growth was initiated. After 1 hour you can easily discover a recombinant carestation.

Example 8

Construction of baculovirus donor plasmid and expression

The purpose of the expression of ceratina in the baculovirus expression system used by the VA system Is Something You™ supplied by the company Gibco Life Technologies. In order to obtain a system of selection, with the gene of saratin poured leading sequence Meletina honey bees, and to introduce restriction sites 5' BamHl and 3' Kpnl held a single PCR using 5'-primer 07 and 3'-primer 08.

The corresponding PCR product was cloned in the vector PCR II (company Invitrogen) and sequenced. The product of the merger of the Meletina with saracino using restriction sites 5'BamHl and 3'Kpnl cloned in the vector pFastBac getting pTD13 (Figure 4). The formation of recombinant baculoviruses and expression of saracina was carried out using the expression system, You You. Donor plasmid pTD13 transformed in competent cells DHIOBac containing bacmid with site-targeted mini-attTn7 and plasmid helper. In the presence of transposition proteins from plasmids assistant element of the mini-Tn7 donor plasmid was transpositionally in site-target mini-attTn7 on backside. Colonies containing recombinant bacmid identified by gap gene lacZ. Selected E. coli clones containing recombinant bacmid received low-molecular-weight mini-DNA, which is then used for transfection of insect cells. More all explained in the manual set for the expression.

Example 9

Adhesion of platelets to collagen under conditions of flow (dynamic analysis)

In the process of analyzing the adhesion of platelets whole human blood is passed through the flow chamber, evaluating the adhesion platelet activity against collagen-covered last top glass under shear flow (simulating the conditions under which the perfusion in arteries in vivo), as described in Sakariassen and others (Mette. Enz., 1988, 169, 37-70). Collagen from human placenta, type III, Sigma)dissolved in 50 mmol/l acetic acid, using retouching airbrush sprayed on a clean cover glass (18 mm × 18 mm). Last night at 4°it was kept in the copolymer of styrene and butadiene.

Before using fresh whole human blood (as anticoagulant was administered low molecular weight heparin; 20 units/ml) was heated at 37°within 10 minutes the Preparations of the proteins of the present invention using a pipette was applied to a glass cover (30 µl per cover glass) and, before being placed in a perfusion chamber, incubated for 10 min in a chamber with a humid environment at room temperature. The blood is then passed through the perfusion chamber (37°C for 5 min with a shear rate of 1300-1).

Then the cover glass was removed, washed copolymer of styrene and butadiene, and within 30 minutes were fixed in 0.25%for Mr. paterova the aldehyde and stained by the method of ay-Grü nwald Giemsa (Figure 9). In untreated control surfaces are painted platelets spread on a very large area, whereas the comparative surface pre-treated saracino, there was a sharp decrease (80%) ability to bind. Quantitative analysis of adhesion of platelets was performed using an optical microscope (Figure 5, 1000-fold increase)connected to the image analyzer control computer (Leica). The results were expressed in percentage units of platelets covered from the last surface.

The inhibitory action of saliva was compared with that of the purified protein (Figure 6). Inhibition of untreated saliva (#616) adhesion of platelets on cover glasses coated with collagen type III, at the shear rate of 1300-1compared with the control was approximately 48%. When using purified protein (#607; saratin) normalized concentrations deposition of platelets was blocked by approximately 81%. Inhibitory effect of saratin increased with increasing dose and increasing concentrations of the protein (Figure 7).

Example 10

Immunization and antibody

After receiving the first batch of the natural protein of high purity, immediately began to immunization of animals. Immune serum was isolated from rabbits. In n the existence had reagents with high titer, allowed to conduct further screening. Additional antisera was made possible with the completion of the peptide sequence of the complete protein. Synthesized three peptides (amino acid sequence 83-103, 13-30, 58-69), using the standard linker associated with KLH, which are then used for immunization. Received three serum against the N-terminal peptide segment and two against C-terminal peptides. The immune serum with high titer allowed to manage the process of purification of natural and recombinant protein, as well as to carry out quantitative analysis of the purified substances by the method of ELISA. Thus, there is no need for laborious and time consuming analysis of inhibition of platelet resorted solely to verify the final purified protein of the potential inhibitor.

Example 11

Immunological testing binding of saratin

Acidified collagen Horm, Nycomed) was coated 96-cellular microtiter plates (firm Nunc) (50 μl of a solution of collagen at a concentration of 20 μg/ml, incubation over night). Before analysis, tablets, three times washed copolymer of styrene and butadiene, in order to avoid nonspecific adhesion, incubated in a solution of serum albumin bovine RMS is a (1%). Then in serial dilution was added to 50 μl of saratin and incubated for one hour. Before proceeding to the analysis using antibodies against saracina, tablets, three times washed. After additional incubation, the excess antibody was removed and continued the test with a second antibody labeled with Biotin. The reading was carried out by conducting a color reaction with catalysis streptavidin-POD with substrates, such as tablets ODB (firm Dako) (wavelength 490 nm).

