Composition, inhibiting platelet aggregation

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology, namely, to obtaining inhibitors of adhesion and/or aggregation of platelets, and can be used in medicine. A polypeptide, used as a component of a pharmaceutical composition and in sets for screening of the inhibitors of platelet adhesion or aggregation, is obtained in a recombinant way with the application of a matrix of the salivary gland cDNA of Anopheles stephensi.

EFFECT: invention makes it possible to obtain the polypeptide, possessing inhibiting activity with respect to platelet aggregation and/or inhibiting activity with respect to platelet adhesion.

10 cl, 4 dwg, 5 ex

 

The technical field TO WHICH the INVENTION RELATES

The present invention relates to a polypeptide possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, or pharmaceutical compositions, for example, inhibiting platelet aggregation compositions containing as an active ingredient expressed product (recombinant polypeptide), a murine encoding the specified polypeptide of the polynucleotide.

In addition, the present invention relates to a polypeptide having the ability to bind with collagen, or a pharmaceutical composition containing as an active ingredient specified polypeptide.

The present invention also relates to a method for screening compounds (e.g., agonists) that facilitate inhibitory activity against platelet aggregation, as an active ingredient pharmaceutical compositions. In addition, the present invention relates to a new polypeptide possessing inhibitory activity against platelet aggregation, and to codereuse it to the polynucleotide.

The LEVEL of TECHNOLOGY

Platelets aggregate because of damage to the endothelial cells of blood vessels and various other factors. Occlusion platelet thrombus�m of the coronary vessel, cerebral or peripheral vessel vessel is the cause of myocardial infarction, cerebral infarction or chronic obstruction of the arteries, respectively. Examples of thrombotic diseases which follow such activation (stimulation of aggregation) of platelets include arterial sclerosis, ischemic cerebral infarction, ischemic heart disease, including myocardial infarction and angina, chronic obstruction of the arteries, and venous thrombosis.

Inhibiting platelet aggregation drugs used as medicines for the prevention of ischemic disorders that accompany the above-mentioned various diseases in the form of complications, and as drugs for the prevention of pathological conditions after hypertension, the pulmonary hypertensie, cerebral infarction, pulmonary infarction and subarachnoid hemorrhage. In addition, the above medicines are used for the prevention of blood clots during percutaneous transluminal coronary angioplasty (PTCA) and stent placement, and is also used as agents for the prevention of restenosis after stent deployment by incorporating by applying on the stent or sealing him in such drugs inhibiting platelet aggregation.

IU�do the a mosquito pierces the skin with the help of the jaws of the oral apparatus, performing the function of the needle reaching the peripheral blood vessel during blood sucking, and for the detection of peripheral blood vessel often repeats the behavior in the form of a perforation and extraction of the jaws, which is referred to as "sensing". Suppose that at the same time the mosquito secretes saliva that contains promote vasodilation substance to facilitate detection of the blood vessel. Due to the above-mentioned sensing peripheral blood vessel is often damaged, becoming stagnant. Generally, when damage to a blood vessel collagen tissue beneath the endothelium becomes unprotected from the destroyed cells released adenosine diphosphate (ADP), and activated coagulation factors with the formation of thrombin. Thrombin strongly activates platelets to induce platelet adhesion, platelet aggregation and release of granules and, ultimately, leads to the formation of a stable thrombus by means of coagulation with the formation of fibrin (mechanism of hemostasis). It is known that the mosquito's saliva contains a substance that inhibits the mechanism of hemostasis (see non-patent document 1).

The most studied protein of the salivary gland of the mosquito is apyrase. This enzyme is f�Xia inhibiting platelet aggregation substance, first identified in salivaAedes aegypti. The resulting decomposition of ADP released from damaged endothelial cells of blood vessels, red blood cells and adherent platelets, to AMP (adenosine monophosphate), apyrase inhibits platelet aggregation and exerts antihemostatic action. To explain the different behavior which vampires insects in addition to mosquitoes, apparently, is a significant function analysis of substances in their saliva.

It is predicted that the number of substances of saliva involved in the inhibition of platelet aggregation, and reported, for example, inhibitory activity against platelet aggregation protein derived fromTriatoma infestans(patent documents 1 and 2).

Among derived from the salivary gland proteins ofAnopheles stephensiidentified protein having inhibitory activity against blood clotting, but was not identified protein having inhibitory activity against platelet aggregation (see patent document 3).

In addition, it was reported 33 new proteins cloning the cDNA library of salivary glands ofAnopheles stephensibut the report did not describe the protein having inhibitory activity against platelet aggregation (see non-patent document 2).

The authors of the present invention previously reported about the protein (ARR) with GE (Gly to Glu)-rich �posledovatelnosti, cloned from the salivary glands ofAnopheles stephensi(see non-patent document 3). The protein is encoded by an open reading frame (ORF) of 810 p. O. and is a protein with a M. M. of 28.5 kDa, consisting, according to the calculations of 269 amino acid residues. Subsequently discovered that this protein is similar to the antigen from M. M. 30 kDa (access number in GenBank - AY226454) which is open and non-patent document 2, however, in non-patent documents 2 and 3 describe the effects and the function of the protein.

[Patent document 1] JP 2004-121091-A

[Patent document 2] JP 2004-121086-A

[Patent document 3] JP 2003-116573-A

[Non-patent document 1] Riberio, J. M., J. Exp. Biol., 108, 1-7 (1984))

[Non-patent document 2] Valenzuela, J. G., et al., "Exploring the salivary gland transcriptome and proteome of the Anopheles stephensi mosquito", Insect Biochemistry

[Non-patent document 3] Hiroyuki Watanabe et al., Medical Entomology and Zoology 55 Suppl., pp 41, 19 (2004).

Disclosure of the INVENTION

The present invention provides a new pharmaceutical composition, in particular an inhibitor of platelet aggregation and/or an inhibitor of platelet adhesion.

The present invention, furthermore, provides a new pharmaceutical composition containing as an active ingredient a protein having the ability to bind to collagen.

The present invention also provides a method for screening substances such as an agonist, which is inhibitory asset�spine against platelet aggregation and/or inhibitory activity against adhesion of platelets substances.

As a result of further extensive studies, in addition to studying the protein (AARR), previously reported by the authors of the present invention, the last have discovered a new protein having inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, salivary glandAnopheles stephensisuccessfully isolated and identified the DNA encoding this protein, and recently discovered that the protein has inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets.

The present invention has features that are contained in the following paragraphs 1-14.

Paragraph 1. Pharmaceutical composition containing as an active ingredient at least one of the following polypeptides (a) to(d):

(a) a polypeptide comprising the amino acid sequence of SEQ ID NO:1;

(b) a polypeptide comprising the amino acid sequence comprising one or more deletions, insertions, substitutions or additions of amino acids in the amino acid sequence of the above polypeptide (a), and possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets;

(C) a polypeptide comprising amino acid consisten�etelnost SEQ ID NO:3; and

(d) a polypeptide comprising the amino acid sequence comprising one or more deletions, insertions, substitutions or additions of amino acids in the amino acid sequence of the polypeptide (c), and possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets.

Item 2. Pharmaceutical composition containing as an active ingredient expressed product, which is expressed by at least one of the following polynucleotides (e) to(j):

(e) a polynucleotide comprising the DNA sequence SEQ ID NO:2 or its complement;

(f) a polynucleotide which hybridise under stringent conditions with the above mentioned polynucleotide (e) and capable of expressing a polypeptide possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets;

(g) a polynucleotide comprising a DNA sequence homologous to the above polynucleotide (e) 80% or more, and capable of expressing a polypeptide possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets;

(h) a polynucleotide comprising the DNA sequence SEQ ID NO:4 or its complement;

<> (i) a polynucleotide which hybridise under stringent conditions with the above mentioned polynucleotide (h) and capable of expressing a polypeptide possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets;

(j) a polynucleotide comprising a DNA sequence homologous to the above polynucleotide (h) 80% or more, and capable of expressing a polypeptide possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets.

Item 3. Pharmaceutical composition in accordance with paragraph 1 or 2, in which the indicated inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets is inhibitory activity against platelet aggregation induced by collagen, and/or inhibitory activity against adhesion of platelets to collagen.

Item 4. Pharmaceutical composition in accordance with paragraph 1 or 2, having the ability to bind to collagen.

Item 5. Inhibitor of platelet aggregation and/or adhesion inhibitor of the platelet-containing polypeptide described in paragraph 1, or expressed product, which is expressed by a polynucleotide described in POON�those 2.

Item 6. A method of screening an agonist inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, in which the level of inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets polypeptide described in paragraph 1, measured in the presence or absence of a test substance, and magnitude of that measured in the presence of a test substance, compared with the value measured in the absence of the test substance, for selecting a test substance that increases the inhibitory effect, as an agonist.

Item 7. A method of screening an agonist inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, in which the level of inhibitory activity of the expressed product, which is expressed as described in paragraph 2 of the polynucleotide, measured in the presence or absence of a test substance, and magnitude of that measured in the presence of a test substance, compared with the value measured in the absence of the test substance, for selecting a test substance that increases the inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets,as an agonist.

Item 8. Method for screening substances of the candidate, which is an agonist of the inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets polypeptide described in paragraph 1, expressed or product described in paragraph 2, which includes the following stages(1)-(4):

(1) the stage of preparation of the culture medium containing the cell, transformed expressing vector that expresses described in paragraph 1 polypeptide, or a cell comprising the described in paragraph 2 of the expressed product, and rich in platelets plasma;

(2) the step of adding platelet aggregation agent in the culture medium of the above stage (1) to induce platelet aggregation in the presence or absence of a test substance;

(3) the stage of measuring the level of platelet aggregation in the presence or absence of a test substance on the above stage (2); and

(4) the stage of selecting the test substance as a substance of a candidate, when the value measured in the presence of a test substance, the more the value measured in the absence of the test substance.

Item 9. A kit for screening an agonist of the polypeptide described in paragraph 1, or expressed product, which is expressed as described in paragraph 2 on�nucleotides, characterized in that the components it contains rich in platelets plasma, one of the polypeptides described in paragraph 1, and expressed the product described in paragraph 2, and the agent, platelet aggregation.

Item 10. The selected polypeptide comprising the amino acid sequence of SEQ ID NO:1.

Item 11. The selected polypeptide comprising the amino acid sequence of SEQ ID NO:3.

Item 12. The selected polypeptide comprising the amino acid sequence of SEQ ID NO:5.

Item 13. The polynucleotide comprising the DNA sequence SEQ ID NO:2 or its complement.

Item 14. The polynucleotide comprising the DNA sequence SEQ ID NO:4 or its complement.

Item 15. The polynucleotide comprising the DNA sequence SEQ ID NO:6 or its complement.

For the above-mentioned at least one polypeptide (protein) (a)-(d) is sometimes given below link in the form of "SY-001". At the expressed product of the above-mentioned at least one polynucleotide of (e)-(j) is sometimes given below link in the form "expressed product of the present invention".

BRIEF description of the DRAWINGS

Fig.1 shows the inhibitory activity of SY-001, produced in example 1, 3(1) in respect of platelet aggregation induced by collagen.

Fig.2 shows the inhibitory activity of SY-001, produced in �the reamer 1, 3(2), in respect of platelet aggregation induced by collagen.

Fig.3 shows the inhibitory activity against platelet aggregation SY-001, produced in examples 1 and 3, which inhibits platelet adhesion to collagen in example 5.

Fig.4 demonstrates that SY-001, produced in examples 1 and 3, has the ability to bind to collagen in example 6.

The BEST OPTIONS for carrying out the INVENTION

Presented here is the reduction of amino acids, peptides, base sequences, and nucleic acids are in accordance with the biological nomenclature of IUPAC-IUB, Eur. J. Biochem., 138: 9 (1984) defined by IUPAC-IUB, "Guideline for preparing specifications comprising base sequences and amino acid sequences" (Patent office), and is commonly used in the art symbols.

The polynucleotide (DNA molecule) here includes not only double-stranded DNA but also single-stranded DNA, including the sense chain and antisense chain, which they constitute, and is not limited to its length. Consequently, encoding SY-001 the polynucleotide comprises double-stranded DNA, including genomic DNA, single-stranded DNA (sense circuit), including cDNA, and single-stranded DNA (antisense strand) having a sequence complementary to the sense chain, and synthetic DNA fragments, if not mentioned�recover it otherwise.

The polynucleotide (DNA molecule) is not defined functional area, and may include at least one of the following areas: cupressiforme expression region encoding a region, a leader sequence, an exon and an intron.

The polynucleotide includes RNA and DNA. A polypeptide comprising a specific amino acid sequence, and a polynucleotide comprising a specific DNA sequence, include their fragments, homologues, derivatives and mutants.

Mutants of the polynucleotide (mutant DNA) include naturally occurring allelic mutants naturally occurring mutants and the mutants having a deletion, substitution, addition and insertion. But these mutants encode a polypeptide having essentially the same function as the function of the polypeptide encoded by the polynucleotide prior to the mutation.

Mutation of the polypeptide (modification of amino acid sequence) optionally occurs via occurring, for example, mutation or post-translational modification and may be a mutation is artificially produced using naturally occurring protein (e.g., SY-001). The above mutant polypeptide include allelic variants, homologues and naturally occurring mutants, homologous to the polypeptide prior to the mutation of at least 80%, preferably �and 95% and more preferably 99%.

Homology to the polypeptide or polynucleotide can be analyzed in the measurement using the FASTA program (Clustal, V., Methods Mol. Biol., 25, 307-318 1994)). As an example, the most preferred and easy method of analysis homologues can result in a method in which the sequence is stored on storage media (e.g. floppy, CD, HDD, hard drive disk external communications, digital video disk, etc.) that can be read by the computer, and then a database of known sequences are subjected to a search in accordance with well-known search procedure using the stored sequences. Specific examples of the databases of known sequences include the following:

-The DNA database of Japan (DDBJ) (http://www/ddbj,nig.ac.jp/);

-Genebank (http://www.ncbi.nlm.nih.gov/web/Genebank./Index.htlm); and

-Database of nucleic acid sequences of the European molecular biology laboratory (EMBL) (http:// ebi.ac/uk/ebi docs/embl db.html).

