The protein having the ability to inhibit stimulated collagen platelet aggregation and prevent the interaction between collagen and vwf, method thereof, farmcampsite based on it


(57) Abstract:

The invention relates to biotechnology and medicine and can be used to obtain a protein able to inhibit stimulated collagen platelet aggregation. Protein (Branding) has a molecular mass of about 15 kDa, binds to collagen. The protein inhibits the binding of collagen to platelets and their subsequent activation, which leads to the aggregation of platelets and thrombus formation. Protein prevents binding of the factor a background of Villebranda of collagen. Protein isolated from the crude extracts of the leech Hirudo medicinalis by polyacrylamide gel electrophoresis. Protein suitable for the prevention and treatment of thrombolytic disease. 3 S. and 1 C.p. f-crystals, 2 tab., 6 Il.

The invention relates to a new protein, acting as a strong inhibitor-stimulated collagen activation and aggregation of platelets and, above all, having the ability to prevent the interaction between collagen and the factor a background of Villebranda.

Normal hemostasis in humans is controlled by a complex number of interrelated mechanisms, including both cellular and humoral biochemical components. Beach and activation of coagulation. If the damage to blood vessels one of the first results of this is the influence of endothelial cells on the blood. It is believed that subendothelial collagen is one of the first incentives that lead to adhesion of platelets, followed by a shape change, aggregation and formation of thrombus.

Therefore, a therapeutic effect on the stage of adhesion of platelets and/or platelet aggregation suitable for the prevention or treatment of the majority of thrombotic disorders.

It is known that the saliva of leeches contains several agents capable of inhibiting platelet aggregation.

So, hirudin is a well-known connection (Merck index, 1983, N 4613; FEBS Lett, 165, 180 (1984)). Hirudin prevents thrombin-induced platelet aggregation by binding to thrombin with the formation of a stoichiometric complex (1: 1). This, in turn, inhibits catalyzed by thrombin conversion of fibrinogen to fibrin.

In EP-A-0348208 described low molecular weight antagonist of factor mediawindow receptor activation of platelets obtained from saliva Hirudinidae, with the ability to inhibit platelet aggregation induced by aggregation agents, such as VEILS-acether (dryer through hydrolytic break peptide bonds in helical regions of the collagen molecule, described in WO 87/00860.

Medical leeches (Hirudino medicinalis) to prevent the formation of thrombus at the site of the bite is not only used by hirudin. Very pronounced characteristic bite of this leech is prolonged bleeding for more than 10 hours up to 24 hours, whereas hirudin, apparently washed out of the wound within 15-30 minutes. Therefore, this effect should be responsible other components in addition to hirudin.

Known (WO 92/07005) protein isolated from Hirudo medicinalis, with mol. a mass of 64 kDa, called "Kalina" and is able to prevent stimulated collagen platelet aggregation.

Other low molecular weight (16 kDa) inhibitor-induced collagen platelet aggregation, designated as LAPP found in Haementeria officinalis (EP-A-0480651).

However, none of these proteins does not affect the main feature of the adhesion of platelets, mediawindow factor von Willebrand's disease (vWF).

This factor is a complex multimeric glycoprotein that plays an essential role in platelet function. It is necessary for the proper adhesion of platelets when they are exposed to subendothelial, and for the normal formation of a blood clot is more in diameter, where prevailing conditions of high coefficient of murine shift. Now it is recognized that the mechanisms underlying the function of vWF, provide for interaction with components of subendothelial, as well as with specific receptors on trombotsitnoy membrane (Ruggeri et al., 1992, Meth. Enz., 215, 263-275).

Therefore, the interference of the corresponding protein in the action vWF should provide an additional advantage in therapeutic use.

Thus the present invention is to create a new protein, which is a strong inhibitor-stimulated collagen aggregation and adhesion of platelets and which prevents the interaction between collagen and vWF. The present invention is also developing a method of obtaining pure protein with the above properties and the development of a new pharmaceutical compositions on the basis of this protein, as well as the development of implantable or extracorporeal medical devices used in contact with fluid body, and the surface of which has thromboresistance by covering her immobilized protein.

This object is achieved by a new protein MM = 15 kDa, which is isolated from the text by the molecular mass of 14 15.5 KD, preferably 14 - 15,0 (depending on the applied methods) and prevents the interaction between collagen and vWF. In contrast to these results known LAPP has a molecular mass of 16 kDa and isolated from Haementeria officinalis. The LAPP influence on vWF is not proven so far. In addition, the amino acid sequence of LAPP and the protein of the present invention (branding) different.

