MX2008005795A - Platelet aggregation inhibitor composition - Google Patents

Abstract

The invention provides a pharmaceutical composition comprising at least one polypeptide of the following (a) to (d):(a) a polypeptide comprising the amino acid sequence of SEQ ID NO:1;(b) a polypeptide comprising an amino acid sequence comprising one or more amino acid deletions, insertions, substitutions or additions in the amino acid sequence ofthe above (a) and having a platelet aggregation inhibitory activity and/or a platelet adhesion inhibitory activity;(c) a polypeptide comprising the amino acid sequence of SEQ ID NO:3;and (d) a polypeptide comprising an amino acid sequence comprising one or more amino acid deletions, insertions, substitutions or additions in the amino acid sequence of the above (c) and having a platelet aggregation inhibitory activity and/or a platelet adhesion inhibitory activity, as an active component.

Description

INHIBITORY COMPOSITION OF PLATELET AGGREGATION TECHNICAL FIELD The present invention relates to a polypeptide having a platelet aggregation inhibitory activity and / or platelet adhesion inhibitory activity, or a pharmaceutical composition, for example, a platelet aggregation inhibiting composition comprising an expressed product (recombinant polypeptide ), which is expressed by a polynucleotide that encodes the polypeptide as an active component. The present invention further relates to a polypeptide having a collagen binding capacity, or a pharmaceutical composition comprising the polypeptide as an active component. The present invention also relates to a method for identifying a compound (e.g., an agonist) that facilitates the platelet aggregation inhibitory activity as an active component in the pharmaceutical composition. In addition, the present invention relates to a novel polypeptide having the inhibitory activity of platelet aggregation and a polynucleotide encoding the same.

TECHNICAL BACKGROUND Platelets are added due to vascular endothelial cell injury and several other factors. When a coronary vessel, a cerebral vessel or a peripheral vessel is occluded by this platelet thrombus, myocardial infarction, cerebral infarction or chronic arterial obstruction, respectively, is caused. Examples of thrombotic diseases following such activation (stimulation of aggregation) of platelets include arterial sclerosis, ischemic cerebral infarction, ischemic heart diseases including myocardial infarction and angina, chronic arterial obstruction and venous thrombosis. Medications with the action to inhibit platelet aggregation are used as preventive drugs for ischemic diseases that occur with the various previous diseases as complications, and as preventive drugs for pathological conditions subsequent to hypertension, pulmonary hypertension, cerebral infarction, pulmonary infarction and subarachnoid hemorrhage. In addition, the above drugs are used for the prevention of thrombus formation after percutaneous transluminal coronary angioplasty (PTCA) and stenting, and are also used as restenosis preventive agents after stent placement involving said drug having the action to inhibit platelet aggregation, by applying it on or including it in, the stent itself.

Meanwhile, a mosquito pierces the skin with its sharp oral stylet that reaches the peripheral blood vessel when it sucks blood, and often repeats a puncture behavior inside and outside referred to as sounding, to find the peripheral blood vessel. It is believed that the mosquito simultaneously secretes saliva containing a substance that facilitates vasodilation to make it easy to detect the blood vessel. Due to the previous sounding, the peripheral blood vessel is often damaged until it becomes congested. In general, when the blood vessel is injured, the collagen in the tissue under the vascular endothelium is exposed, adenosine diphosphate (ADP) is released from the dissolved cells, and the coagulation factors are activated to form thrombin. Thrombin strongly activates platelets to induce platelet adhesion, platelet aggregation and granule release, and finally forms a firm thrombus by blood coagulation with fibrin formation (hemostasis mechanism). It has been known that mosquito saliva contains the substance that inhibits said mechanism of hemostasis (see non-patent literature 1). A protein from the salivary glands in the mosquito, which has been studied in more detail, is apyrase. This enzyme is a substance inhibiting platelet aggregation that was first identified in the saliva of Aedes aegypti. Apyrase inhibits platelet aggregation, resulting in the breakdown of ADP released from damaged vascular endothelial cells, erythrocytes, and platelets adhered to AMP (adenosine) monophosphate), and exhibits anti-hemostatic action. To elucidate the vampire behavior of several vampire insects beyond the mosquito, it seems essential to analyze the function of their salivary substances. It is predicted that many salivary substances are involved in the inhibition of platelet aggregation and, for example, an inhibitory activity of platelet aggregation has been reported for the protein derived from Triatoma infestans (see Patent Documents 1 and 2). Among the proteins derived from the salivary glands in Anopheles stephensi, the protein having a blood coagulation inhibitory activity has been identified, but no protein has been identified that has the inhibitory activity of platelet aggregation (see patent document 3). ). In addition, 33 novel proteins have been reported from the cloning of the salivary gland cDNA library in Anopheles stephensi, no protein that has the inhibitory activity of platelet aggregation is described in the report, and the functions in many of them they remain unknown (see non-patent literature 2). The present inventors previously reported a protein (AAPP) having a sequence rich in GE (Gly Glu) cloned from the salivary glands in Anopheles stephensi (see non-patent literature 3). The protein has an open reading frame (ORF) of 810 bases, and it is the 28.5 kDa protein that is inferred to be composed of 269 amino acid residues. It was later found that the protein is similar to an antigen of kDa (accession number of GenBank AY226454) described in non-patent literature 2, but there is no description for the actions and functions (activity) of the protein in non-patent literatures 2 and 3. [Patent Document 1 ] Publication JP 2004-121091 -A [Patent Document 2] Publication JP 2004- 21086-A [Patent Document 3] Publication JP 2003-1 16573-A [Non-Patent Literature 1] Riberío, JM, J. Exp. Biol., 108, 1-7 (1984) [Non-Patent Literature 2] Valenzuela, J. G., et. al., Exploring the salivary gland transcriptome and proteome of the Anopheles stephensi mosquito ", Insect Biochemistry [Non-Patent Literature 3] Hiroyuki Watanabe et al., Medical Entomology and Zoology 55 Suppl., pp. 41, 19 (2004).