The majority of search algorithms for the analysis of homology available skilled in the art specialists. One example includes a program referenced in the BLAST. In this program there are 5 BLAST. Among them, three (BLASTN, BLASTX and TBLASTX) are designed to test nucleotide sequence. The remaining two percent�fools are designed to check the sequence of the protein (Coulson, Trends in Biotechnology, 12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)).

In addition, for the analysis of the identified sequences in the art, there are additional programs, for example, the program sequence alignment and program to identify more distant sequences.

Mutant DNA is silent (no change in amino acid residue encoded by the mutated nucleic acid sequence) or conservative in its encoded amino acid. Examples of conservative amino acid substitutions are shown below.

Initial amino acid
balance
Conservative substituted
acid balance
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Ser
Thr
Trp
Tyr
Val
Ser
Lys
Gln or His
Glu
Ser
Asn
Asp
Pro
Asn or Gln
Leu or Val
Ile or Val
Arg, Asn or Glu
Leu or Ile
Met, Leu or Tyr
Thr
Ser
Tyr
Trp or Phe
Ile or Leu

Typically, one or more codons encoding the Cys residue, affect the formation of disulfide bonds of a specific polypeptide.

Replacement of amino acid residues, which, as usually considered, affects properties�and protein includes the following changes:

(a) the replacement of a hydrophobic residue with a hydrophobic residue such as Leu, Ile, Phe, Val or Ala residue Ser or Thr;

(b) substitution of amino acid residue different from Cys and Pro, the residue is Cys or Pro;

(c) substitution of a residue having a load positively charged side chain, e.g., Lys, Arg or His, bearing a negative charge balance, for example, Glu or Asp; and

(d) replacement of amino acid residues having an extremely large side chain, e.g., Phe, amino acid residue not having a side chain, e.g., Gly.

(1) SY-001

SY-001 includes the amino acid sequence of SEQ ID NO:1 or 3 or an amino acid sequence having one or more deletions, insertions, substitutions or additions of amino acids in the amino acid sequence SEQ ID NO:1 or 3, and has inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, which inhibits platelet adhesion to collagen and/or the ability to bind to collagen.

SY-001 can be a polypeptide that is expressed in the system of protein expression usingEscherichia colior in the protein expression system using baculovirus (AcNPV), demonstrated described in later examples, using the method of gene recombination, or a polypeptide derived chemically� synthesis.

As one specific example of the amino acid sequence of SY-001 can lead to one of SEQ ID NO:1 or 3. Amino acid sequence of SY-001 is not limited to one of SEQ ID NO:1 or 3 and can be sequences, having with them a certain homology, (homologous sequences). Homologous sequences can include polypeptides comprising amino acid sequence having one or more deletions, insertions, substitutions or additions of amino acids in the amino acid sequence SEQ ID NO:1 or 3, and possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, which inhibits platelet adhesion to collagen and/or the ability to bind to collagen.

Inhibitory activity against platelet aggregation, which has a SY-001, includes the effects of inhibition or blocking condition in which the blood vessel (in particular, in coronary arteries, the aorta and cerebral arteries) increases the amount of the substance inducing platelet aggregation, or blood vessel facilitates platelet aggregation, or the state in which, when the damage of a blood vessel excessive platelet aggregate at the injury site.

Inhibitory activity against platelet aggregation can be detected by inhibition (suppression) of platelet aggregation induced platelet aggregation agent, rich in platelets plasma (PRP) with the addition of SY-001 in the plasma in the in vitro experiment.

In more detail, the inhibitory activity against platelet aggregation SY-001 can be measured using the following method. I.e. first using centrifugation from the whole blood of the person preparing PRP. Subsequently, this prepared PRP pre-incubated with a solution, e.g. a solution of PBS containing SY-001, and for platelet aggregation to this solution was then added to the agent, platelet aggregation. Examples of platelet aggregation agent include ADP (adenosine diphosphate), collagen, CRP (related to collagen peptide), convulxin, TRAP (peptide activator of thrombin receptor), epinephrine, arachidonic acid, U-46619 (an analog of thromboxane A2, TXA2 analogue) and A (calcium ionophor). The rate of platelet aggregation in the resulting solution is measured using the turbidimetric thromboangiitis with the transmission of light (MCM HEMA TRACER 313M supplied MC Medical), and the rate of inhibition with the help of SY-001 of platelet aggregation is calculated from the values of a dimension based on the values in the control not containing SY-001 So it is possible to detect the inhibitory activity against platelet aggregation SY-001.

You can also determine the inhibitory activity against adhesion of platelets SY-001 with the following method.

A solution of PBS containing SY-001 installed in concentrations that are added to the wells of 96-well plates coated with collagen solution and incubated for 30 minutes at room temperature. After incubation, the well was added a suspension of platelets and incubated for 45 minutes at room temperature. After incubation the incubation solution was removed from the wells using a pipette, and the hole was washed with PBS.

In the hole was added a solution of PBS containing 1% SDS, and after swinging and mixing of the hole is subjected to air drying. Then, the well was added distilled water, the amount of protein in each hole was measured using a kit for analysis of protein Dc (BIO-RAD Laboratories). The rate of inhibition of platelet adhesion using SY-001 is calculated from the values of a dimension based on the values in the control not containing SY-001, and inhibitory activity against adhesion of platelets SY-001 is calculated from the curve of platelet adhesion. Thus, it is possible to detect the inhibitory activity against adhesion of platelets SY-001, which inhibit platelet adhesion to collagen.

You can also define str�Amnesty SY-001 contact collagen using the following method.

To each well of 96-well plates with or without collagen coating add 300 ál of blocking solution and incubated for 1 hour. After removal from each well of the incubation solution to each well was added 100 μl of a solution containing SY-001 installed in concentrations, and incubated for 1 hour at room temperature. After removal from each well of the incubation solution to each well was then added 200 µl of 2% sucrose and incubated for 5 minutes at room temperature. After removal from each well of the incubation solution, the wells are dried in each well add 100 ál of reconstituted solution Nickel-HRP (KPP: Kirkegaard&Perry Laborator, Ltd) and incubated for 30 minutes at room temperature. After rinsing with buffer for leaching to each well add 100 ál of substrate for peroxidase ABTS (KPL Ltd.), and 96-well plate gently shake.

After completion of the reaction in each well was added 100 μl of 1% SDS, and then the hole is measured using reader Micro Plate Reader with the variation of the absorbance at 405-410 nm.

The ability of SY-001 at defined concentrations to contact the collagen calculated from the obtained values of OD (optical density) on the basis of the magnitude in the control not containing SY-001, and the ability of SY-001 contact collagen calculated from the curve svyazyvanie� collagen. Thus, it is possible to detect the ability of SY-001 contact collagen.

You can also define antiplatelet activity, which has a SY-001, using the following method. I.e. PRP is prepared by centrifugation of whole blood, obtained by sampling with a syringe with an anticoagulant blood sample from a healthy donor. Then, the resulting PRP was diluted in appropriate buffer, e.g., Tyrode-Hepes (134 mm NaCl, 0,34 mm Na2HPO4, 2.9 mm KCl, 12 mm NaHCO3, 20 mm Hepes, 5 mm glucose, 1 mm MgCl2, pH of 7.3), and this serial dilution add SY-001 installed in concentrations and pre-incubated. To this solution was added fluorescently labeled antibody against selectin P, antibody (supplied by Becton Dickinson), which learns RAS-1 (GPIIb/GPIIIa), fibrinogen and annexin V, and subsequently for platelet activation add platelet aggregation agent such as ADP, collagen or TRAP. Therefore, the assessment activity (inhibitory activity against activation) SY-001 in relation to platelet activation measured the fluorescence intensity of activated platelets using flow cytometry. This inhibitory activity against platelet activation is inhibitory activity against platelet activation.

In the above method, when used�and fluorescently labeled antibodies, the platelet and leukocyte, you can also assess the effect of compounds on the interaction of platelet and leukocyte (platelet-leukocyte adhesion).

For details of the method for measuring the inhibitory activity of SY-001 in respect of platelet aggregation, which uses a turbidimetric thrombogram with the transmission of light, in the present invention reference is made, for example, Born, G. V. R., "Aggregation of blood platelets by adenosine diphosphate and its reversal, Nature, 1962, 194, 927-8 and Sudo, T., et al., "Potent effects of novel anti-platelet aggregatory cilostamide analogues on recombinant cyclic nucleotide phosphodiesterase isozyme activity", Biochem. Pharmacol., 2000, 59, 347-56. With regard to the measurement of platelet activation in the present invention reference is made, for example, Ito, H., et al., "Cilostazol inhibits platelet-leukocyte interaction by suppression of platelet activation", Platelets, 2004, 15, 293-301.

Consequently, there is a likelihood of SY-001, having inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, as a therapeutic agent against the spread or prevention-related blood diseases and their complications, exerting an action on the blood and blood vessel are mammals animals with inhibition or prevention of thrombus formation or embolus.

Accordingly, it is assumed the effective use of SY-001, having inhibito�ing activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, as a therapeutic agent or prophylactic agent for pathological conditions after diseases and their complications (e.g., myocardial infarction, embolism of cerebral vessels, chronic obstruction of the arteries, arterial sclerosis, ischemic stroke, angina, venous thrombosis, hypertension, pulmonary hypertensie, cerebral infarction, pulmonary infarction, heart failure, nephritis, renal failure and subarachnoid hemorrhage caused by thrombus formation or embolus.

It also assumes the effective use of SY-001, having inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets for the prevention of thrombus formation during PTCA and stent placement and as an agent for preventing restenosis after stent deployment by incorporating by applying on the stent or sealing him in medicines containing SY-001.

The degree and position of modification, i.e. deletions, insertions, substitutions or additions" of amino acid residues in SY-001, presented in the form of the above-mentioned polypeptide (b) and (d) is not particularly limited, provided that the polypeptide (equivalent to a polypeptide) comprising a modified amino acid sequence, has �usesto the same activity, the activity of the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 3. Preferably, this modification is usually raised from about one to several amino acid residues.

In the present invention deletions, insertions, substitutions or additions of multiple amino acid residues" refer to the fact that deleteroute, inserted, substituted or added 2 or more and 20 or less amino acids. Preferably, the set of amino acids is 2 or more and 10 or less amino acids, more preferably 2 or more and 7 or less and even more preferably 2 or more and 5 or less. This is a modified amino acid sequence homologous to the amino acid sequence SEQ ID NO:1, or 3, for example, approximately 70% or more, preferably about 80% or more, more preferably about 95% or more, and even more preferably about 98% or more.

Specific examples of the polypeptide (SY-001), which is an active component of the pharmaceutical composition of the present invention, shown described in later examples.

SY-001 has inherent inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets.

SY-001 has �RISSA him the ability to bind to collagen.

Therefore, the pharmaceutical composition of the present invention containing as the active component SY-001, applicable as a therapeutic agent or prophylactic agent for pathological conditions after diseases and their complications (e.g., acute coronary syndrome, myocardial infarction, embolism of cerebral vessels, chronic obstruction of the arteries, arterial sclerosis, ischemic stroke, angina, venous thrombosis, hypertension, pulmonary hypertensie, cerebral infarction, pulmonary infarction, heart failure, nephritis, renal failure and subarachnoid hemorrhage caused by thrombus formation or embolus. Pharmaceutical composition of the present invention is also applicable for the prevention of thrombus formation during PTCA and stent placement and as an agent for preventing restenosis after the placement of the exhibition stand, drawing on the stent or sealing him in a pharmaceutical composition.

(2) the Polynucleotide (DNA molecule) encoding SY-001

One specific example of a polynucleotide encoding SY-001, (which is sometimes referred as "the DNA molecule of SY-001") may include a polynucleotide (DNA molecule) comprising a DNA sequence SEQ ID NO:2 or 4 or its complement.

Another example of a DNA molecule of SY-001 includes �olignucleotides, hybridizes under stringent conditions with a polynucleotide comprising the complement of the DNA sequence SEQ ID NO:2 or 4, and capable of expressing a polypeptide possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets.

"Stringent conditions" herein may be conditions under which hybridization occurs in 2×SSC containing 0.1% SDS, at 50°C and terminated after washing in 1×SSC containing 0.1% SDS, at 60°C.

In addition, another example of a DNA molecule (polynucleotide) SY-001 includes a polynucleotide comprising a DNA sequence homologous to the most closely related sequences among the polynucleotides comprising the DNA sequence SEQ ID NO:2 or 4, or its complement, 80% or more, preferably 95% or more and more preferably 98% or more, and capable of expressing a polypeptide possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets.

Essential requirement for DNA molecules (which are sometimes given in the form of modified DNA molecules), capable of expressing having the desired effects of the polypeptides shown as other examples, is that the polypeptide with the amino acid n�the sequence, encoded by this DNA molecule could exhibit inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets (in particular, inhibitory activity against platelet aggregation induced by collagen, and/or inhibitory activity against adhesion of platelets to collagen). In other words, the requirement is that the transformants transformed with the recombinant expressing the vector in which the polynucleotide is integrated (modified DNA molecule) that can Express the protein having inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, as its expressed product.

The above modified DNA molecule includes DNA molecules comprising DNA sequences that encode the amino acid sequence SEQ ID NO:1 or 3, in particular amino acid sequence (modified amino acid sequence) having one or more deletions, insertions, substitutions or additions of amino acids, and DNA sequences that encode its complement. Modified DNA can be sequences that can be used to detect DNA molecules of the present invention prior to its modification.

<> DNA molecule homologous to the polynucleotide (DNA SY-001 or its fragments) included in the DNA molecule of SY-001 and comprising the DNA sequence SEQ ID NO:2 or 4, means a set of related DNA molecules, between which and the DNA sequence of SEQ ID NO:2 or 4 there is a homology of sequences which are recognized according to their structural characteristics, their common patterns of expression and the similarity of their biological functions as one of a family of DNA-molecules.