The protein of the present invention includes variants described purified protein that retain the activity of a selected protein, including fragments, subunits, existing in the nature of mutations and randomly generated artificial mutants. Also included hybrid proteins, such as fused proteins derived from the described protein.

Protein isolate and purify, if necessary, one-step purification by preparative electrophoresis, from the saliva of medicinal leeches Hirudo medicinalis. A typical method according to the present invention includes the reconstruction of liofilizovannyh source of saliva Hirudo medicinalis in conventional buffer system at a pH of 7.5-8.5 and cleaning at least one conventional chromatographic stage, preferably gel chromatography.

A new protein according to the present invention can IP the you in accordance with the invention may contain as the active component of this protein, as well as additional active ingredients such as anticoagulants such as hirudin or heparin or thrombolytic agents, such plasminogen activator or Gemeenten.

New protein and pharmaceutical preparations of the present invention, respectively, can be used for treatment of various thromboembolic disorders, including venous thrombosis, peripheral arterial thrombosis, cerebrovascular thrombosis and myocardial infarction, as well as for the treatment of patients with arteriovenous anastomosis or patients who left coronary bypass anastomosis. Drugs can also be used to treat autoimmune diseases, including lupus erythematosus, rheumatoid arthritis and polyarteritis nodosa. The preparations of the present invention is also suitable for stimulation of the phenomenon of prolonged bleeding, which happens after being bitten by leeches, they can, therefore, replace, fully or partially, leeches in cases where there are indications for treatment with leeches.

A new protein in accordance with the invention can form pharmaceutically suitable salts with any non-toxic organic or inorganic acid. Inorganic acids javlyautsya acid phosphate and sodium acid sulphate of potassium. Examples of organic acids are mono-, di - and tricarboxylic acids, such as acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleimide, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic acid, and sulfonic acids, such as methanesulfonate. Salt having end carboxypropyl amino acid parts include non-toxic salts of carboxylic acids formed with any suitable inorganic or organic bases. These salts include, for example, salts of alkali metals such as sodium or potassium, alkaline earth metals such as calcium and magnesium, light metals of group IIIA including aluminum; and organic primary, secondary and tertiary amines, such as trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethenamine, N,N'-dibenziletilendiaminom, dehydroabietylamine and N-alkylpiperidines.

The preparations in accordance with the invention can be introduced in the form of standardized doses containing conventional non-toxic pharmaceutically suitable carriers, diluents, auxiliaries and fillers, which are typical for parenteral introduction is jakcie and infusion. A uniform dose in accordance with the invention can contain daily amount of protein of the present invention or can be divided into several parts, which together form the desired dose. Optimal therapeutically suitable dose for a given patient (mammal, including humans) depends on various factors, such as activity used specifically active material, age, weight, sex, diet, time and route of administration, rate of excretion of the active material from the body and so on, Therefore, the concentration in the blood, which inhibits the stimulation of platelet aggregation by collagen, preferably, is between 0.1 mg/l 50 mg/l

As mentioned above, the term "pharmaceutically suitable carrier" refers to an inert, non-toxic solid or liquid filler, diluent or kapsulirujushchej material that does not interact with the active compound or substance of the body of the patient. Suitable, preferably, liquid carriers, well known in pharmacy, such as sterile water, saline, aqueous dextrose, solutions of sugars, ethanol, glycols and oils, including butter oil, animal, rastitelni carriers in accordance with the invention is also used pharmaceutically suitable gels or creams, suitable for application to application, medical device, which are typically made from plastics and synthetic fibers.

The object of the present invention is also developing implantable or extracorporeal medical device, which is used in contact with fluid body and the surface of which has thromboresistance, coating her immobilized protein mentioned above. The protein according to the invention immobilized on the medical device so as to give the surface biocompatibility and thromboresistant. Such devices sometimes have the wetted surface, which is usually educere aggregation of platelets, which is not used for implantable and extracorporeal devices, when applied in contact with blood or other body fluids. Examples of such devices are prostheses, artificial organs, suture material, synthetic segments of blood vessels, catheters, dialyzers, pipes and vessels that carry blood.

The formation of coatings on medical devices and immobilization of proteins carried out by standard methods (for example, US 4885207).