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a novel pharmaceutical composition, in particular an inhibitor of platelet aggregation and / or a platelet adhesion inhibitor. The present invention further provides a novel pharmaceutical composition comprising a protein, which has a collagen binding capacity, as an active component.

The present invention also provides a method for identifying a substance such as an agonist, the method of identification that determines an inhibitory activity of platelet aggregation and / or an inhibitory activity of the platelet adhesion of the substance. As a result of an additional extensive study separate from the protein study (AAPP) previously reported by the present inventors, the present inventors have found a novel protein having a platelet aggregation inhibitory activity and / or a platelet adhesion inhibitory activity. in the salivary glands of Anopheles stephensi, they have successfully isolated and identified a DNA encoding it, and recently found that the protein has platelet aggregation inhibitory activity and / or platelet adhesion inhibiting activity. The present invention provides inventions that represent the following items 1 to 14. Item 1 A pharmaceutical composition comprising at least one polypeptide of the following (a) to (d): (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1; (b) a polypeptide comprising an amino acid sequence comprising one or more deletions, insertions, substitutions or additions of amino acid in the amino acid sequence of (a) above, and it has an activity inhibiting platelet aggregation and / or an activity inhibiting platelet adhesion; (c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3; and (d) a polypeptide comprising an amino acid sequence comprising one or more deletions, insertions, substitutions or additions of amino acid in the amino acid sequence of subsection (c) above, and having an inhibitory activity of platelet aggregation and / or an inhibitory activity of platelet adhesion as an active component. Item 2. A pharmaceutical composition comprising an expressed product, which is expressed by at least one polynucleotide of subsections (e) to (j) below: (e) a polynucleotide comprising the DNA sequence of SEQ ID NO: 2 , or a complement to it; (f) a polynucleotide that hybridizes with the polynucleotide of part (e) above under a severe condition, and is capable of expressing a polypeptide having an inhibitory activity on platelet aggregation and / or a platelet adhesion inhibiting activity; (g) a polynucleotide comprising the DNA sequence having 80% or more of homology with the polynucleotide of part (e) above, and which is capable of expressing the polypeptide having a platelet aggregation inhibitory activity and / or a inhibitory activity of platelet adhesion; (h) a polynucleotide comprising the DNA sequence of SEQ ID NO: 4, or a complement thereof; (i) a polynucleotide that hybridizes with the polynucleotide of part (h) above under the severe condition, and that is capable of expressing the polypeptide having the platelet aggregation inhibitory activity and / or a platelet adhesion inhibiting activity; and (j) a polynucleotide comprising the DNA sequence having 80% or more homology to the polynucleotide of part (h) above, and which is capable of expressing the polypeptide having the platelet aggregation inhibitory activity and / or an activity inhibiting platelet adhesion, as an active component. Item 3. The pharmaceutical composition according to item 1 or 2, wherein said inhibitory activity of platelet aggregation and / or an inhibitory activity of platelet adhesion, is the platelet aggregation inhibitory activity induced by the collagen and / or the inhibitory activity on platelet adhesion (ie, adhesion of platelets) to collagen. Item 4. The pharmaceutical composition according to item 1 or 2, wherein said pharmaceutical composition has the ability to bind collagen. Item 5. An inhibitor of platelet aggregation and / or a platelet adhesion inhibitor, comprising the polypeptide described in item 1 or the expressed product, which is expressed by the polynucleotide described in item 2. Item 6. A method for identifying an agonist for an inhibitory activity of platelet aggregation and / or a platelet adhesion inhibiting activity, wherein a level of the inhibitory activity of platelet aggregation and / or a platelet adhesion inhibiting activity of the polypeptide described in item 1 is measured in the presence or absence of a subject substance, and a measurement value in the presence of the subject substance is compared to a measurement value in the absence of the subject substance to select the subject substance that increases the inhibitory action as the agonist. Item 7. A method for identifying an agonist for an inhibitory activity of platelet aggregation and / or a platelet adhesion inhibiting activity, wherein a level of the inhibitory activity of the expressed product, which is expressed by the polynucleotide described in item 2, is measured in the presence or absence of a subject substance, and a measurement value in the presence of the subject substance is compared to a measurement value in the absence of the subject substance to select the subject substance that increases the inhibitory activity of the platelet aggregation and / or an inhibitory activity of platelet adhesion such as the agonist. Item 8. A method for identifying a candidate substance that serves as an agonist for platelet aggregation inhibitory activity and / or a platelet adhesion inhibitory activity of the polypeptide described in item 1 or the expressed product described in item 2, comprising the steps (1) to (4) below: (1) a step for preparing a culture medium comprising a cell transformed with an expression vector, which expresses the polypeptide described in item 1 or a cell comprising the expressed product described in item 2 and platelet-rich plasma; (2) a step for adding a platelet aggregant agent to the culture medium of subsection (1) above, to induce platelet aggregation in the presence or absence of a subject substance; (3) a step to measure a level of platelet aggregation in the presence or absence of the substance subject in subsection (2) above; and (4) a step for selecting the subject substance as the candidate substance when a measurement value in the presence of the subject substance is greater than a measurement value in the absence of the subject substance. Item 9. A kit for identifying an agonist for the polypeptide described in item 1 or the product expressed by the polynucleotide described in item 2, characterized in that it contains platelet-rich plasma, either one of the polypeptide described in item 1 and the expressed product described in item 2, and a platelet aggregant agent as components. Item 10. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1. Item 1 1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 3.