Such homologous DNA molecule can be molecules, artificially obtained on the basis of naturally occurring DNA molecules (e.g., DNA fragment SY-001). Examples of such an artificial method of obtaining include genetic engineering techniques such as site-directed mutagenesis [Methods in Enzymology, 154, 350, 367-382 (1987); ibid 100, 468 (1983); Nucleic Acids Res., 12, 9441 (1984); "Zoku Seikagaku Jikken Kouza 1", "Idenshi Kenkyuho II" edited by the Japanese Biochemical Society, p 105 (1986)], the methods of chemical synthesis, such as phosphocreatine method and phosphoramidite method [J. Am. Chem. Soc., 89, 4801 (1967); ibid 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981); ibid. 24, 245 (1983)] and their combinations. More specifically, the DNA molecule can also be synthesized using phosphoramidite method or phosphotriesterase method, and also can be synthesized using commercially available automated synthesizer of oligonucleotides. The double-stranded fragment mo�but to obtain by synthesis of the complementary strand and annealing the complementary strand to chemically synthesized single-stranded chain with a corresponding condition or adding the complementary strand to chemically synthesized single-stranded chain using DNA polymerase with an appropriate primer.

The DNA sequence of SEQ ID NO:2 or 4, which is a specific variant of the DNA molecule of SY-001 is one of the examples of the combinations of codons encoding amino acid residues of the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 3. The DNA molecule of SY-001 is not limited to a DNA molecule having a specific DNA sequence, and can have a combination of optimal codons and DNA sequence selected for each amino acid residue. The codon can be selected in accordance with the standard method. At this time you can take into account the frequency of use of codons used in the host [Nucl Acids Res., 9, 43(1981)].

(3) Obtaining a DNA molecule of SY-001

The DNA molecule of SY-001 is easy to produce and receive using a synthesis based on the open here information about nucleic acid sequence of the polynucleotide encoding SY-001, or direct synthesis of the DNA molecule corresponding to the nucleic acid sequence encoding the amino acid sequence, on the basis of information about the amino acid sequence of SY-001 (chemical DNA synthesis). For getting you can apply a General genetic engineering techniques [see, for example, Molecular Cloning 2d Ed, Cold Spring Harbor Lab. ress (1989); "Zoku Seikagaku Jikken Kouzs 1", "Idenshi Kenkyuho II" edited by the Japanese Biochemical Society (1986)].

As an example of a method of chemical synthesis of DNA, by which is directly synthesized DNA molecule of SY-001 can result in solid-phase synthesis method using phosphoramidite method. For this method of synthesis can be used automatic synthesizer.

Obtaining the DNA molecule of SY-001 with the help of genetic engineering methods can more specifically be done via the preparation of a cDNA library from the appropriate source in which expressives DNA molecule of SY-001, in accordance with the standard method and selecting from the library of the desired clone, using the appropriate probe or specific in relation to the DNA molecule of SY-001 antibody [Proc. Natl. Acad. Sci., USA., 78, 6613 (1981); Science, 222, 778 (1983)].

In the above method as an example of the source cDNA can lead to various cells and tissues in which to Express the DNA molecule of SY-001, and extend from them cultivated cells. In particular, it is desirable to obtain salivary glandAnopheles stephensithat is insect-terrorist. All of the following: extraction and isolation of total RNA from the source, the separation and purification of mRNA and obtaining and cloning of cDNA can be carried out in accordance with standard methods.

The DNA molecule of SY-001 can be obtained using libraries� salivary gland cDNA Anopheles stephensiobtained by extraction, separation and purification of mRNA salivary glands. In addition, the DNA molecule of SY-001 can also be obtained using a library of phages prepared by extraction of the above mRNA salivary glands, addition of poly-A to RNA, then collecting the RNA with poly-A, obtain cDNA using reverse transcriptase and subsequently adding sites of restriction enzymes at both ends of the cDNA that is inserted into the phage.

A method for screening a DNA molecule of SY-001 of the cDNA library is not particularly limited, and can be developed in accordance with standard methods. As an example of a specific method, you can lead the method in which the corresponding cDNA clone selected using immunological screening using antibodies (e.g., antibodies against salivaAnopheles stephensi) specific against cDNA produced a protein, the method, the plaque hybridization using a probe that selectively binds to the target DNA sequence, the method of hybridization of the colonies and their combinations.

The probe used in each of the above methods of hybridization, is, as a rule, the DNA fragment is chemically synthesized on the basis of information about the DNA sequence of the DNA molecule of SY-001. As the above probe can be advantageously used�SQL already obtained the DNA molecule of SY-001 and its fragment. In addition, as probes for the above screening can also use a semantic primer and antisense primer, obtained on the basis of information about the DNA sequence of the DNA molecule of SY-001.

Used as a probe DNA (nucleotides) represents the partial DNA (nucleotides) corresponding to the DNA sequence of SY-001 and includes at least 15 consecutive DNA, preferably at least 20 consecutive DNA and more preferably at least 30 consecutive DNA. As a probe, you can use the clone positive in relation to the said product DNA molecules of the present invention.

After receiving the DNA molecule of SY-001 can appropriately use a method of amplification of DNA/RNA by PCR method [Science, 230, 1350 (1985)]. In particular, from the library when it is difficult to get full-size cDNA, can be appropriate to use the RACE method [rapid amplification of cDNA ends; Jikken Igaku, 12(6), 35(1994)], in particular the 5'-RACE method [M. A. Frohman, et al., Proc. Natl. Acad. Sci., USA., 8, 8998 (1988)].

Used for PCR-primer method can optionally be constructed based on the sequence information of the DNA molecule of SY-001, as shown in the later examples, and synthesize in accordance with the standard method. As such a primer can �use DNA (primer for the SP6 promoter and primer for the T7 terminator), added at both ends of the cDNA plasmid vector into which the cDNA is integrated SY-001, as shown in described later example.

Amplificatory PCR method, the DNA fragment or RNA can be isolated and cleaned in accordance with standard methods, for example, using gel electrophoresis.

The DNA molecule of SY-001 and its various DNA fragments obtained as described above can be sequenced in accordance with standard method, for example, dideoxy-method [Proc. Natl. Acad. Sci., USA., 74, 5463 (1977)] or by the method of Maxima-Gilbert [Methods in Enzymology, 65, 499 (1980)] or simply using a commercially available kit for sequencing.

(4) Obtaining methods genetic engineering expressed product of the present invention

Expressed product (recombinant SY-001) of the present invention can easily and stably be obtained in large quantities expressed in the form of product DNA molecules or protein containing SY-001, using sequence information of the DNA molecule of SY-001, in accordance with the General methods of genetic engineering [e.g., Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990 (1983)]. More specifically, the expressed product of the present invention can be obtained by preparing a recombinant DNA (expression vector) that is capable of expressing the DNA �odorous desired protein, in the host cell, transforming the host cell with this vector with obtaining transformant, cultivation transformant and collecting the target protein from the resulting culture.

When you receive the expressed product of the present invention as a host cell, you can use any of prokaryotic organisms and eukaryotic organisms. For example, prokaryotic organisms as a host can be any ofEscherichia coli, Bacillus subtilisand the like, usually used. Fittingly, if you useEscherichia coliin particular strain K12Escherichia coli. The host cell eukaryotic organisms include cells of vertebrates and yeast. Examples of suitable vertebrate cells include COS cells monkeys [Cell, 23: 175 (1981)], the cells are Chinese hamster ovary and their line with a deletion of the gene reductase enzyme dihydrofolic acid [Proc. Natl. Acad. Sci., USA., 77: 4216 (1980)], as examples of suitable yeast cells include cells belonging to the genus Saccharomyces. Of course, the host cell is not limited to them.

When the host is used prokaryotic cell with the use of a vector capable of replicating in the host cell can appropriately be used expressing plasmid, obtained by tting the promoter and SD sequence (sequence Shine-Dalgarno) and iniziare�to the next codon (for example, ATG) necessary for initiation of protein synthesis, 5' from the gene of the present invention so that the gene could be expressed in this vector. As the above often use vector plasmids such as pET-16b, pET-32, pBR322, pBR325, pUC12 and pUC13 derived fromEscherichia coli. Not limited to, possible to use various known vectors. Examples of commercially available vectors used for expressing system usingEscherichia coliinclude pGEX-4T (Amersham Pharmacia Biotech), pMAL-C2, pMAL-P2 (New England Biolabs), pET-16, pET-32, pET-21, pET-21/lacq (Invitrogen) and pBAD/His (Invitrogen).

When used as host cells of the spinal expression vector includes vectors usually contain a promoter, the site of RNA splicing, polyadenylation site and transcription termination sequence located 5' from a murine gene of the present invention. They may in addition contain start replication if required. Specifically, examples of the expression vector include pSV2dhfr [Mol. Cell. Biol., 1: 854 (1981)], having an early SV40 promoter. In addition to the above it is possible to use various known commercially available vectors. Examples of commercially available vectors used for expressing system using animal cells include such vectors as pEGFP-N, pEGFP-C (Clontrech), pIND (Invitrogen) and with pcDNA3.1/His (Invitrogen), for animal cells�x are vectors as pFastBac HT (GibcoBRL), pAcGHLT (PharMingen), pAc5/V5-His, pMT/V5-His and pMT/Bip/V5-His (Invitrogen) insect cells.

As an example of a vector for insect cells can result in a baculovirus vector (Takara), which is embedded in cDNA SY-001. Specifically expressed product of the present invention can be obtained by introducing a baculovirus expressing vector into which the cDNA is integrated SY-001, cultivated in BmN4 cells or larvae of the silkworm (Bombyx mori) using the nuclear polyhedrosis virus (BmNPV) of the silkworm expression system and discharge from the culture medium or liquid mass of the silkworm using chromatography. The expressed product of the present invention can also be obtained by embedding cDNA SY-001 of the nuclear polyhedrosis virus (AcNPV)Autographa californicaexpression in Sf9 cellsSpodoptera frugiperdaor Tn5 cellsTrichoplusia niand also purification from the culture supernatant using chromatography.

When used as the host cells are yeast specific examples of expression vectors include pAM82 [Proc. Natl. Acad. Sci., USA., 80: 1 (1983)], having the gene promoter of acid phosphatase. Examples of commercially available expression vectors for yeast cells include pPICZ (Invitrogen) and pPICZα (Invitrogen).

The promoter is not particularly limited. When used as a host bacterium, belonging�nd the genus Escherichia, you can preferably use a tryptophan (trp) promoter, lpp-promoter, lac promoter, recA promoter and PL/RL-promoter. When the host belongs to the genus Bacillus, are preferred SP01-promoter, SP02-promoter, penP-promoter, etc. When using as a host yeast can appropriately use pH05-promoter, a PGK-promoter, GAP promoter, ADH-promoter, etc. When used as a host cell of the animal, the preferred promoters are the promoter derived from SV40, promoter of retrovirus, a promoter of metallothionein, heat shock promoter, cytomegalovirus promoter, SRα-promoter, etc. When used as the host cells of an insect can cite as an example the promoter of baculovirus P10, promoter poledrini, etc.

As expressing the DNA molecule of SY-001 vector can preferably be used expressing a protein vector. Specific examples of the vector include pGEX (Promega) for expression in the form of a fusion with glutathione-S-transferase (GST) protein.

As an example, the polynucleotide sequences corresponding to the encoding the Mature polypeptide sequence that helps the expression and secretion of a polypeptide from a host cell, can result in a secretory leader sequence and the sequence. These sequences include mark�rnye sequence (exegetically marker, histidinemia marker), for example, the marker in the case of animal cells, used for cleaning are fused to the Mature polypeptide from the bacterial host.

The method of introducing desired recombinant DNA (expression vector) into a host cell and a method of transformation using this DNA is not particularly limited, and can be used by various common methods.

The resulting transformants can be cultured in accordance with standard methods, and the cultivation of a target protein (expressed product) encoded by the DNA molecule of SY-001, constructed as required, is expressed and produced (accumulated and secreted) intracellularly or extracellularly or on the cell membrane transformant.

The medium used for cultivation, can be arbitrarily selected and used depending on the host cell usually different in your environment.

The thus obtained expressed product (recombinant protein) of the present invention can be separated and cleaned using various separation techniques [see Biochemistry Data Book II, pages 1175-1259, 1stedition 1stprinting published by Tokyo Kagaku Dojin on June 23, 1980; Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987)], using physical and chemical nature of the product, as required.

In �astnosti, such methods include ordinary programmirovaniya processing, processing (salting out) using a protein precipitating agent, centrifugation, osmotic shock, destruction by ultrasound, ultrafiltration, various liquid chromatography, such as chromatography on a molecular sieve (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography and liquid chromatography high resolution (HPLC), dialysis and combinations thereof. A particularly preferred method involves affinity chromatography using the column, which is associated with antibody specific against SY-001.

When designing a polynucleotide encoding SY-001, you can use the DNA sequence of the DNA molecule of SY-001 SEQ ID NO:2. In this consistently also possible to freely select, change and use the codon for each amino acid residue, as required.

To the amino acid sequence of SY-001, when a part of the amino acid residues or amino acid sequence modified by substitution, insertion, deletion or addition, you can use various methods, such as site-directed mutagenesis described above.

(5) the Expressed product of the present invention, an active component of the pharmaceutical composition of the present invention

Expressed product (and expressed the same product used in the screening method of the present invention) of the present invention, an active component of the pharmaceutical composition of the present invention can be obtained using genetic engineering methods presented in the above section (4).

The inhibitory activity of the expressed product of the present invention against platelet aggregation is the same inhibitory activity against platelet aggregation defined for SY-001 and described above in the above section (1). This inhibitory activity can be determined according to the publicly known method of testing platelet aggregation, for example, using the method for determining the aggregation is rich in platelets plasma (PRP) using the turbidimetric thromboangiitis with the transmission of light.

More specifically, the inhibitory activity against platelet aggregation expressed product of the present invention can be determined and evaluated by measuring the level of platelet aggregation when added platelet aggregation agent in PRP prepared from human blood, in the presence or absence of SY-001, using the turbidimetric thrombogram with the transmission of light, and RA�couple of speed of inhibition using SY-001 platelet aggregation curve of platelet aggregation.

The inhibitory activity of the expressed product of the present invention against platelet adhesion is the same inhibitory activity against adhesion of platelets (i.e., inhibits platelet adhesion to collagen) defined for SY-001 and described above in the above section (1). This inhibitory activity can be determined according to the publicly known method of testing the adhesion of platelets to collagen, for example, using the method for determining the adhesion of platelets to collagen using a kit for analysis of protein Dc (BIO-RAD Laboratories).