The present invention relates shall prevent stimulated collagen platelet aggregation and binding of vWF to collagen in vitro, in vivo and in vitro processing.

Brief description of drawings

Fig. 1. Cleaning brandina from the saliva of leeches gel chromatography. Details are given in example 2. Dark shaded areas indicate the active fraction. Number (3-14) under the curve denote the number of factions.

Fig. 2. Cleaning brandina electrophoresis gel in the presence of dodecylsulfonate sodium. For details, refer to example 4. Numbers are indicated fractions (8-12) and markers (M). Fractions 10 and 11 give a positive result when tested as in example 1.

Fig. 3. A standard curve collagenolytic activity, measured by asokaramR. For details, refer to example 4. Vertical axis: optical density (OD) at 560 nm, the horizontal axis: collagenase activity (IU/sample).

Fig. 4a. A dose-dependent inhibition of stimulated collagen platelet aggregation. The details outlined in example 5. Vertical axis: % aggregation, the horizontal axis: concentration brandina (nm).

Fig. 4b. Collagen specificity of inhibiting the aggregation of platelets. Details are given in example 5. Vertical axis: % of aggregation, the horizontal axis: 1 = Supervisory experience, 2 = collagen, the re 6. Vertical axis: optical density (OD) at 405 nm, the horizontal axis: concentration of purified protein (nm) of purified protein;........ control w/o protein.

The following examples describe the present invention in more detail.

Example 1.

The activity of the protein of Hirudo medicinalis in vitro as an inhibitor of stimulated collagen platelet aggregation.

In the original (raw) saliva Hirudo medicinalis can detect activity, which inhibits dependent dose-dependent manner platelet aggregation in platelet-rich plasma (PRP), caused by the presence of collagen.

Blood taken from volunteers, the final concentration of sodium citrate 0,38%. Enriched and platelet-poor plasma receive differential centrifugation.

Platelet aggregation is carried out in agrometer PAP-4 (Biodata) the addition of various concentrations of corporate collagen hormR(Hormonchemie). As an option, use the maximum extinction after the addition of collagen. Antithrombotic increase size.

The analysis described above, is used throughout the following protein purification and to obtain characteristics of the purified protein of the invention.

Example 2.2with pH 8.0 (TC buffer) at a concentration of 20 mg/ml Saliva injected into a column of CM-fractogelR(E. Merck, Darmstadt, FRG) and elute NaCl gradient in the same buffer. Erwerbende faction active in the analysis of inhibition of platelet described in example 1 does not have activity of hirudin. Final cleaning is performed gel chromatography on a column with modified diola in the silicon dioxide in 20 mm Tris-HCl, 10 mm CaCl2, 200 nm NaCl with pH 8.0 (TCN-buffer). A typical example of this stage of treatment is shown in Fig. 1. The yield of purified protein relative to the amount of protein in the original saliva is 5%. In other experiments, the output is between 2 and 7%.

Example 3.

One-step protein purification by preparative electrophoresis.

According to other variant protein can successfully provide in a single phase preparative electrophoresis. In the device "Prep-cellR(Biorad, Munich, FRG) cylindrical polyacrylamide gel usually with 10% acrylamide will polimerizuet according to the manufacturer's instructions. The sample is injected into the buffer for electrophoresis. In the process of electrophoresis buffer moves perpendicular to the direction of electrophoresis, providing transfer blueraven is under dodecylsulfonate sodium and combined in accordance with the molecular weight of 15.0 kDa. In another modified experiment a certain molecular weight is 14.5 kDa.

Example 4.

Characterization of the protein.

Purified protein having activity in accordance with example 1 has a molecular mass of approximately 15000 D defined by polyacrylamide gel electrophoresis in the presence of dodecylsulfonate sodium in terms of recovery. In Fig. 2 shows the active fraction obtained typical gel chromatography described in example 2. Protein bands on the gel visualize silver staining.

Any proteolytic activity of the purified protein using the preferred method of determination described below is not installed. Casein, covalently modified resolutionR(Boehringer, Mannheim, FRG), used as a substrate with proteinase K as a positive control. 50 μl of a solution of 0.4% casein add-labeled resorufin 50 μl of 200 mm Tris, 20 mm CaCl2with pH 7.8 and 100 μl of solution samples. After incubation for up to 24 hours at 37oC the reaction is interrupted by addition of 450 μl of a 5% solution of trichloroacetic acid. The reaction mixture was centrifuged and 400 μl of the supernatant are transferred into CUV what it shows significant proteolytic activity in the initial saliva, whereas in the case of purified protein observe the magnitude of the absorption is slightly lower than the background level (table 1).