Item 12. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 5. Item 13. A polynucleotide comprising the DNA sequence of SEQ ID NO: 2, or a complement thereof. Item 14. A polynucleotide comprising the DNA sequence of SEQ ID NO: 4, or a complement thereof. Item 15. A polynucleotide comprising the DNA sequence of SEQ ID NO: 6, or a complement thereof. At least one polypeptide (protein) of items (a) to (d) above is sometimes referred to hereafter as "SY-001". The product expressed by at least one polynucleotide of parts (e) to (j) above, is sometimes referred to as the "expressed product of the present invention".

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the inhibitory activity of the platelet aggregation of SY-001 produced in example 1, 3 (1), induced by the stimulation with collagen; Figure 2 shows the inhibitory activity of the platelet aggregation of SY-001 produced in example 1, 3 (2), induced by the stimulation with collagen; Figure 3 shows the inhibitory activity of platelet adhesion of SY-001 produced in Examples 1 and 3, which inhibits platelet adhesion to collagen in Example 5; and Figure 4 shows that SY-001 produced in Examples 1 and 3, has the ability to bind collagen in Example 6.

BEST MODES TO CARRY OUT THE INVENTION The representation herein by abbreviations of amino acids, peptides, base sequences and nucleic acids, accesses the IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984) defined by IUPAC-IUB, "Guideline for preparing specifications comprising base sequences and amino acid sequences" (Patent Office), and notes commonly used in the art. A polynucleotide (DNA molecule) encompasses not only double-stranded DNA, but also single-stranded DNA that includes sense strands and antisense strands that comprise them, and is not limited to a length thereof. Therefore, the polynucleotide encoding SY-001 includes double-stranded DNA that includes genomic DNA and single-stranded DNA (sense strand) that includes cDNA and single-stranded DNA (antisense strand) that has the complementary sequence to the sense chain and synthetic DNA fragments thereof, unless otherwise mentioned.

The polynucleotide (DNA molecule) is not defined herein by a functional region, and may include at least one of a region of expression suppression., a coding region, a guiding sequence, an exon and an intron. The polynucleotide also includes RNA and DNA. The polypeptide comprising the certain amino acid sequence, and the polynucleotide comprising the certain DNA sequence, include fragments, homologs, derivatives and mutants thereof. Mutants of the polynucleotide (mutant DNA) include naturally occurring allelic mutants, non-naturally occurring mutants, and mutants that have deletion, substitution, addition, and insertion. However, these mutants encode the polypeptide which has substantially the same function as the function of the polypeptide encoded by the polynucleotide before the mutation. It is not necessary for the polypeptide mutation to occur (modification of the amino acid sequence) by the mutation of natural occurrence, eg, mutation or post-translational modification, and may be those artificially made using the naturally occurring protein (e.g., SY-001). The above mutants of the polypeptide include allelic variants, homologs and natural mutants having at least 80%, preferably 95% and more, preferably 99% homology to the polypeptide prior to mutation.