More specifically, inhibitory activity against adhesion of platelets expressed product of the present invention against platelet adhesion to collagen can be determined and evaluated by measuring the adhesion of platelets to collagen when you add a suspension of platelets prepared from human blood, in the presence or absence of SY-001, using a set of Dc protein analysis, and calculation of inhibitory activity against adhesion of platelets SY-001 curve of platelet adhesion.

The ability of the expressed product of the present invention to contact with collagen is the same ability to bind to collagen (i.e., SY-001 binds to collagen) defined for SY-001 and described in�above in the above section (1). This ability to bind with collagen can be defined in accordance with a publicly known method of testing binding to collagen, for example, by the method of determining ability to bind with collagen, using the card reader Micro Plate Reader with the variation of the absorbance at 405-410 nm.

More specifically, the ability of the expressed product of the present invention to contact with collagen can be determined and evaluated by measuring the level of binding of SY-001 with collagen with the addition of SY-001 installed in concentrations in the presence or absence of collagen, using the card reader Micro Plate Reader with the variation of the absorbance at 405-410 nm, and calculating the ability of SY-001 contact collagen from the curve of binding to collagen.

The composition of the present invention is applicable as a therapeutic agent or prophylactic agent for pathological conditions after diseases and their complications (e.g., acute coronary syndrome, myocardial infarction, embolism of cerebral vessels, chronic obstruction of the arteries, arterial sclerosis, ischemic stroke, angina, venous thrombosis, hypertension, pulmonary hypertensie, cerebral infarction, pulmonary infarction, heart failure, nephritis, renal failure and subarachnoid crevoisier�I, due to the formation of thrombus or embolus, according to the inhibitory activity against platelet aggregation and/or platelet adhesion expressed product of the present invention, is included as an active ingredient. The composition of the present invention is also applicable for the prevention of thrombus formation during PTCA and stent placement and as an agent for preventing restenosis after placement of the stand by inclusion with application to stent or sealing him in this composition.

(6) Chemical synthesis of SY-001, which is an active component of the pharmaceutical composition of the present invention

Polypeptide (SY-001), which is an active component of the pharmaceutical composition of the present invention can also be obtained using the General method of chemical synthesis in accordance with the information about the amino acid sequence SEQ ID NO:1 or 3. The method includes methods for synthesis of peptides using conventional liquid phase method or solid phase method. Methods of synthesis of peptides include the so-called stepwise elongation method in which each amino acid is sequentially synthesized and linked one after the other for chain elongation and condensation method fragments, which are synthesized fragments, including several amino acids, and then sootvetstvujusjie connect. SY-001 can be synthesized by one of these two methods.

Condensation method used for peptide synthesis can also be performed in accordance with standard methods. Examples of standard methods include Isigny method, the method using a mixed anhydrate carboxylic acids, DCC method, the method using an active ester method, oxidation and reduction, DPPA (diphenylphosphoryl)-method, method using DDC + additive (1-hydroxy-benzotriazole) method using N-hydroxysuccinimide, N-hydroxy-5-norborene-2,3-dicarboximide and the Woodward method.

When the above reaction in the synthesis of peptide amino acid not involved in the reaction, or a carboxyl group in the peptide can be protected in the form of ester of lower alkyl alcohols such as methyl ester, ethyl ester and the ester tert-butyl alcohol, and Arakelova ester such as benzyl ester, para-methoxybenzyloxy ester and para-nitrobenzyloxy ester, typically by esterification.

Hydroxyl group in the amino acid such as tyrosine residue having a functional group in the side chain can be protected with acetyl group, benzyl group, benzyloxycarbonyl group or a tert-butyl group, but the protection is not always required. To�ome, for example, guanidinium in the balance of arginine may be protected with suitable protective groups, such as the nitro group, Casilina group, para-methoxybenzenesulfonyl group, methylene-2-sulfonylurea group, benzyloxycarbonyloxy group, isobutylacetophenone group or adamantanecarbonyl group.

The reaction of removing protection with these protective groups having a protective group of the amino acid, peptide and polypeptide, which is the active component of the resulting, ultimately, the pharmaceutical compositions of the present invention, can be performed in accordance with commonly used methods, such as contact recovery method and a method using liquid ammonia/sodium, hydrogen fluoride of bromovalerate, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonate, etc.

The thus obtained SY-001, which is the active component of the pharmaceutical compositions of the present invention can arbitrarily be cleaned in accordance with different methods, such as methods using ion exchange resins, distribution chromatography and gel chromatography and the method of distribution by the countercurrent method, commonly used in the chemistry of peptides.

(7) the Pharmaceutical composition of the present izobreteny�

It is important that the pharmaceutical composition of the present invention contain as the active component SY-001 or expressed product of the present invention. Pharmaceutical composition useful as a pharmaceutical composition, in particular as an inhibitor of platelet aggregation for inhibiting or blocking condition in which the blood vessel (in particular, in coronary arteries, the aorta and cerebral arteries) increases the substance inducing platelet aggregation, or blood vessel facilitates platelet aggregation, or the state in which, when the damage of a blood vessel excessive platelet aggregate at the injury site.

Pharmaceutical composition is also applicable, in particular, as an inhibitor of adhesion of platelets for inhibiting or blocking the adhesion of platelets to the substance in the form of an aggregating agent, such as collagen, a condition in which a blood vessel (in particular, in coronary arteries, the aorta and cerebral arteries) increases the substance inducing platelet adhesion, or in a blood vessel is facilitated by the ability of platelets to the adhesion, or the state in which, when the damage of a blood vessel excessive platelet aggregate at the site of damage�Oia.

Pharmaceutical composition of the present invention is applicable as a therapeutic agent or prophylactic agent for pathological conditions after diseases and their complications (e.g., acute coronary syndrome, myocardial infarction, embolism of cerebral vessels, chronic obstruction of the arteries, arterial sclerosis, ischemic stroke, angina, venous thrombosis, hypertension, pulmonary hypertensie, cerebral infarction, pulmonary infarction, heart failure, nephritis, renal failure and subarachnoid hemorrhage caused by thrombus formation or embolus, through the use of its inhibitory activity against platelet aggregation and/or platelet adhesion.

Pharmaceutical composition applicable for the prevention of thrombus formation during PTCA and stent placement to prevent restenosis after stent deployment by incorporating by applying on the stent or sealing him in the song, relying on the inhibitory activity of SY-001 against platelet aggregation and/or platelet adhesion.

SY-001 or expressed product of the present invention, an active component of the pharmaceutical composition of the present invention has inhibitory activity against Agra�ation of platelets and/or platelet adhesion and can be used for manipulation of the disease associated with platelet aggregation and/or adhesion of platelets, the cell or the target tissue through the use of its effect or activity. Examples of target cells, the influence of such platelet aggregation and/or adhesion of platelets may include blood cells, and platelets. Examples of the tissues comprising these cells can include a coronary blood vessel, cerebral blood vessel, the blood vessel of the neck, blood vessel, venous vessel, peripheral blood vessel, peripheral venous vessel, kidney, blood vessel, renal and hepatic arterial vessel.

In accordance with inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, or the effect of inhibition of platelet aggregation and/or the effect of inhibiting adhesion of platelets in the target cell, which(s) has the pharmaceutical composition of the present invention, it is possible to treat or prevent pathological conditions after diseases and their complications (e.g., myocardial infarction, embolism of cerebral vessels, chronic obstruction of the arteries, arterial sclerosis, ischemic stroke, angina, venous thrombosis, hypertension, pulmonary hypertensie, cerebral infarction, pulmonary infarction, cardio� failure, nephritis, renal failure and subarachnoid hemorrhage caused by thrombus formation or embolus.

In accordance with inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets SY-001 against platelet aggregation and/or adhesion of platelets may prevent the formation of thrombus during PTCA and stent placement to prevent restenosis after stent deployment by incorporating by applying on the stent or sealing him in the pharmaceutical compositions of the present invention.

The pharmaceutical composition of the present invention for the prevention of restenosis after placement of a stent can be used in the form of a cyclodextrin clathrate. The pharmaceutical composition of the present invention can also be used when applying (applying a thick layer or sputtering) on a biodegradable plastic which is the material of the stent, or the sealing of the stent.

SY-001 or expressed product, which is the active component of the pharmaceutical compositions of the present invention, also includes its pharmaceutically acceptable salt. Examples of such salts include non-toxic salts of alkaline metals such as sodium, potassium, lithium, calcium, magnesium, barium, and ammonium salts, salts of alkaline earth metals and ammonium�nye salt. These salts can be prepared in accordance with standard methods. The above salts are, in addition, include non-toxic acid additive salts obtained by the reaction of SY-001 or expressed product of the present invention with an appropriate organic or inorganic acid. Examples of typical representatives of the non-toxic acid additive salts include hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, malate, para-toluensulfonate (tosylate), citrate, fumarate, succinate, tartrate, sulfonate, glycolate, ascorbate, benzolsulfonat and napsylat.

The pharmaceutical composition of the present invention is prepared in the form of pharmaceutical preparation, getting SY-001, the expressed product of the present invention or its salt as an active ingredient, and it contains a pharmaceutically effective amount of the active ingredient with appropriate pharmaceutical carrier or diluent.

As an example, the pharmaceutical carrier used for the preparation of pharmaceutical compositions, fillers and diluents, such as fillers, thickeners, binders, wetting agents, disintegrators, surface activators and lubricants. Them you arbitrarily�playing and use depending on the form of standard doses of the resulting composition. Particularly preferred pharmaceutical composition is prepared optionally with the use of different ingredients, for example, stabilizer, antibacterial agent, buffer, isotonic agent, a chelating agent, adjusting the pH of the agent, a surfactant, etc., used for conventional protein compositions.

Examples of the stabilizer in the above composition include human serum albumin, normal L-amino acids, saccharides and cellulose derivatives. They can be used separately or in combination with surface-active substance. In particular, by using this combination in some cases further increases the stability of the active component.

L-amino acid is not particularly limited, and can be any amino acid from glycine, cysteine, glutamic acid, etc.

Sugars are not particularly limited. For example, you can use the monosaccharides, such as glucose, mannose, galactose and fructose, SharePort, such as mannitol, Inositol and xylitol, disaccharides such as sucrose, maltose and lactose, polysaccharides such as dextran, hydroxypropylmethyl, chondroitin sulfate and hyaluronic acid, and derivatives thereof.

Surfactant is not particularly limited, and you can use any of nonionic surface-�active substances and non-ionic surfactants. Their specific examples include surfactants based polyoxyethyleneglycol, sorbitan alkyl ether, polyoxyethylene alkyl ether, sorbitan manatiling ester and glycerides of fatty acids.

Cellulose derivative is not particularly limited. You can use methylcellulose, ethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose and sodium carboxymethylcellulose, etc.

The added amount of the above saccharides is approximately 0,0001 mg or more, and preferably about 0.01 to 10 mg, about 1 μg of the active component. The added amount of surfactant is about 0,00001 mg or more, and preferably approximately of 0.0001-0.01 mg relative to 1 μg of the active component. Add the amount of human serum albumin is approximately 0,0001 mg or more, and preferably approximately 0,001-0,1 mg, about 1 μg of the active component. Suitably, if the added amount of L-amino acid is about 0.001 to 10 mg, about 1 μg of the active component. The added amount of cellulose derivative is approximately 0,0001 mg or more, and preferably approximately 0,001-0,1 mg, about 1 μg of the active component.

To�icesto active component, contained in the pharmaceutical composition of the present invention, arbitrarily chosen from a wide range. Suitably, the composition amount of the active component was typically about 0,00001-70% in weight ratio, and preferably approximately of 0.0001-5% in weight ratio.

The pharmaceutical composition can be added various additives such as buffers, isotonic agents and hepatoblastoma agents. Examples of the buffer include boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid and/or salts thereof (e.g. alkali metal salts, such as salts of sodium, potassium, calcium and magnesium, and salts of alkaline earth metals). Examples of the isotonic agent include sodium chloride, potassium chloride, saccharides and glycerol. Examples of chelating agent include sodium edetate and citric acid.

The pharmaceutical composition can be prepared in the form of a solution and, in addition, lyophilized dosage form, obtained by lyophilization of pharmaceutical composition, which is prepared for use in appropriate concentration by dilution in a buffer containing salt.

The form of a standard dose of the pharmaceutical composition can be arbitrarily selected depending on therapeutic purpose. Primaryie typical representatives include solid dosage forms, such as tablets, coated tablets, powder medicines, powders, granules and capsules and dosed liquid forms such as solutions, suspensions, emulsions, syrups and elixirs. They further depending on ways of introduction are classified on oral agents, parenteral agents, nasal agents, vaginal agents, suppositories, sublingual agents, and ointment, and you can combine them, to form and be prepared in accordance with the General methods. If required, in the pharmaceutical compositions of the present invention can also contain coloring agents, preservatives, flavoring agents, corrigenda and sweeteners and other pharmaceuticals.

The method of administration of the pharmaceutical composition is not particularly limited and is determined depending on various forms of composition, age, sex, other conditions and severity of illness of the patient. For example, a tablet, a dragee, liquid, suspension, emulsion, granule and capsule is administered orally. Injectable agent is administered separately or in a mixture with conventional substitute fluid, such as glucose and amino acid, intravenously, if necessary, intramuscularly, intradermally, subcutaneously or intraperitoneally separately. The suppository is introduced intrarectally. Vaginal agent is injected into the vagina, nasal agent is administered in the nose, sublingual agent is injected into the ro�new cavity, and ointment, acting transdermal, topical use.

Dose of pharmaceutical composition is not particularly limited, and it randomly select from a wide range depending on the desired therapeutic effect, the route of administration, time of treatment, age, sex and other conditions of the patient. As a rule, preferably when the dose is determined so that the amount of the active component is typically from 0.01 μg to 10 mg, preferably from about 0.1 μg to 1 mg per kg of body weight per day. The composition can be administered using separate introductions from one to several times daily or intermittently.

(8) Screening compounds (agonists) that facilitate inhibitory activity against platelet aggregation and/or adhesion of platelets

The present invention also provides a method of screening of candidate compounds that promote inhibitory activity against platelet aggregation and/or platelet adhesion.