Example 12

Competitive analysis of screening inhibitors

The ability of recombinant labeled ceratina (His label) of Example 7 to bind with collagen was compared with that of unlabeled native of saratin. Tablets coated with acidified collagen Horm, Nycomed) was prepared as described in Example 11. Detection was performed using antibodies against saracina rabbit. Labeled and unlabeled sartini showed identical to the coupling. Alternatively, unlabeled, saratin modified by biotinidase (Pierce firm, set for biotinidase) and compared with the unmodified saracino. Both proteins have the same ability to bind with collagen. Next conducted experiments with cross-competition of biotinylated saracina with the unmodified saracino, peptides, saracino, isolated from saliva, saliva or antibodies against saracina. Analysis of the binding of various "competing" with collagen was carried out by evaluating the coupling of biotinylated ceratina (reaction with the substrate ODB using conjugate streptavidin-POD). This experiment with the biotinylated saracino typically used to determine the concentration of ceratina in saliva (750 µg/l), mapping of antigenic determinants of the antibodies against saracina, evaluation of biologically active and mutated saracina. To better explore the opportunities when conducting this experiment, was used as a blocking and non-blocking antibodies against saracina obtained using specific peptides of saratin.

Example 13

The expression vector and expression in yeast

You used the expression system pichia multi copy (firm Invitrogen), a typical example of expression in yeast. Construction of yeast expression vector is depicted in Figure 8. When creating the expression vector of saratin resorted to amplification by PCR, allowing to form the ends of the restriction (5' EcoR I, 3' Not I), which can be ligitamate in an appropriate vector (RSC). Used 5'-primer 09 and 3'-primer 10.

Before transforming spheroplasts Pichia expression vector was linearizable Sal I. to ensure the integration of the gene is of saratin, with colonies held a screening on the subject of His+Mut+positive mutants. Growing conditions: 28-30°C, the optical density to 2-6. Expression was induced by re-suspension was centrifuged cells in the environment and adding methanol at a final concentration of 0.5%. These conditions were maintained, usually within 24 hours. After 6 days fermentation product was collected from the supernatant and were analyzed by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate and ELIZA.

1. The polypeptide isolated from N. medicinalis, characterized by a molecular weight of about 12±1 KD, biological activity of the inhibitor-induced collagen adhesion of platelets and the amino acid sequence shown in SEQ ID NO:1, its natural option, functionally active fragment or mutant, characterized conservative substitutions of amino acids.

2. The polypeptide according to claim 1 with an isoelectric point at pH of 3.7±0.5 in.

3. The polypeptide according to claim 1 or 2 with six molecules of cysteine can form a-S-S connection.

4. The polypeptide according to any one of claims 1 to 3, having the amino acid sequence shown in SEQ ID NO:2.

5. The polypeptide according to any one of the claim 1 to 4, intended for the manufacture of a medicinal product for the treatment of thromboembolic diseases.

6. The polypeptide according to any one of claims 1 to 4, designed for use ex vivo as coatings for artificial surfaces.

7. The polypeptide according to any one of claims 1 to 4, is designed to modify ex vivo intraocular lenses.

8. The polypeptide according to any one of claims 1 to 4, intended for contact with the surface of the lens.

9. The polypeptide according to any one of claims 1 to 4, designed for covalent cross-stitching ex vivo material used for the manufacture of lenses.

10. Selected polynucleotide that encodes a polypeptide with the activity of the inhibitor-induced collagen adhesion of platelets, which is characterized by a nucleotide sequence selected from the group comprising a) a nucleotide sequence that determines the amino acid sequence of the polypeptide according to claim 1; b) a nucleotide sequence essentially corresponding to the DNA sequence shown in SEQ ID NO:1; C) a nucleotide sequence complementary to b), and d) a nucleotide sequence representing the sequence of the DNA along its entire length by at least 80% identical, is shown in SEQ ID NO:1.

11. The expression vector containing the selected polynucleotide of claim 10.

12. Pic is b obtain a polypeptide with the activity of the inhibitor-induced collagen adhesion of platelets, comprising culturing a host transformed by the vector according to claim 11, under conditions sufficient to obtain the above-mentioned polypeptide and isolating the polypeptide from the supernatant culture fluids or cellular precipitate.

13. Immunospecific antibody obtained to the polypeptide SEQ ID NO:2 or its immunogenic part.

14. The antibody according to item 13, designed to detect polypeptide according to claims 1-4.

15. Pharmaceutical composition for the treatment of thromboembolic diseases, containing the polypeptide according to claims 1-4, and a pharmaceutically acceptable carrier or excipient.

16. The pharmaceutical composition according to item 15, containing more drugs selected from the group including aspirin, heparin, streptokinase, and combinations thereof.



 

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Fumaric acid amides // 2290946

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