The homology of the polypeptide or the polynucleotide can be analyzed by measurement using the FASTA program (Clustal, V., Methods Mol. Biol., 25, 307-318 (1994)). As the most preferable and simplest method for homology analysis, it is possible to exemplify the method in which the sequence is stored in a medium (for example, floppy disk, CD-ROM, hard disk drive mechanism, drive control mechanism). external disk, DVD, etc.), capable of being measured by computer, and then the known sequence database is searched according to a well-known search procedure, using the stored sequence. Specific examples of the known sequence database include the following: DNA Datábase of Japan (DDBJ) (http://www.ddbj.nig.ac.jp/); Genebank (http: //www.ncbi.nlm.nih.gov/web/Genebank/lndex.htlm); and - the European Molecular Biology Laboratory Nucleic Acid Sequence Datábase (EMBL) (http://www.ebi.ac.uk/ebi docs / embl db.html). Many search algorithms for homology analysis are available to those skilled in the art. An example thereof includes a program referred to as a BLAST program. There are 5 BLAST procedures in this program. Among them, three (BLASTN, BLASTX and TBLASTX) have been designated to verify the nucleotide sequence. The remaining two have been designated to verify the sequence of proteins (Coulson, Trends in Biotechnology, 12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)). In addition, additional programs, for example, a sequence alignment program and a program for identifying more distant sequences, are available in the art to analyze the identified sequence. The mutant DNA is silent (no change in an amino acid residue encoded by a mutated nucleic acid sequence), or conservative for the amino acid encoded by it. Examples of conservative amino acid substitutions are shown below.

Original amino acid residue Reserved conservative amino acid residue Ala Ser Arg Lys Asn Gln or His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn or Gln He Leu or Val Leu lie o Val Lys Arg, Asn or Glu Met Leu or He Phe Met, Leu or Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp or Phe Val He or Leu In general, one or more codons coding for a Cys residue affect a disulfide bond of the polypeptide particular. The substitution of the amino acid residue that is generally thought to affect the characteristics of the protein, includes the following: a) the substitution of a hydrophobic residue with a hydrophilic residue, for example, the substitution of Leu, lie, Phe Val or Ala with Ser or Thr; b) the substitution of the amino acid residue minus Cys and Pro with Cys or Pro; c) the substitution of the residue having an electrically positive side chain, for example, Lys, Arg or His with an electrically negative residue, for example, Glu or Asp; and d) the substitution of the amino acid residue having an extremely long side chain, for example, Phe with the amino acid residue that does not have the side chain, for example, Gly. (1) SY-001 SY-001 comprises the amino acid sequence of SEQ ID NO: 1 or 3 or the amino acid sequence having one or more deletions, insertions, substitutions or additions of amino acid in the amino acid sequence of SEQ ID NO. : 1 or 3, and has the inhibitory activity of platelet aggregation and / or the inhibitory activity of platelet adhesion, which inhibits platelet adhesion to collagen and / or the ability to bind to collagen. SY-001 can be the polypeptide expressed in a protein expression system using Escherichia coli or a protein expression system using baculovirus (AcNPV) shown in the examples described below by gene recombination technology, or the polypeptide obtained by synthesis chemistry. As a specific example of the amino acid sequence of SY-001, it is possible to exemplify one of SEQ ID NO: 1 or 3. The amino acid sequence of SY-001 is not limited to one of SEQ ID NO: 1 or 3, and it can be those (homologous) that have a certain homology with it. Homologous sequences can include polypeptides comprising the amino acid sequence having one or more deletions, insertions, substitutions or amino acid additions in the amino acid sequence of SEQ ID NO: 1 or 3, and having the aggregation inhibitory activity platelet and / or inhibitory activity of platelet adhesion, which inhibit platelet adhesion to collagen and / or collagen binding capacity.

The inhibitory activity of platelet aggregation, which SY-001 has, includes actions that inhibit or block the condition in which the substance that induces platelet aggregation increases in the human blood vessel (in particular in the coronary artery, aorta and cerebral artery ), or a platelet aggregation capacity is facilitated in the blood vessel, or the condition in which damage has occurred in the blood vessel, and the platelets aggregate excessively at the damaged site. This inhibitory activity of platelet aggregation can be detected by inhibiting (suppressing) platelet aggregation induced by the platelet aggregant agent in platelet-rich plasma (PRP), by adding SY-001 in the plasma in the in vitro experiment. More particularly, the inhibitory activity of platelet aggregation of SY-001 can be determined by the following method. That is, first, PRP is prepared from human whole blood by centrifugation. Then, this prepared PRP is pre-incubated with a solution, for example, PBS solution containing SY-001, and the platelet aggregant agent is further added to this solution to add the platelets. Examples of the platelet aggregant include ADP (adenosine diphosphate), collagen, CRP (collagen-related peptide), convulxin, TRAP (thrombin receptor activating peptide), epinephrine, arachidonic acid, U-46619 (thromboxane analogue A2, analog of TXA2) and A23187 (calcium operon). A rate of platelet aggregation of the resulting solution is measured using a turbidimetric platelet aggregometer with transmittance of light (MCM HEMA TRACER 313M: provided by MC Medical), and an inhibitory rate of platelet aggregation of SY-001 is calculated from a measurement value obtained based on the value in the control that does not contain SY-001. In this way, the inhibitory activity of platelet aggregation of SY-001 can be detected. The inhibitory activity of platelet adhesion of SY-001 can also be determined by the following method. PBS solution containing SY-001 at determined concentrations is added to a 96-well plate coated with collagen solution, and incubated for 30 minutes at room temperature. After incubation, the platelet suspension is added to the well, and incubated for 45 minutes at room temperature. After incubation, the incubation solution is removed with a pipette from the well, and the well is washed with PBS. PBS solution containing 1% SDS is added to the cavity, and the cavity is air-dried after agitation. Then, the distilled water is added to the cavity, and the amount of protein in each cavity is measured using a De protein test kit (BIO-RAD Laboratories). The rate of inhibitory activity of platelet adhesion of SY-001 is calculated from a measurement value obtained based on the value in the control that does not contain SY-001, and calculating the platelet adhesion inhibitory activity of SY- 001. 001 from its platelet adhesion curve.