The method is characterized by measuring the inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 3, or a polypeptide comprising the amino acid sequence having one or more deletions, stavo�, substitutions or additions of amino acids in the amino acid sequence SEQ ID NO:1 or 3, and possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, in the presence or absence of a test substance and selecting a test substance that affect the inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets as an agonist, by comparing the value measured in the presence of a test substance, with the value measured in the absence of the test substance.

The level of inhibitor in relation to platelet aggregation effect can be determined by the rate of inhibition of platelet aggregation, calculated from the values of the measurements obtained using the turbidimetric thromboangiitis with the transmission of light. More specifically, a sample of human blood we take from a syringe with an anticoagulant, and is taken from whole blood using centrifugation to prepare rich in platelets plasma (PRP). To PRP, which is then pre-incubated at 37°C, was added a solution of purified SY-001, previously dissolved in PBS. Subsequently, the aggregation of platelets thereto was added a solution of collagen (supplied by NYCOMED) as an aggregating trom�ocity agent, and incubation was continued for a set period of time. Then, using the turbidimetric thrombogram with the transmission of light (MCM HEMA TRACER 313M supplied MC Medical), measure the transmittance of a solution curve of platelet aggregation. The rate of inhibition of platelet aggregation can be calculated from this curve in each case SY-001 or SY-001 + test substance. As above aggregating agent in addition to collagen can be used ADP (adenosine diphosphate), CRP (related to collagen peptide), convulxin, TRAP (peptide activator of thrombin receptor), epinephrine, arachidonic acid, U-46619 (an analog of thromboxane A2, TXA2 analogue), A (calcium ionophor), etc.

The level of inhibitory activity against adhesion of platelets can also be defined by the rate of inhibition of platelet adhesion, calculated from the values of the measurements obtained using the test kit Dc protein (Lowry method [Lowry, O. et al., J. Biol. Chem., 193, 265 (1951)] (BIO-RAD Laboratories). More specifically, a sample of human blood we take from a syringe with an anticoagulant, and a suspension of human platelets prepared from PRP obtained by centrifugation of whole blood. Solution of purified SY-001, previously dissolved in PBS, was added to the wells of 96-well plates, coated with collagen and incubated for 30 minutes at to�room temperature. After incubation, the well was added a suspension of platelets and incubated for 45 minutes at room temperature. After incubation the incubation solution was removed from the wells using a pipette, and the hole was washed with PBS.

In the hole was added a solution of PBS containing 1% SDS, and after swinging and mixing of the hole is subjected to air drying. Then, the well was added distilled water, the amount of protein in each hole was measured using a kit for analysis of protein Dc (BIO-RAD Laboratories), and the rate of inhibition of platelet adhesion using SY-001 is calculated from the values of a dimension based on the values in the control not containing SY-001, and inhibitory activity against adhesion of platelets SY-001 is calculated from the curve of platelet adhesion. The rate of inhibition of platelet adhesion can be calculated from this curve in each case SY-001 or SY-001 + test substance. As the above-mentioned inducer of platelet adhesion can be used collagen.

The present invention also provides a method for screening substances candidate that increase the inhibitory activity against platelet aggregation SY-001, which includes the following stages(1)-(4):

(1) the stage of preparation of a culture medium containing a cell expressing transformed SY-001 vector, and rich in otnosheniya.rabotayu plasma (PRP);

(2) the stage of induction of platelet aggregation by adding an aggregating platelets substances into the culture medium of the above stage (1) in the presence or absence of a test substance;

(3) the stage of measuring the levels of inhibition of platelet aggregation in the presence of a test substance and the absence of a test substance on the above stage (2); and

(4) the stage of selecting the test substance as a substance of a candidate, when the value measured in the presence of a test substance, the more the value measured in the absence of the test substance.

The present invention also provides a method for screening substances candidate that increase the inhibitory activity against platelet aggregation expressed product of the present invention, which includes the following stages(1)-(4):

(1) the stage of preparation of the culture medium containing the cell containing the expressed product of the present invention, and is rich in platelets plasma (PRP);

(2) the stage of induction of platelet aggregation by adding an aggregating platelets substances into the culture medium of the above stage (1) in the presence or absence of a test substance;

(3) the stage of measuring the levels of inhibition of platelet aggregation in the presence of the investigated�about substances and the absence of a test substance on the above stage (2); and

(4) the stage of selecting the test substance as a substance of a candidate, when the value measured in the presence of a test substance, the more the value measured in the absence of the test substance.

The present invention also provides a method for screening substances of the candidate, which determines inhibitory activity against adhesion of platelets SY-001, which includes the following stages(1)-(4):

(1) the stage of preparation of the culture medium (or wells) containing the cell, transformed expressing SY-001 vector, and well plate coated platelet adhesion substance (i.e., collagen);

(2) the step of inducing platelet adhesion by adding a suspension of platelets in the culture medium of the above stage (1) in the presence or absence of a test substance;

(3) the stage of measuring the levels of inhibition of platelet adhesion in the presence of a test substance and the absence of a test substance on the above stage (2); and

(4) the stage of selecting the test substance as a substance of a candidate, when the value measured in the presence of a test substance, the more the value measured in the absence of the test substance.

The present invention also provides a method for screening substances candidate, using�u which determines inhibitory activity against adhesion of platelets expressed product of the present invention, which includes the following stages(1)-(4):

(1) the stage of preparation of the culture medium (or wells) containing the cell containing the expressed product of the present invention, a well plate and covered with platelet adhesion substance (i.e., collagen);

(2) the step of inducing platelet adhesion by adding a suspension of platelets in the culture medium of the above stage (1) in the presence or absence of a test substance;

(3) the stage of measuring the levels of inhibition of platelet adhesion in the presence of a test substance and the absence of a test substance on the above stage (2); and

(4) the stage of selecting the test substance as a substance of a candidate, when the value measured in the presence of a test substance, the more the value measured in the absence of the test substance.

Method for screening of the present invention can be performed virtually using technology vysokomarochnogo for screening. In accordance with the practical application of this technology, for example, when skanirovanii on the catalyst inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets drug (including SY-001 and labeled ligand polypeptide) were incubated with any of the following: a synthetic reaction mixture, to�emochnoy fraction, fraction of blood, for example, is rich in platelets plasma, or suspension of platelets in the presence or absence of a test substance, under skanirovanie. Whether the test substance is an agonist of SY-001 or antagonist of SY-001, determined by the decrease in the amount of the bound labeled ligand. The level of activity of SY-001 can be determined by using a reporter system colorimetric marker (not limited to), embedding a reporter gene that responds to changes in the activity of the polynucleotide or polypeptide, or analysis of binding, publicly known in the art.

For screening the compound candidate as a catalyst inhibitory activity against platelet aggregation you can use competitive analysis, in which SY-001 or other mutant [e.g., SY-001(151-269), SY-001(21-269)] and another inhibitor of platelet aggregation (for example, acetylsalicylic acid, Cilostazol) combined with a compound that binds to them.

For screening the compound candidate as a catalyst inhibitory activity against adhesion of platelets can be used for competitive analysis in which SY-001 or other mutant [e.g., SY-001(151-269), SY-001(21-269)] and another inhibitor of platelet adhesion (e.g., ticlopidine) combined with the connection that links�recover it with them.

The test substance (compound candidate), proskanirovanno using the screening method of the present invention is a high probability that agonist SY-001 in the form of catalyst inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets.

The agonist can be a substance that increases the inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets when his presence in the system of screening of the present invention.

Specific examples of these substances under study include oligopeptides, proteins, antibodies, RNA molecules, miRNAs (small interference RNA), non-peptide compounds (synthetic compounds) fermented products, cell extracts (plant extracts, animal extracts and plasma. These substances may be substances, newly developed, or known substances.

Examples of substances inhibiting platelet aggregation, and/or a substance inhibiting adhesion of platelets include inorganic or organic low-molecular compounds, naturally occurring or synthetic peptides and polypeptides or peptides and polypeptides, obtained by gene recombination technology, which increase the activity of SY-001 by tie�tion with him. Semantic DNA molecule coding for SY-001 DNA molecules injected in vivo directly or input built-in in the recombinant vector of the form are also included in the above matter, inhibiting platelet aggregation, and/or a substance inhibiting platelet adhesion.

The substance is a candidate increases the inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, proskanirovanno in accordance with the screening method of the present invention is evaluated as follows. I.e. the substance candidate, which increases the inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, or promotes such activity to approximately 20% or more, preferably about 30% or more and more preferably about 50% or more compared with the control (with no test substance), can be estimated as compounds that promote inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets.

I believe that the connection that facilitate inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, applicable in quality�e therapeutic agent or prophylactic agent for pathological conditions of diseases and their complications for example, myocardial infarction, embolism of cerebral vessels, chronic obstruction of the arteries, arterial sclerosis, ischemic stroke, angina, venous thrombosis, hypertension, pulmonary hypertensie, cerebral infarction, pulmonary infarction, heart failure, nephritis, renal failure and subarachnoid hemorrhage caused by thrombus formation or embolus.

Among substances that promote inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets obtained by the screening method of the present invention is thought, there is also substance, itself possessing inhibitory activity against platelet aggregation induced platelet aggregation substance in vivo, and/or inhibitory activity against adhesion of platelets induced by platelet adhesion substance in vivo, and such a substance, believed to be applicable as an inhibitor of platelet aggregation and/or inhibitor of platelet adhesion in various fields, including the pharmaceutical field.

(9) a Kit for screening

The present invention also provides a kit for screening an agonist SY-001, characterized in that as component parts it contains rich in platelets plasma�, SY-001 (including the expressed product of the present invention) and platelet aggregation agent.

A kit for screening of the present invention as essential ingredients contains (1) SY-001 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO:1 or 3) or expressed product of the present invention (for example, the expressed product of the encoding SY-001 DNA molecules, DNA sequence SEQ ID NO:2 or 4), (2) cell culture medium, comprising rich in platelets plasma, and (3) platelet aggregation agent. As other optional ingredients set, similar to the kits for screening of this type may contain various reagents, such as cell culture medium, reaction diluents, coloring agents, buffers, fixing solutions and solutions used for washing.

Specific examples of the kit of the present invention include kits containing the following component parts 1-3: a part 1: cells (expressing SY-001 cells, cultured cells, including cells ofEscherichia colior insect cells transformed with a polynucleotide (DNA molecule), encoding SY-001) cultured in 60 mm Cup at 37°C in 5% CO2,at 0.5-1×105cells/cell, using MOPS buffer containing EGTA-Na; part 2: a substance (e.g., collagen,ADP), inducing platelet aggregation; part 3: rich in platelets plasma.

In the method of screening using the kit for screening of the present invention the rate of inhibition of platelet aggregation is determined in a rich in platelets plasma in the visible area of a single cell, which added check the substance (test substance, drug substance candidate). Then determine the rate of inhibition of platelet aggregation in the cell in which you check the substance was not added. Subsequently validate a significant difference between the first speed and second speed. These measurement and evaluation can be performed in accordance with standard methods.

The present invention also provides a kit for screening an agonist SY-001, characterized in that as component parts it contains platelet adhesion agent (i.e. collagen), SY-001 (including the expressed product of the present invention) and a suspension of platelets.

A kit for screening of the present invention as essential ingredients contains (1) SY-001 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO:1 or 3) or expressed product of the present invention (for example, the expressed product of the encoding SY-001 DNA molecules, D�To sequence SEQ ID NO:2 or 4), (2) platelet adhesion agent (i.e. collagen) and (3) a suspension of platelets. As other optional ingredients set, similar to the kits for screening of this type may contain various reagents, such as cell culture medium, reaction diluents, coloring agents, buffers, fixing solutions and solutions used for washing.

Specific examples of the kit of the present invention include kits containing the following component parts 1-3: a part 1: cells (expressing SY-001 cells, cultured cells, including cells ofEscherichia colior insect cells transformed with a polynucleotide (DNA molecule), encoding SY-001) cultured in 60 mm Cup at 37°C in 5% CO2,at 0.5-1×105cells/well, using PBS-buffer; part 2: platelet adhesion substance (e.g., collagen, ADP) to induce platelet adhesion; and a part 3: a suspension of platelets.

In the method of screening using the kit for screening of the present invention, the rate of inhibiting adhesion of platelets to determine the level of adhesion of platelets to collagen on the visible area of a single hole in which it is added check the substance (test substance, drug substance candidate). Then determine the rate of inhibition of adhesion of platelets in lung�, which check the substance was not added. Subsequently validate a significant difference between the first speed and second speed. These measurement and evaluation can be performed in accordance with standard methods.

EXAMPLES

Further, the present invention is described in more detail with reference to the following examples. These examples are given only for the purpose of explanation, an example and do not limit the present invention

Example 1

1. Preparation of cDNA library from the salivary glands ofAnopheles stephensi

Female ofAnopheles stephensi(strain SDA500) on 3-7 day after hatching forced to suck the blood of mice (strain BALD/c, purchased from CLEA Japan Inc.). After 6 hours of sucking the blood of a mosquito removed the wings, the legs, the head part and the abdominal part, and only the breast area, including the salivary gland, kept in liquid nitrogen. At that time, when he collected brisket from 300 mosquitoes was extracted with RNA using the kit RNeasy Midi Kit (QIAGEN). The library was obtained by collecting cobordism-added RNA, obtaining cDNA using reverse transcriptase, adding sites for restriction enzymes (sites for EcoRI and HindIII) on both ends and can be embedded into phages using the sets λ cloning SCREEN-1 Cloning Kits (Novagen).

2. The immunity screening cDNA library of salivary glands

Escherichia coli(ER-1647, Takara) were infected to�each of the above phage library and skanirovali using antibodies against the salivary glands of Anopheles stephensias a probe. Antibody against salivary glandAnopheles stephensiwere obtained by rabbit immunization with salivary gland homogenateAnopheles stephensitogether with the adjuvant of frand.