In addition to the proteolytic activity described above, the original saliva has also collagenolytic activity. The preferred method of measuring such activity involves the use of Azolla, non-specific chromogenic substrate collagenase, as follows. AsacolR(Calbiochem) suspension in 50 mm Tris, 200 mm NaCl, 0.1 Brij 35,0,01% NaN3with pH 8.0 (buffer A) with a concentration of 20 mg/ml and washed twice by centrifugation, aspiration and resuspension. 100 ál of sample or control buffer is transferred by pipette into the wells of microtiter plate with 96 wells in a series of dilution in buffer A, then add 100 µl of the suspension Azolla. This mixture is incubated at 37oC usually within 18 hours. To terminate the reaction, unreacted substrate precipitated by centrifugation at 1000 Hg for 10 minutes. Supernatant transferred to a new microtiter tablet and measure the absorption at 560 nm. To obtain the standard curves (Fig. 3) use collagenase from Clostridium histolyticum (Sigma).

Table 2 shows the results of typical measurements primene show activity in the analysis asokaram, unexpectedly, it was shown that the purified protein of the present invention has no significant collagenolytic activity above background values.

Example 5.

A dose-dependent inhibition of platelet aggregation purified protein.

Platelet-rich plasma is incubated for 2 minutes at 37oC with the test compound and aggregate by adding collagen to the final concentration of 1.25 µg/ml Protein of the present invention (branding) had IC50about 15 nm. In other experiments receive the results between 0.5 and 100 nm. The crude extract of saliva shows the ability to inhibit stimulated collagen and stimulated ADP platelet aggregation. On the contrary, branding ineffective when the most possible high concentrations. Details can be seen in Fig. 4a and 4b.

Example 6.

Inhibition of vWF binding to collagen in vitro with purified protein.

In addition to the direct inhibition of the interaction of platelets with collagen purified protein of the present invention has an unexpected additional ability to inhibit the binding of vWF.

It shows the coating of microtiter plate with 96 locaility for 18 hours using a phosphate buffer with a pH of 7.2. After incubation for 1 hour at 37oC wells blocked with 250 μl of bovine serum albumin in saline solution with phosphate buffer and washed three times with saline phosphate buffer without albumin. Add 25 ál of sample containing the protein according to the invention, and then 75 μl of human plasma, diluted in the ratio 1:80 saline phosphate buffer containing 5 mm ethylenediaminetetraacetic acid, 0.1% bovine serum albumin, 0.001% of tween 80 and has a pH of 7.4, and incubated 2 hours at 37oC. After another washing associated vWF determined using 110 ál of diluted 1/4000 polyclonal rabbit anti-vWF-anticorodal, conjugated with horseradish peroxidase (Dako, Copenhagen, Denmark). Anticigarette incubated 1 hour at 37oC. Manifestation of color spend ABTS for 30 minutes at 37oC, measured at 405 nm. In some experiments, conducted with purified factor vWF (Serbio, Remagen, FRG) instead of human plasma get such results comparable with experiment with plasma.

The binding of vWF from plasma and purified form can be prevented by brandylina, purified protein according to the present invention, dependent on dose. 50%="ptx2">

Linking brandina collagen is achieved not only in the inhibition of the interaction of platelets with collagen, but also the binding of factor a background of Villebranda with this subendothelial matrix component.

1. Substantially pure protein having the ability to inhibit stimulated collagen platelet aggregation and prevent the interaction between collagen and the factor a background of Villebranda (vWF), derived from the saliva of medicinal leeches Hirudo medicinalis and having a molecular weight of 14 to 15.5 kDa.

2. A method of obtaining a protein having the ability to inhibit stimulated collagen platelet aggregation and prevent the interaction between collagen and the factor a background of Villebranda, characterized in that the protein isolated from the saliva of the medicinal leech Hirudo medicianalis, cleaning is performed in one stage by preparative polyacrylamide gel electrophoresis and protein fraction having a molecular weight of 14 - 15.5 KD, unite.

3. Pharmaceutical composition containing as active ingredient a protein under item 1, in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

4. The pharmaceutical composition of the medical device before use.


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