In this way, the inhibitory activity of platelet adhesion of SY-001, which inhibits platelet adhesion to collagen, can be detected. The binding capacity of SY-001 to the collagen can also be determined by the following method. A blocking solution of 300 μ? it is added to each 96-well plate with or without a collagen coating, and incubated for 1 hour. After removing the incubation solution from each well, 100 μ? of the solution containing SY-001 at certain concentrations are added to each well, and incubated for 1 hour at room temperature. After removing the incubation solution from each well, 200 μ? of 2% sucrose are then added to each well, and incubated for 5 minutes at room temperature. After removing the incubation solution from each cavity and after the cavities are dried, 100 pl of the reconstituted Ni-HRP solution (KPP: Kirkegaard &; Perry Laborator, Ltd.) are added to each well, and incubated for 30 minutes at room temperature. After washing with washing pH regulator, 100 μ? of ABTS peroxidase substrate (KPL Ltd.) are added to each well, and the 96-well plate is shaken gently. After the reaction ends, 100 μ? of SDS at 1% are added to each well, and the well is then measured using a microplate reader with a change in absorbance at 405-4 0 nm. The binding capacity of SY-001 to collagen at determined concentrations is calculated from a DO value obtained based on the value in the control vector that does not contain SY-001, and calculating the binding capacity of SY-001 to the collagen from its collagen binding curve. In this way, the binding capacity of SY-001 to the collagen can be detected. An antiplatelet activity that SY-001 has, can also be determined by the following method. That is, PRP is prepared from the whole blood obtained by collecting a blood sample using a syringe with an anticoagulant from a healthy donor by centrifugation. Then, the prepared PRP is diluted with a suitable pH regulator, for example, Tyrode-Hepes (34 mM NaCl, 0.34 mM Na2HP0, 2.9 mM KCI, 12 mM NaHCO3, 20 mM Hepes, 5 mM glucose, 1 mM MgCl2, pH 7.3), and SY-001 at certain concentrations is added to this serial dilution, and pre-incubated. To this solution are added fluorescence-labeled anti-selectin P antibody, an antibody (provided by Becton Dickinson) which recognizes PAC-1 (GPIIb / GPIIIa complex), fibrinogen and annexin V, and then the platelet aggregant agent, for example , ADP, collagen or TRAP, is added to activate the platelets. In this way, the fluorescent intensity of the activated platelets is measured by flow cytometry to evaluate the activity (inhibitory activity on activation) of SY-001 on the activation of platelets. This inhibitory activity on the activation of platelets is an activity inhibiting the activation of platelets. In the above method, if fluorescently labeled antibodies are used that recognize the platelet and the leukocyte, it is also possible evaluate the action of the compound on an interaction of the platelet and the leukocyte (platelet-leukocyte adhesion). For details of the method for measuring the inhibitory activity of SY-001 on platelet aggregation using the turbidimetric platelet aggregometer with light transmittance, the present invention relates to, for example, Born, GVR, "Aggregation of blood platelets by adenosine diphosphate and its reversal ", Nature, 1962, 194, 927-9, 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. For the measurement of platelet activation, the present invention also makes reference to, for example, Ito, H., et al. , "Cilostazol inhibits platelet-leukocyte interaction by suppression of platelet activation", Platelets, 2004, 1 5, 293-301. Therefore, SY-001 having the inhibitory activity on platelet aggregation and / or the inhibitory activity on platelet adhesion, has a possibility as a therapeutic agent for the occurrence or prevention of blood-related diseases and complications thereof. , acting on blood and blood vessel in mammalian animals, to inhibit or prevent the formation of thrombi or emboli. Accordingly, it is thought that SY-001 having the inhibitory activity on platelet aggregation and / or the inhibitory activity on platelet adhesion has a possibility of being useful as the agent Therapeutic agent or the preventive agent for the pathological conditions subsequent to the diseases and the complications thereof, for example, myocardial infarction, cerebral embolism, chronic arterial obstruction, arterial sclerosis, ischemic cerebral infarction, angina, venous thrombosis, hypertension, pulmonary hypertension , cerebral infarction, pulmonary infarction, heart failure, nephritis, renal dysfunction and subarachnoid hemorrhage, caused by the formation of thrombi or emboli. It is also thought that SY-001 having the inhibitory activity on platelet aggregation and / or the inhibitory activity on platelet adhesion, has a possibility of being useful for the prevention of thrombus formation after PTCA and stent placement, and as the restenosis preventive agent after stent placement, by wrapping the medicine containing SY-001, applying it or including it in the stent itself. A degree and a position of the modification, that is, the "deletion, insertion, substitution or addition" of the amino acid residues in SY-001 represented as items (b) and (d) above are not particularly limited, while the polypeptide (one equivalent) comprising the modified amino acid sequence has substantially the same activity as that of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3. It is preferable that the above modification is typically performed in about one to several amino acid residues.