Escherichia coliER-1647 infected positive clone obtained by screening, and the phages were amplified. Then the phages were removed, and the embedded portion was cloned in the plasmid pSCREEN-1b(+) (Novagen) using the cut from the plasmid CreMediated Plasmid Excision (Novagen). The sequence of bases built part sequenced using DNA parts (primer for the promoter SP6: SEQ ID NO:10, code number of the product - TKR3867, Takara Bio, and primer for the T7 terminator: SEQ ID NO:11, code number of the product - NV432, Novagen), added to both ends pSCREEN, using the analyzer 310 Genetic Analyzer supplied by ABI.

The analysis of the sequence of bases is found that the fragment of the cloned gene contains a stop codon, but it lacks the initiating codon. To obtain the missing 5'region of cDNA SY-001 has performed 5'-RACE method [Frohman, M. A., et al., PNASC, 8, 8998-9002 (1988)] using the DNA of phage λSCREEN extracted from cDNA library of salivary glands, as template and the primers of the primer SP6 promoter (SEQ ID NO:10) and primer pAnS-1 (SEQ ID NO:12) for cloning of the DNA fragment of about 470 p. O. This DNA fragment overlaps the DNA fragment obtained in the above screenshot�Nghe, and, in addition, contained the initiating codon. Two of the DNA fragment was ligated to determine the complete sequence of bases of DNA molecules encoding SY-001.

The open reading frame found in cDNA encodes protein with the expected M. M. of 28.5 kDa of 269 amino acid residues and contained 807 p. O. This protein, as predicted, is a protein, secretively in the form of saliva, as predicted from the derived amino acid sequence, the protein is an acidic protein with a pI=3,8, 21 amino acids at N-end are hydrophobic and exhibit a similar signal peptide sequence, and the end is not attached to the membrane area.

This deduced amino acid sequence shown in SEQ ID NO:5. The sequence of bases of the DNA molecule encoding the amino acid sequence shown in SEQ ID NO:6.

3. The expression of SY-001 using recombinant DNA and cleaning

(1) the Receipt of SY-001 (22-269)

A DNA fragment encoding amino acid residues in positions 22-269 SY-001 (SEQ ID NO:5), were amplified by PCR. As the matrix used cDNA salivary gland presented above in 1. Used primer pAnSG-F7 (SEQ ID NO:13) was the site for NcoI (CCATGGG) in 3 to 8 positions from 5'-end, and used primer pAnSG-R1 (SEQ ID NO:14) was the site for NotI (GCGGCCGC) at 2-9 Polo�relations from the 5'-end. Then the DNA fragment was cloned into pENTR/D-TORO (Invitrogen) to construct the plasmid pENTR-SY-001-exon 1-4.

Subsequently, a DNA fragment of SY-001 (approximately 760 BP), obtained by cleavage of the plasmid pENTR-SY-001-exon 1-4 using NcoI and NotI, was built into the site NcoI/NotI pET32-b(+) (Novagen) to construct the plasmid pET32-SY-001-exon 1-4.

Escherichia coliBL21 (DE3) transformed with this plasmid pET32-SY-001-exon 1-4, and the resulting transformants were cultured with shaking in 6 ml LB-medium (LBA) containing 50 μg/ml ampicillin, for 15 hours at 37°C. Then the culture medium was added to 600 ml of LBA, which was then cultured with shaking for 4 hours at 37°C. Subsequently, the culture medium was added 6 ml of 100 mm IPTG, and the medium was further cultured with shaking for 4 hours at 37°C. the resulting culture medium was centrifuged at 6000×g for 15 minutes and supernatant was discarded. The resulting precipitate was literally by adding 40 ml of 6 M guanidine hydrochloride. The resulting bacterial solution was centrifuged at 30000×g for 25 minutes, and supernatant was collected. Thereto was added 1.8 ml Nickel-NTA (QIAGEN), then the mixture was stirred for 15 hours at 4°C. the Mixture was centrifuged at 3000×g for 3 minutes, supernatant was discarded and collected a Nickel-NTA. The collected n�Kel-NTA was added a solution of TBS (150 mm NaCl, 50 mm Tris-HCl [pH 7,5]) containing 6 M urea and 10 mm imidazole. The resulting mixture was centrifuged at 3000×g for 3 minutes and supernatant was discarded. The resulting Nickel-NTA was Packed in a column.

Protein SY-001-exon 1-4 were suirable with columns as follows. I.e. through the column successively passed 2 ml TBS-solutions containing 6 M urea and 20 mm, 50 mm, 100 mm or 200 mm imidazole, respectively, and collected corresponding fractions. A portion of each fraction was subjected to electrophoresis in 12% SDS-page and then stained with the dye Kumasi to identify fractions containing protein SY-001-exon 1-4. This fraction were dialyzed against PBS for 48 hours. The amount of protein was determined using BSA Protein Assay Kit (PIERCE), and the yield was 5 mg.

Amino acid sequence of the recombinant protein obtained in this way is shown in SEQ ID NO:7. Amino acid sequence in positions 1-162 and amino acid sequence in positions 410-420 are amino acid sequences derived from protein thioredoxin and plasmids pET32-b (+) containing the sequence His-marker, respectively. Amino acid sequence of SY-001 (22-269) of the present invention resides in the provisions 162-409.

(2) preparation of SY-001 (148-269)

PCR using as matrix of PLA�the foreign ministries of the pET32-SY-001-exon 1-4, prepared as described above in (1), was performed using primers pAnSG-F8 (SEQ ID NO:15) and pAnSG-R1 (SEQ ID NO:14). The resulting DNA fragment (382 BP) was cloned into pENTR/D-TORO (Invitrogen) to construct the plasmid pENTR-SY-001-exon 3-4. The plasmid pENTR-SY-001-exon 3-4 were digested with NcoI/NotI to obtain the DNA fragment size 372 p. O. This fragment embedded in sites NcoI/NotI pET32-b(+) (Novagen) to construct the plasmid pET32-SY-001-exon 3-4.

Escherichia coliBL21 (DE3) transformed with this plasmid pET32-SY-001-exon 3-4, and the resulting transformants were cultured with shaking in 6 ml LB-medium (LBA) containing 50 μg/ml ampicillin, for 15 hours at 37°C. Then the culture medium was added to 600 ml of LBA, which was then cultured with shaking for 4 hours at 37°C. Subsequently, the culture medium was added 6 ml of 100 mm IPTG, and the medium was further cultured with shaking for 4 hours at 37°C. the resulting culture medium was centrifuged at 6000×g for 15 minutes and supernatant was discarded. The resulting precipitate was literally by adding 40 ml of 6 M guanidine hydrochloride. The resulting bacterial solution was centrifuged at 30000×g for 25 minutes, and supernatant was collected. Thereto was added 1.8 ml Nickel-NTA (QIAGEN), then the mixture was stirred for 15 hours at 4°C. the Mixture was centrifuge�grown at 3000×g for 3 minutes, the supernatant was discarded and collected a Nickel-NTA. The collected Ni-NTA was added a solution of TBS (150 mm NaCl, 50 mm Tris-HCl [pH 7,5]) containing 6 M urea and 10 mm imidazole. The resulting mixture was centrifuged at 3000×g for 3 minutes and supernatant was discarded. The resulting Nickel-NTA was Packed in a column.

Protein SY-001-exon 3-4 was suirable with columns as follows. I.e. through the column successively passed 2 ml TBS-solutions containing 6 M urea and 20 mm, 50 mm, 100 mm or 200 mm imidazole, respectively, and collected corresponding fractions. A portion of each fraction was subjected to electrophoresis in 12% SDS-page and then stained with the dye Kumasi to identify fractions containing protein SY-001-exon 3-4. This fraction were dialyzed against PBS for 48 hours. The amount of protein was determined using BSA Protein Assay Kit (PIERCE), and the output was 1.8 mg.

Amino acid sequence of the recombinant protein obtained in this way is shown in SEQ ID NO:8, and this sequence contains 293 amino acid residues. In this amino acid sequence amino acid sequence in positions 1-160 and amino acid sequence in positions 283-293 are amino acid sequences derived from protein thioredoxin and plasmids pET32-b (+) containing posledovatelno�ü His-marker respectively. Amino acid sequence of SY-001 (148-269) is in the provisions 161-282.

Example 2

The preparation of recombinant SY-001 (21-269) using baculovirus expression system

(1) the Receipt of SY-001 (21-269) of baculovirus

A DNA fragment encoding the polypeptide in the provisions 21-269 SY-001 (SEQ ID NO:1), were amplified by PCR using as template cDNA salivary gland presented above in example 1 1. Used primer pAnSG-F10 (SEQ ID NO:16) was the site for BamHI (GGATCC) in 5-10 positions from 5'-end and a FLAG sequence in 12-41 positions. Used primer pAnSG-R1 (SEQ ID NO:14) was the site for NotI (GCGGCCGC) at 2-9 positions from 5'-end. The DNA fragment was cloned into pENTR/D-TORO (Invitrogen) to construct the plasmid pENTR-SY-001-exon 1-4.

Subsequently, a DNA fragment of SY-001 (780 BP), obtained by cleavage of the plasmid pENTR-SY-001-exon 1-4 with BamHI/NotI, embedded in sites BamHI/NotI pBACgus-1 (Novagen) to construct a vector plasmid for baculovirus transfer pBACgus-SY-001-exon 1-4.

Recombinant baculovirus was obtained using the kit for the preparation of recombinant baculovirus (BacVector-2000 Transfection Kit, Novagen) via cotransfection Sf9 cells using the above vector plasmids for baculovirus transfer pBACgus-SY-001-exon 1-4 and DNA BacVector-2000. The obtained recombinant baculovirus outlined in the form of AcNPV-SY-001-e�zones 1-4.

Ie Sf9 cells were cultured at 1×107cells per 150 mm Petri dish and was infected with AcNPV-SY-001-exon 1-4 at a multiplicity of infection of approximately 5. After 3-4 days to collect approximately 250 ml of culture supernatant from 10 to 150 mm Petri dishes, and thereto was added 1.5 ml Ni-NTA (QIAGEN), then the mixture was stirred for 15 hours at 4°C. the Mixture was centrifuged at 3000×g for 3 minutes, supernatant was discarded. Assembled Nickel-NTA, and to it was added 50 ml of a solution of TBS (150 mm NaCl, 50 mm Tris-HCl [pH 7,5]) containing 10 mm imidazole. The resulting mixture was centrifuged at 3000×g for 3 minutes and supernatant was discarded. The resulting Nickel-NTA was Packed in a column.

Protein SY-001-exon 1-4 were suirable with columns as follows. I.e. through the column successively passed 2 ml TBS-solutions containing 20 mm, 50 mm, 100 mm or 200 mm imidazole, respectively, and collected corresponding fractions. A portion of each fraction was subjected to electrophoresis in 12% SDS-page and then stained with the dye Kumasi to identify fractions containing protein SY-001-exon 1-4. This fraction were dialyzed against PBS for 48 hours. The amount of protein was determined using BSA Protein Assay Kit (PIERCE), and the yield was 0.8 mg.

Amino acid sequence of the recombinant protein SY-001 (21-269) thus obtained with�special, shown in SEQ ID NO:9. In this sequence the sequence in positions 1-10 is a FLAG sequence, amino acid sequence in positions 11-259 is a sequence of SY-001 (21-269), and consistency in the provisions 260-270 is a sequence originating from pBACgus-1, containing the sequence His-token.

Example 3

Inhibitory effect of SY-001 in respect of platelet aggregation was evaluated by measuring platelet aggregation in a rich in platelets plasma (PRP) using a turbidimetric thrombogram with the transmission of light.

In General, the method is the following. I.e. the first sample of blood was taken from healthy donor with a syringe with an anticoagulant, and is rich in platelets plasma (PRP) prepared by centrifugation of whole blood taken. The prepared PRP was mixed and pre-incubated with a solution of SY-001 [PBS solution, which is dissolved in SY-001, having amino acid SEQ ID NO:7 prepared in example 1 3-(1)] or PBS as a control, and subsequently, the platelet aggregates when adding platelet aggregation agent. As platelet aggregation agent used ADP (supplied by Sigma), collagen (supplied by NYCOMED), CRP (supplied by Peptide Institute Inc.), convulxin(supplied by lexis), TRAP (supplied Sawaday Technology Co.), epinephrine (supplied by Daiichi Pharmaceutical Co., Ltd.), arachidonic acid (supplied by Sigma), U-46619 (Cayman supplied) or A (supplied by Sigma), respectively.

Then, using the turbidimetric thrombogram with the transmission of light (MCM HEMA TRACER 313M supplied MC Medical), measured the speed of platelet aggregation for 5 minutes, and the maximum rate of aggregation was obtained when measured within 5 minutes. The rate of inhibition with the help of SY-001 of platelet aggregation (%) was calculated according to the following formula.

The rate of inhibition of platelet aggregation (%)=(1-As/Ac)×100

As: the maximum rate of platelet aggregation in PRP with SY-001

Ac: the maximum rate of platelet aggregation in PRP in one quality control

The details of the above procedures are presented in the following paragraphs (a) to(C).

(a) First, 60 ml of blood was taken from healthy donor with a syringe with 6 ml of 3.8% sodium citrate as anticoagulant. Subsequently, the blood was centrifuged at 1100 rpm/min for 10 minutes, layer rich in platelets plasma (PRP) in the form of the upper layer was transferred into another test tube. The remaining part of the lower layer was centrifuged at 3000 rpm/min for 10 minutes, and the resulting upper transparent yellow layer (poor in relation to Tr�of Mazitov plasma, PPP) was transferred to another test tube. A mixture in which the number of platelets increased to 3×108/ml, obtained by mixing PRP and PPP, obtained as described above, and used for subsequent measurements. To this mixture, reference is made in the form of "PRP sample for measurements in subsequent measurement.

For the velocity of platelet aggregation of thromboangiitis found that the transmission of light in the RRR is 100% the speed of platelet aggregation, light transmission in PRP is 0% the speed of platelet aggregation.

(b) determining the concentration of collagen and measurement of inhibitory activity against platelet aggregation

First, in thrombogram established for aggregation cuvette, to which was added 200 μl of PRP-sample for measurement, not containing SY-001, and were incubated for 2 minutes at 37°C. Subsequently added to 22.2 μl of collagen solution (supplied by NYCOMED GmBH, Moriya Sangyo), and the rate of platelet aggregation continuously measured for 5 minutes at 37°C. At the maximum speed of platelet aggregation took the highest speed of platelet aggregation within 5 minutes. Used the concentration of the solution of collagen comprising 5-20 mcg/ml (final concentration in PRP-sample for measurement was 0.5-2 µg/ml) in submaximal concentrations are inducere�if 70% of the maximum speed of platelet aggregation.