In the present invention, "deletions, insertions, substitutions or additions of multiple amino acids" refer to 2 or more and 20 or fewer amino acids have been deleted, inserted, substituted or added. The multiple amino acids are preferably 2 or more and 10 or less, more preferably 2 or more and 7 or less, and even more preferably 2 or more and 5 or less. This modified amino acid sequence has, for example, about 70% or more, preferably about 80% or more, more preferably about 95% or more, and even more preferably about 98% or more homology, with the amino acid sequence of either of SEQ ID NO: 1 or 3. Specific examples of the polypeptide (SY-001) which is the active component of the pharmaceutical composition of the present invention, are as shown in the examples described below. SY-001 has the inhibitory activity of platelet aggregation and / or the inhibitory activity of the inherent platelet adhesion. SY-001 also has the ability to bind to collagen, inherent. Therefore, the pharmaceutical composition of the present invention containing SY-001 as the active component, is useful as the therapeutic agent or the preventive agent for the pathological condition subsequent to the diseases and complications thereof, for example, acute coronary syndrome, myocardial infarction, cerebral embolism, chronic arterial obstruction, arterial sclerosis, ischemic cerebral infarction, angina, venous thrombosis, hypertension, pulmonary hypertension, cerebral infarction, pulmonary infarction, heart failure, nephritis, renal dysfunction and subarachnoid hemorrhage, caused by the formation of thrombi or emboli. The pharmaceutical composition of the present invention is also useful for the prevention of thrombus formation after PTCA and stent placement, and as the restenosis preventive agent after stent placement, by applying it on the stent itself. (2) Polynucleotide (DNA molecule) encoding SY-001 A specific example of the polynucleotide encoding SY-001 (sometimes referred to as a "SY-001 DNA molecule"), may include the polynucleotide DNA) comprising the DNA sequence of SEQ ID NO: 2 or 4, or the complement thereof. Another example of the DNA molecule of SY-001 includes the polynucleotide that hybridizes with the polynucleotide comprising the complement of the DNA sequence of SEQ ID NO: 2 or 4 under the severe condition, and is capable of expressing the polypeptide having the inhibitory activity of the platelet aggregation and / or the platelet adhesion inhibitory activity. The "severe condition" may include in the present the condition in which the hybridization occurs in 2 x SSC containing 0.1% SDS at 50 ° C, and is not separated by washing in 1 x SSC containing 0.1% SDS at 60 ° C.