(C) Then, in each cell, to which was added 200 μl of PRP-sample for measurement was added (i) SY-001 (22-269) [having the amino acid sequence of SEQ ID NO:7 prepared in example 1 3-(1)] prepared in a concentration of 30 nm, 100 nm and 300 nm, respectively, (ii) SY-001 (148-269) [having the amino acid sequence of SEQ ID NO:8 prepared in example 1 3-(2)], prepared at a concentration of 100 nm, 300 nm and 1000 nm, respectively, or (iii) PBS (137 mm NaCl, 2.7 mm KCl, 8.1 mm Na2HPO4, 1.5 mm KH2HPO4) as a control. Each mixture was determined in thrombogram and incubated for 2 minutes at 37°C. Subsequently added to 22.2 μl of the collagen solution in prescribed concentrations defined above, and the rate of platelet aggregation was measured during 5 minutes of the addition of a solution of collagen.

Fig.1 shows the result of inhibitory activity of SY-001 (22-269) against platelet aggregation induced by collagen. Fig.2 shows the result of inhibitory activity of SY-001 (148-269) against platelet aggregation induced by collagen.

In each of these figures the horizontal axis represents the time course of (-0.5 to 5 minutes), starting 30 seconds prior to the addition of collagen, and the vertical axis represents the rate of platelet aggregation (%).

Fig.1 curve (1) demo�strirred the result in case of addition of SY-001 (22-269) at a concentration of 300 nm, curve (2) shows the result when adding a SY-001 (22-269) at a concentration of 100 nm, curve (3) shows the result when adding a SY-001 (22-269) at a concentration of 30 nm, and curve (4) shows the result of control.

Fig.2 curve (1) shows the result when adding a SY-001 (148-269) at a concentration of 1000 nm, curve (2) shows the result when adding a SY-001 (148-269) at a concentration of 300 nm, curve (3) shows the result when adding a SY-001 (148-269) at a concentration of 100 nm, and curve (4) shows the result of control.

From the results shown in these figures, it is obvious that SY-001 of the present invention, as shown, reduces platelet aggregation induced by collagen, with increase of the added amount, i.e. SY-001 has inhibitory activity against platelet aggregation.

The same experiments as that of the above, performed using recombinant protein SY-001 (21-269), produced in a baculovirus expressing the system in example 2. In the result, we obtained essentially the same results as the results presented in Fig.1. Therefore, it is obvious that SY-001 of the present invention has inhibitory activity against platelet aggregation.

In experiments using the above SY-001 (21-269) speed Agra�ation of platelets obtained when changing the concentrations of SY-001 (21-269) from 3 nm to 1000 nm. Then, using the SAS software (SAS Institute, Japan, version 8.1) performed an analysis of log-logit transformation of inhibitory activity against platelet aggregation (IC50) SY-001 (21-269). In the calculated result that the IC50SY-001 (21-269) is 25 nm.

On the basis of the above results suggested that the epitope, which is present on the C-terminal section between the provisions 148-269 amino acid sequence of SY-001 SEQ ID NO:5, may contribute to the inhibitory activity against platelet aggregation SY-001 of the present invention.

Example 4

Synthesis of mutant SY-001

SY-001 synthesized below using the method of solid-phase synthesis using Fmoc (9-fluorenylmethoxycarbonyl) method.

Synthetic polypeptide having the desired number of amino acid residues from N-Terminus to C-end amino acid sequence SEQ ID NO:5, can be obtained by the implementation of the reaction in the continuous flow model using as the active reagents TBTU [2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate] and HOBt [1-hydroxy-benzotriazole hydrate].

If necessary, the resulting synthetic polypeptide can be dissolved in dimethylsulfoxide and further diluted with acetonitrile.

Example 1 composition

(1) a Pharmaceutical composition of the present izobreteny� in injectable form can be prepared by adding and mixing 100 μg/ml of SY-001 (having the amino acid sequence of SEQ ID NO:8), 0.01 mg/ml tween-80 (polyoxyethylene(20) sorbitan monooleate; Polysorbate 80), 15 mg/ml of dextran 40, 0.1 mg/ml cysteine and 1 mg/ml HSA (zavorotnova of human albumin) in 0.01 M buffer, citric acid - sodium citrate (pH 6.0), by filtration of the mixture (using a membrane filter of 0.22 μm), then the distribution of sterile filtrate to 1 ml in a vial and lyophilization. The composition can be used by dissolving in 1 ml of salt solution used.

(2) the Pharmaceutical composition of the present invention in the injectable form can be prepared by adding 10 μg/0.1 ml SY-001 (having the amino acid sequence of SEQ ID NO:8), 5 mg cysteine acid and 1 mg of HSA (zavorotnova of human albumin) in a vial in distilled water for injection, filtering the resulting solution to 1 ml in one ampoule and its lyophilization.

The effect of AAR on platelet adhesion to collagen

A solution of SY-001 (50 μl) at a concentration of 3, 10, 30, 100, 300, 1000 or 3000 nm were added to wells of 96-well plates, coated with 40 μg/ml collagen (NYCOMED GMBH) and incubated for 30 minutes at room temperature. After incubation, to each well was added 50 μl of a suspension of platelets (6×108cells/ml) and incubated for 45 minutes at room temperature. After incubation of each well was removed the solution using a pipette and the wells were washed with 200 ál of BS. Subsequently, to each well was added 20 μl of PBS containing 1% SDS, then stirred pokazyvaet and subjected to air drying at 45°C. Then, to each well was added 5 μl of distilled water, and the protein concentration in the wells was measured using a kit for analysis of protein Dc (BIO-RAD).

The results shown in Fig.3.

As can be seen in Fig.3, it is determined that SY-001 of the present invention inhibits the adhesion of platelets to collagen dose-dependent manner, and that SY-001 exhibits a strong inhibitory effect against adhesion of platelets, especially in doses of 300 μg/ml or more.

The ability of SY-001 contact collagen

To each well of 96-well plates (NUNC, 152038), coated with collagen, or 96-well plates (NUNC, 260895) not covered by collagen, was added to the blocking solution (300 μl) and incubated for 1 hour at room temperature. From each well was removed the solution, and to each well was added 100 μl of the protein solution SY-001 at concentrations of 3, 10, 30, 100 or 300 nm and incubated for 1 hour at room temperature. From each well was removed the solution, and in the hole there was added 200 µl of 2% sucrose and incubated for 5 minutes at room temperature. From each well was removed the solution, the hole was dried. Subsequently, to each well was added 100 µl of reconstituted solution Nickel-HRP (ExpressDetector Nickel-HRP, set�aemy KPL) and incubated for 30 minutes at room temperature. After rinsing with buffer for leaching to each well was added 100 μl of substrate for peroxidase ABTS and gently mixed by rocking. After the reaction was added 100 μl of 1% SDS, and the absorbance was measured at a wavelength of 405-410 using reader Micro Plate Reader.

The results shown in Fig.4. The triangles represent the reaction curve of an empty vector as a control. As shown in Fig.4, it is determined that SY-001 has the ability to bind to collagen.

As indicated above, were able to determine that SY-001 of the present invention has not only an inhibitory effect against platelet aggregation, but also inhibitory activity against adhesion of platelets to collagen. Also was able to determine that SY-001 of the present invention has the ability to bind to collagen.

On the basis of these results a pharmaceutical composition comprising as an active ingredient SY-001 of the present invention may be a pharmaceutical composition, useful as a therapeutic agent and a preventive agent for myocardial infarction, embolism of the vessels of the lungs, cerebral infarction, etc.

Inhibitory against platelet adhesion effect and the ability to bind to the recombinant collagen of AARR

Fig.3: ARR ingibiruyuthee of platelets to collagen.

Fig.4: ARR has the ability to bind to collagen.

The list of sequences, free text

SEQ ID NO:10 is the sequence of primer for the SP6 promoter, SEQ ID NO:11 is the sequence of primer for the T7 terminator, SEQ ID NO:12 is the sequence of primer pAnS-1, SEQ ID NO:13 is the sequence of primer pAnSG-F7, SEQ ID NO:14 is the sequence of primer pAnSG-R1, SEQ ID NO:15 is the sequence of primer pAnSG-F8, and SEQ ID NO:16 is the sequence of primer pAnSG-F10.

INDUSTRIAL APPLICABILITY

In accordance with the present invention can provide a pharmaceutical composition containing as an active component SY-001 or expressed product of the present invention. Believe that the composition of the present invention is applicable as a therapeutic agent or prophylactic agent for pathological conditions after various diseases, e.g., diseases or complications caused by thrombus or embolus, for example, acute coronary syndrome, myocardial infarction, embolism of cerebral vessels, chronic obstruction of the arteries, arterial sclerosis, ischemic stroke, angina, venous thrombosis, hypertension, pulmonary hypertensie, cerebral infarction, pulmonary infarction, cere�echnol failure, nephritis, renal failure and subarachnoid hemorrhage that involves SY-001 and the polynucleotide (DNA molecule) encoding a polypeptide SY-001.

In addition, it is believed that the composition of the present invention is applicable for the prevention of blood clots during PTCA and stent placement to prevent restenosis after stent placement by enabling SY-001 by applying SY-001 on the stent or sealing SY-001 of the stent.

Using SY-001 and encoding SY-001 DNA molecule provided by the present invention can be skanirovat agonist polypeptide or of a product expressed from the DNA molecule (expressed product of the present invention), i.e. to skanirovat substance that promotes inhibitory activity inherent in these proteins, against platelet aggregation and/or adhesion of platelets, as a matter candidate.

1. The selected polypeptide possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets and comprising the amino acid sequence SEQ ID NO: 3.

2. The polynucleotide encoding a polypeptide possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, where the polynucleotide consists of the DNA-consisten�Telenesti SEQ ID NO: 4 or its complement.

3. Composition possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, where the composition contains as an active ingredient a polypeptide according to claim 1 and a carrier.

4. A composition according to claim 3, which is used for inhibition of platelet aggregation.

5. A composition according to claim 3, which is used for inhibiting platelet adhesion.

6. Composition possessing inhibitory activity against platelet aggregation and/or inhibitory activity against adhesion of platelets, where the composition contains as an active ingredient a polypeptide which is expressed by a polynucleotide consisting of the DNA sequence SEQ ID NO: 4, and the media.

7. A composition according to claim 6, which is used for inhibition of platelet aggregation.

8. A composition according to claim 6, which is used for inhibiting platelet adhesion.

9. A kit for screening substances inhibiting platelet aggregation comprising components (1) to(3):
(1) the polypeptide according to claim 1,
(2) cell culture medium containing plasma, rich in platelets, and
(3) the agent, platelet aggregation.

10. A kit for screening substances inhibiting platelet adhesion comprising components (1) to(3):
(1) the polypeptide according to claim 1,
(2) substance, adhesion of thrombocy�s, and
(3) a suspension of platelets.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to dentistry, and can be used in diagnostics of dental caries by method of infrared spectroscopy. For this purpose sample of patient's saliva is preliminarily dries, dry residue is crushed and suspended in vaseline oil. IR-spectroscopy is carried out in spectrum area 1200-800 cm-1 and height of peaks of absorption bands with maximums 1070, 1017, 960, 860 cm-1 is determined. After that, values of four ratios of peak height with maximum at 1070 cm-1 to peak height with maximum 1017 cm-1, peak height with maximum at 1070 cm-1 to peak height with maximum 960 cm -1, peak height with maximum at 1070 cm-1 to peak height with maximum 860 cm -1, ratio of peak height with maximum at 1017 cm-1 to peak height with maximum 860 cm-1. Dental caries is diagnosed at the following values of ratios of peak heights 1070/1017, equal to 11.64±0.14, peak heights 1070/960, equal to 1.08±0.12, peak heights 1070/860, equal to 6.40±0.41, and peak heights 1017/860, equal to 4.15±0.44.

EFFECT: invention provides self-descriptiveness of diagnostics and increases its accuracy.

3 ex

FIELD: medicine.

SUBSTANCE: method consists in production of cryostat sections of nerve, washing them in two portions of maleate buffer, incubation in incubation medium. Incubation is carried out for 30-60 minutes and incubation medium has the following composition: acetylcholine iodide 10 mg; 0.1 M Na-maleate buffer 2 ml; 0.1 M sodium citrate 4 ml; 0.03 M copper sulphate 4 ml; 0.005M potassium ferricianide 4 ml; medium pH constitutes from 7.4 to 7.6 and temperature is from 50 to 55°C.

EFFECT: method by invention makes it possible to evaluate viability of peripheral nerves in short term, which is necessary during reconstructive-restoring and highly technological neurosurgical operations.

2 ex

FIELD: medicine.

SUBSTANCE: functional activity of neutrophilic granulocytes is studied by means of hemiluminiscent analysis, calculated is an index of formation of active forms of oxygen (IFAFO), representing the ratio of the area under the curve of luminol-dependent hemiluminiscence of the neutrophilic granulocytes, induced by a live bacterial suspension of staphylococcus aureus, to the time of achieving the maximum of luminol-dependent hemiluminiscence of the neutrophilic granulocytes, induced by the live bacterial suspension of staphylococcus aureus. If IFAFO value is higher than 100 o.u./s, sensitisation to staphylococcus aureus in patients with allergic rhinitis is diagnosed.

EFFECT: application of the claimed method makes it possible to diagnose staphylococcal allergy in case of allergic rhinitis before beginning therapy and contributes to the optimal selection of the patients' treatment.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention represents a method for determining a probability of preserving the myocardium following infarction by creating an admission examination-based data array of 7 peripheral blood parameters, 11 biochemical analysis parameters and 6 parameters of standard 12-lead electrocardiogram in 200 patients with the Q-myocardial infarction and 200 patients without the myocardial infarction. The parameters are stratified with respect to 7 intervals, wherein values related to the probability of preserving the myocardium following infarction are derived by calculating a ratio of the patients without myocardial infarction to all the patients with acute coronary syndrome. The probability is evaluated in a specific patient by analysing the above parameters, searching the respective intervals and values related to the probability of preserving the myocardium in the data array. Summing up the derived values enables calculating an integrated index, which is normalised, and a dimension from 0 to 100% is reduced.