In addition, another example of the DNA molecule (polynucleotide) of SY-001, includes the polynucleotide comprising the DNA sequence having 80% or more, preferably 95% or more, and more preferably about 98% or more homology , with the polynucleotide most closely related among the polynucleotides comprising the DNA sequence of SEQ ID NO: 2 or 4 or the complement thereof, and capable of expressing the polypeptide having the platelet aggregation inhibitory activity and / or the inhibitory activity of platelet adhesion. For DNA molecules (sometimes referred to as modified DNA molecules) capable of expressing the polypeptides shown as these other examples having the desired action, it is an essential requirement that the polypeptide of the amino acid sequence encoded by the DNA molecule can expressing the inhibitory activity of platelet aggregation and / or the inhibitory activity of platelet adhesion (in particular, the inhibitory activity on platelet aggregation induced by collagen and / or the inhibitory activity on platelet adhesion to collagen). In other words, the requirement is that a transformant transformed with a recombinant expression vector in which the polynucleotide (modified DNA molecule) has been inserted, can express the protein having the inhibitory activity of platelet aggregation and / or the inhibitory activity of the platelet adhesion, as the product expressed therein. The above modified DNA molecule includes DNA molecules that include the DNA sequences encoding the sequence of amino acids of SEQ ID NO: 1 or 3, in particular the amino acid sequences (modified amino acid sequences) having one or more deletions, insertions, substitutions or additions of amino acids, and the DNA sequences coding for the complement thereof . The modified DNA may be that which can detect the DNA molecule of the present invention prior to modification using it. A DNA molecule homologous to the polynucleotide (DNA of SY-001, or fragments thereof) included in the DNA molecule of SY-001 and comprising the DNA sequence of SEQ ID NO: 2 or 4, means a series of molecules of related DNAs having sequence homology with the DNA sequence of SEQ ID NO: 2 or 4 and recognized as a family of DNA molecules by their structural characteristics, and as a group in their expression patterns and similarity in their biological functions. Said homologous DNA molecule can be those obtained artificially based on the naturally occurring DNA molecule (for example, DNA fragment of SY-001). Examples of this artificial method include genetic engineering techniques such as site-specific mutagenesis [Methods, 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)], chemical synthesis methods such as the phosphotriester method and the 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 combinations thereof. More specifically, the DNA molecule can also be synthesized by the phosphoramidite method or the phosphotriester method, and can also be synthesized using a commercially available automatic oligonucleotide synthesizer. A double stranded fragment can be obtained by synthesizing a complementary strand, and attaching the complementary strand to a single strand synthesized chemically under a suitable condition, or by adding the strand complementary to the single strand synthesized chemically using DNA polymerase with suitable primers. The DNA sequence of SEQ ID NO: 2 or 4, which is a specific modality of the DNA molecule of SY-001, is an example of a combination 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 the DNA molecule having said particular DNA sequence, and may have the combination of optional codons and the DNA sequence selected for each residue of amino acid The codon can be selected according to the standard method. On that occasion, a frequency of use of codons in a used host may be considered [Nucleic Acids Res., 9, 43 (1981)]. (3) Production of the DNA molecule of SY-001 The DNA molecule of SY-001 can be produced and obtained easily by synthesis, based on the information of the nucleic acid sequence of the polynucleotide encoding SY-001 described in present, or by directly synthesizing the DNA molecule corresponding to the nucleic acid sequence encoding the amino acid sequence based on the amino acid sequence information of SY-001 (chemical synthesis of DNA). General genetic engineering techniques can be applied to this production [see, for example, Molecular Cloning 2a. ed., Cold Spring Harbor Lab. Press (1989); "Zoku Seikagaku Jikken Kouza 1", "Idenshi Kenkyuho II", edited by the Japanese Biochemical Society (1986)]. As a method of chemical synthesis of DNA in which the DNA molecule of SY-001 is directly synthesized, it is possible to exemplify a solid phase synthesis method by a phosphoramidite method. An automatic synthesizer can be used for this synthesis method. The production of the DNA molecule of SY-001 through the technique of genetic engineering, can be carried out more specifically by preparing a cDNA library from a suitable source in which the DNA molecule of SY-001 has been expressed according to the standard method, and selecting a desired clone from the library using a suitable probe or specific antibody for the DNA molecule of SY-001 [Proc. Nati Acad. Sci., USA., 78, 6613 (1981); Science, 222, 778 (1983)].