EFFECT: invention enables increasing the prediction accuracy of preserving the myocardium in the patients with acute coronary syndrome.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to immunology, and can be used in laboratory diagnostics, as a test system and a method for measuring interferon alpha (IFN-α) antiviral activity in human blood serum. The test system for measuring IFN-α activity in the human blood serum comprises diploid cells, a virus-containing fluid and standard human interferon-α (IFN-α). As the diploid cells, the test system comprises cells of the characterised line of the diploid cells - human M-20 fibroblasts at 20-33 passages cultured in the medium with added 10% fibrinolytically active plasma (FAP). As the virus, the system contains the A vesicular stomatitis virus (VSV) adapted to M-20 cells, the Indiana strain; the test system additionally contains a viral dye based on two fluorochromes - acriflavine and rhodamine C. The group of inventions also involves a method for measuring IFN-α activity in the human blood serum with the use of the developed system.

EFFECT: using the given inventions enables the quantitative good-reproducible measurement of IFN-α activity in the examined blood serum sampled by the luminescent microscopy of the vital preparations coloured in a fluorochrome-based vital dye.

3 cl, 4 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention describes a diagnostic technique for infected pancreonecrosis with establishing the indications for a surgical intervention by examining a patient, wherein acetic, propionic, butyric and isovaleric acids are measured in the blood by gas chromatography, and if the acetic acid concentration is more than 0.11 mmole/l enables stating infected pancreonecrosis, and if the concentration of any of three acids: propionic more than 0.095 mmole/l, butyric more than 0.0035 mmole/l, isovaleric acid more than 0.0003 mmole/l enables stating infected pancreonecrosis with active development of an anaerobic infection requiring one of versions of the surgical intervention.

EFFECT: invention enables providing the objective and accurate diagnosing of pancreonecrosis transformation into the stage of infection by using the qualitative parameters with no undesired side effects.

2 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to a method of predicting a probability of reduction of the glomerular filtration rate (GFR) after 3 months of observation after aortocoronary bypass surgery without artificial blood circulation (ACBS without ABC). The essence of the method consists in the fact that the concentration of a kidney injury molecule of type 1 (KIM-1) is determined in blood serum, the ratio of the biomarker KIM-1 concentrations in two time points after 48 hours and 7 days after the operation is calculated and if its value is higher than 1.5, a conclusion about the probability of (GFR) reduction in the remote period after ACBS without ABC is made.

EFFECT: application of the claimed method makes it possible to predict the probability of the glomerular filtration rate (GFR) reduction after 3 months of observation after aortocoronary bypass surgery without artificial blood circulation in an efficient and accurate way.

1 tbl, 1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method includes determination of a titre of antibodies to cytomegalovirus, content of 2,3 DPG (2,3-diphosphoglycerate), and oxyhaemoglobin in erythrocytes. If the titre of the antibodies to cytomegalovirus increases to 1:1600, DPG increases to 6.7±0.3 mcmole/ml, content of HbO2 is 95.0±1.7%, with the reduction of the specific optical density of haemoglobin to 0.70±0.01, it is concluded that a threat of anaemia development is formed.

EFFECT: method makes it possible to study the character of haemoglobin oxygenation impairment by means of determination of the specific optical density.

FIELD: medicine.

SUBSTANCE: neurovisualisation examination of brain is carried out, Cirs comorbidity index and Kaplan-Feinstein comorbidity index are determined, cochleovestibular syndrome, eye-moving impairments, type of diabetes mellitus are identified. Value of discriminate function (D) is calculated. If D value is higher than zero, diagnosed are consequences of ischemic brain stroke (IBS) with hyperhomocysteinemia (HH), if D is lower than zero, consequences of IBS without HH are diagnosed.

EFFECT: method makes it possible to increase reliability of diagnostics of IBS consequences, which is achieved due to complex analysis of said parameters.

2 ex

FIELD: veterinary science.

SUBSTANCE: method includes detection of leukocytes, protein, haemoglobin in faeces, and PH-reaction of faeces. A sample of faeces is dissolved in distilled water and applied drop by drop onto appropriate test fields of a test strip designed for urine testing. By variation of colour within 1 minute the reaction is assumed as positive. If pH is less than 7.0 or more than 7.5, and faeces contain soluble protein, haemoglobin and leukocytes at the same time, availability of the inflammatory process in the intestines is confirmed.

EFFECT: invention makes it possible to quickly and accurately diagnose inflammatory process in intestines.

2 cl, 2 ex

FIELD: medicine.

SUBSTANCE: method is based on measuring a flavonoid amount by differential spectral photometry, calculated as Cinaroside at wave length 400 nm, extracting in water and alcohol and using 70% ethanol as an extraction fluid; the total flavonoid amount calculated as Cinaroside and an absolutely dry raw material in percentage (X) is calculated by formula.

EFFECT: method enables measuring the total flavonoid amount as biologically active components having a basic therapeutic action.

3 ex, 14 dwg, 2 tbl

FIELD: chemistry.

SUBSTANCE: method of Novocain extraction from water solutions includes the preparation of an aqueous salt solution of Novocain by its dissolution in a saturated solution of a salting-out agent, extraction and analysis of an equilibrium water phase, with the application as an extragent of a solvotrophic reagent solution in chloroform with the concentration of 10 wt %, for which purpose the aqueous salt solution of Novocain with pH 8.0±0.5 is prepared due to the application of a saturated ammonium sulphate solution as the salting-out agent and addition of an ammonium buffer solution, Novocain is extracted for 5-7 minutes with the solution of the solvotrophic reagent in chloroform with the ratio of volumes of the aqueous salt solution of Novocain and extragent of 5:1, then the aqueous saline phase is separated from the organic one and analysed by a method of UV-spectrophotometry at a wavelength of 291 nm, the concentration of Novocain in the water solution is found by means of a calibrating graph; the coefficient of distribution (D) and the degree of extraction (R, %) of Novocain is are calculated by formulae.

EFFECT: method for the extraction of Novocain from the water solution, which is characterised by expressiveness, makes it possible to realise the practically complete single extraction of Novocain from the aqueous salt solution and can be applied in the analysis of Novocain-containing water solutions.

1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry and can be used in a system for monitoring content of sodium thiosulphate in solutions. The method of determining sodium thiosulphate in solutions is characterised by adding an analysed sample into a reaction vessel containing a corresponding amount of photogenerated iodine, obtained by blowing with air for 1-2 minutes and irradiating the reaction mixture with a stabilised light source, the mixture consisting of 0.5 M potassium iodide solution, an acetate buffer solution with pH 5.6 and a sodium eosinate sensitising agent, by detecting change in current in a cell and upon achieving constant current, re-blowing the reaction mixture with air for 2-3 minutes and re-irradiating with the stabilised light source until achieving the initial amount of iodine in the vessel, measuring the iodine generation time spent on achieving the decrease thereof, determining the amount of sodium thiosulphate from a calibration curve from the change in current and generation time.

EFFECT: invention provides a simple method of determining sodium thiosulphate in solutions and avoids use of expensive equipment.

10 tbl, 5 dwg

FIELD: medicine.

SUBSTANCE: invention describes a method for lipoic acid measurement in biologically active additives by cathode voltammetry involving transferring a substance from a sample into a solution and taking voltammetric measurement; the cathode voltammetry is performed on a mercury-film electrode at potential -0.373 V of a relatively saturated silver-chloride electrode with borate buffer solution pH 9.18 at continuously current potential trace at 0.06 V/s with the determined lipoic acid content range from 4.5·106 to 1.1·10-3 mole/l.

EFFECT: improving sensitivity and expressivity of the method for measuring lipoic acid in tabletted BAAs by cathode voltammetry.

1 tbl, 1 ex, 3 dwg

FIELD: agriculture.

SUBSTANCE: in agar plate which is in a Petri dish and inoculated with one of the fungal species of the genus Fusarium, circular holes are cut with the diameter of 8 mm, 0.05 ml of the working solution of the test preparation is applied into them, and placed in a thermostat at a temperature of 24.5-25.0°C, after 2-5 days depending on the type of fungus the diameter of the zone of growth inhibition of fungal mycelium is measured and the activity of the preparations is calculated according to the formula , and it is considered that when A=1 the fungicide is ineffective - the zone of growth inhibition is not formed (D=d), when A=2-3 the preparation activity is low, when A=4 it is average, and when A≥5 it is high.

EFFECT: invention provides accuracy of determining the activity of seed disinfectants and fungicides used in agricultural production.

1 tbl

FIELD: medicine.

SUBSTANCE: invention represents a method for preclinical study of cardiotropic antiarrhythmic drugs, involving determining the bioelectric parameters in isolated multicellular perfused preparations and measuring an action potential duration, differing by the fact that the isolated multicellular perfused preparations are presented by rat's pulmonary vein myocardium; the parameters are measured in three operation modes of the multicellular preparations; a resting potential is additionally measured; varying APD 90%, related APD 50%/APD 90%, a spontaneous shear velocity of the resting potential, the most positive membrane potential in the resting preparation, a spontaneous activity train repetition rate, spontaneous action potential train repetition and variability frequency, post-depolarisation number and intensity, as well as a shear membrane potential corresponding to the beginning of train activity are used to evaluate the signs of antiarrhythmic and arrhythmogenic action.

EFFECT: more reliable prediction of the antiarrhythmic action of the potential pharmacological agents and reduction of experimental phase time.

3 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to method of determining platelet resistance to acetylsalicylic acid (ASA) by impedance analysis of aggregation function of platelets in vivo, in which aggregation activity is analyses after incubation of biological material sample with ASA with application of aggregation inducer, and aggregation of platelets is induced by collagen in optimal concentration 2 mg/ml, and simultaneously with measurement of impedance determination of dynamics of platelet granules release is performed by luminescent method, where before carrying out aggregation samples are calibrated by means of adenosine triphosphate (ATP) standard, value of aggregation amplitude are determined in Ohms by obtained aggregatograms and obtained values are given points: values ≤6 correspond to 0 points, values 7-9 correspond to 1 point, values 10-12 correspond to 2 points, values >12 correspond to 3 points; after that, intensity of ATP release from platelet granules is determined in nmoles and obtained values are given points: values <0.5 correspond to 0 points, values 0.5-1.0 correspond to 1 point, values 1.0-1.5 correspond to 2 points, values 1.5 correspond to 3 points, and then resistance index (RI) is calculated by formula, value of IR parameter points to presence of aspirin-resistance of platelets.

EFFECT: carrying out fast complex diagnostics of platelet resistance to ASA with estimation of functional state of platelet granules before beginning treatment and possibility of preventing development of undesirable thrombotic or hemorrhagic complications.

2 dwg, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: claimed is application of fat emulsion for parenteral feeding as solvent for compounds which are poorly soluble in water. Fat emulsion contains in 1 l of solution: 30 g of refined soybean oil, 30 g of triglycerides with the average chain length, 25 g of olive refined oil, 15 g of purified fish oil.

EFFECT: obtaining solvent for compounds, poorly soluble in water, which makes it possible to determine parameters and spectrum of biological activity of novel compounds of chemical nature at the stages of pre-clinical and clinical tests, which does not change basic biological constants and possesses biological inertness.

2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biology, forensic and analytical chemistry and specifically to methods of determining procaine in blood plasma. The method comprises adding sodium fluoride to blood plasma containing procaine to achieve concentration of 10 mg/ml; treating the obtained mixture with acetone; separating the extract from the precipitate by filtering; evaporating acetone from the filtrate in an air current at room temperature; diluting the aqueous residue by adding water; saturating the obtained solution with ammonium sulphate; alkalising with an ammonium buffer solution to pH 9.0-9.5; extracting twice with portions of an organic extraction agent in the form of 30% camphor solution in methyl acetate, with ratio of the aqueous to the organic phase of 1:1 by volume; separating the organic extracts; combining; evaporating the solvent from the combined extract in an air current at room temperature; chromatographing the residue in a thin layer of silica gel STKH-1A on Sorbfil PTSKH-AF-A-UF plates, using a dichloromethane-ethanol mobile phase in ratio of 6:4 by volume; developing the chromatogram in UV light; eluting the analysed substance from the sorbent with a mixture of acetonitrile-methanol-0.025 M potassium dihydrogen phosphate solution with pH 3.0 in ratio of 10:10:90 by volume; chromatographing by HPLC method using a reversed-phase sorbent Nucleosil C18, a polar mobile phase acetonitrile-methanol-0.025 M potassium dihydrogen phosphate solution with pH 3.0 in ratio of 10:10:90 by volume and a UV detector; measuring optical density at wavelength 298 nm and calculating the amount of the analysed compound from the area of the chromatographic peak.

EFFECT: method improves sensitivity of determination.

3 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry. The method is characterised by electrochemically concentrating benzoic acid on the surface of a graphite electrode for 90 s at electrolysis potential of (-0.500) V on a background of 0.1 mol/l sodium hydrogen phosphate, recording polarisation curves with linear potential sweep rate of 25 mV/s and determining concentration of benzoic acid from the peak height in potential range of 0.5-1.6 V relative to a silver chloride electrode.

EFFECT: method provides highly sensitive and rapid determination of benzoic acid in medicinal drugs.

3 ex, 6 tbl

FIELD: biotechnology.

SUBSTANCE: method comprises the steps: collecting from the bile ducts of liver of domestic and/or wild animals infected with fasciolas of only live adult F. hepatica. Placement them into individual tubes with filtered and centrifuged bile diluted with isotonic solution of sodium chloride 1:1. Exposure of tubes at t = 38-39°C if F. hepatica is from cattle, and t = 39-40°C if F. hepatica is from sheep and/or goats, under conditions of thermostat for 5 hours. Subsequent washing eggs in isotonic solution of sodium chloride.

EFFECT: invention enables to obtain up to 100 percent of fertilised eggs of Fasciola of species Fasciola hepatica and can be used for study in the laboratory or field experiments in solving fundamental and applied scientific tasks in the field of epizootiology, treatment and prevention of fascioliasis of domestic or wild animals.

2 ex

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