In the foregoing, as the source of cDNA, it is possible to exemplify several cells or tissues in which the DNA molecule of SY-001 is expressed, and cultured cells derived therefrom. In particular, it is desirable to obtain the salivary glands of Anopheles stephensi, which is a killer insect that serves as the source. All steps of extracting and isolating the total RNA from the source, separating and purifying the messenger RNA, and acquiring and cloning cDNA, can be carried out according to standard methods. The DNA molecule of SY-001 can be produced using the cDNA collection from the salivary glands of Anopheles stephensi obtained by extracting, separating and purifying the messenger RNA from the salivary glands. In addition to this, the DNA molecule of SY-001 can also be produced using a phage collection prepared by extracting the previous messenger RNA from the salivary glands, adding poly A to the RNA, then collecting the RNA with poly A, producing cDNA using reverse transcriptase , and then adding restriction enzyme sites at both ends of the cDNA, which is incorporated into a phage. The method for identifying the SY-001 molecule from the cDNA library is not particularly limited, and can be carried out according to standard methods. As the specific method, it is possible to exemplify the method in which a corresponding cDNA clone is selected by immunological identification using an antibody (e.g., anti-saliva antibody from Anopheles stephensi) specific for the protein produced by cDNA, a method of plaque hybridization using the probe that is selectively linked to the target DNA sequence, a method of colony hybridization, and combinations thereof. The probe used in each hybridization method above is generally the chemically synthesized DNA fragment based on the information for the DNA sequence of the DNA molecule of SY-001. The DNA molecule of SY-001 and the fragment thereof already obtained can be used advantageously as the previous probe. In addition, a sense primer and an antisense primer obtained based on the DNA sequence information for the DNA molecule of SY-001 can also be used as the probes for the above identification. The DNA (nucleotides) used as the probe, is the partial DNA (nucleotides) corresponding to the DNA sequence of SY-001, and comprises at least 15 consecutive nucleotides of DNA, preferably at least 20 nucleotides of consecutive DNA , and more preferably at least 30 consecutive nucleotides of DNA. The positive clone itself for the previous production of the DNA molecule of the present invention, can be used as the probe. When the DNA molecule of SY-001 is obtained, a DNA / RNA amplification method can be suitably used by the PCR method [Science, 230, 1350 (1985)]. In particular, when it is difficult to obtain full-length cDNA from the collection, the RACE method [rapid amplification of cDNA ends; Jikken Igaku, 12 (6), 35 (1994)], in particular the RACE 5 'method [M. A. Frohman, et al., Proc. Nati Acad. Sci., USA., 8, 8998 (1988)]. The initiator used for the PCR method can optionally be designed based on the sequence information of the DNA molecule of SY-001 demonstrated in the example described below, and synthesized according to the standard method. As this primer, it is also possible to use portions of DNA (SP6 promoter initiator and T7 terminator primer) added at both ends of the cDNA of a vector plasmid in which the SY-001 cDNA has been incorporated, as shown in FIG. example described later. The DNA / RNA fragment amplified by the PCR method can be isolated and purified according to standard methods, for example, a gel electrophoresis method. The DNA molecule of SY-001 and several DNA fragments thereof obtained as above, can be sequenced according to the standard method, for example, a dideoxy method [Proc. Nati Acad. Sci., USA., 74, 5463 (1977)] or a Maxam-Gilbert method [Methods in Enzymology, 65, 499 (1980)], or simply using a commercially available sequencing kit. (4) Genetically engineered production of the expressed product of the present invention The expressed product (recombinant SY-001) of the present invention can be produced easily and stably in a large amount as the expressed product of the DNA molecule or the protein containing the same, using the sequence information of the DNA molecule of SY-001 according to common genetic engineering techniques [see, for example, Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc. Nati Acad. Sci., USA., 80, 5990 (1983)]. More particularly, the expressed product of the present invention can be obtained by preparing a recombinant DNA (expression vector) capable of expressing the DNA encoding the desired protein in a host cell, transforming the host cell with this vector to obtain a transformant, culturing the transformant, and collecting the target protein from the resulting culture. In the production of the expressed product of the present invention, any of prokaryotic organisms and eukaryotic organisms can be used as the host cell. For example, the prokaryotic organism as the host can be any of Escherichia coli, Bacillus subtilis, and the like, generally used. Conveniently, Escherichia coli, in particular the K 2 strain of Escherichia coli, can be used. The host cells of eukaryotic organisms include vertebrate and yeast cells. The first examples include conveniently monkey COS cells [Cell, 23: 175 (1981)], Chinese hamster ovary cells, and a dihydrofolic reductase acid deletion line thereof [Proc. Nati Acad. ScL, USA., 77: 4216 (1980)], and the latter examples conveniently include yeast cells belonging to the genus Saccharomyces. The host cells are not in fact limited thereto. When the prokaryotic cell is used as the host, using a vector capable of showing replication in the host cell, it is possible to conveniently use an expression plasmid obtained by incorporating a promoter and an SD (Shine-Dalgarno) sequence and a start codon ( for example, ATG) required for the initiation of protein synthesis at the 5 'end of the gene of the present invention, so that the gene can be expressed in this vector. As the above vector, plasmids such as pET-16b, pET-32, pBR322, pBR325, pUC12 and pUC13 derived from Escherichia coli are generally used frequently. Without being limited thereto, various known vectors may be used. Examples of the commercially available vector used for an expression system using Escherichia coli, include pGEX-4T (Amersham Pharmacia Biotech), pMAL-C2, pMAL-P2 (New England Biolabs), pET-16, pET-32, pET-21 , ??? - 21 / lacq (Invitrogen) and pBAD / His (Invitrogen). The expression vector when a vertebrate cell is used as the host, includes those which typically have the promoter, an RNA splice site, a polyadenylation site and a sequence of termination of the transcription located at the 5 'end of the gene of the present invention that will be expressed. These may also have an origin of replication, if necessary. Specifically, examples of the expression vector include pSV2dhfr [Mol. Cell. Biol., 1: 854 (1981)] which has an SV40 early promoter. In addition to the above, several commercially available, known vectors can be used. Examples of commercially available vectors used for the expression system using the animal cell, include vectors such as pEGFP-N, pEGFP-C (Clontech), pIND (Invitrogen) and pcDNA3.1 / His (Invitrogen) for animal cells, and vectors such as pFastBac HT (GibcoBRL), pAcGHLT (PharMingen), pAc5 / V5-His, ???? / 5-His and pMT / Bip / V5-his (Invitrogen) for insect cells. As the vector for the insect cells, it is possible to exemplify a baculovirus vector (Takara) in which SY-001 cDNA has been incorporated. Specifically, the expressed product of the present invention can be obtained by introducing the baculovirus expression vector in which SY-001 cDNA has been incorporated into cultured BmN4 cells or silkworm larvae (Bombyx mori), using the polyhedrosis virus Nucleus (BmNPV) of the silkworm for expression, and isolating from a culture medium, or a body fluid of the silkworm by chromatography. The expressed product of the present invention can also be obtained by incorporating the SY-001 cDNA into the nuclear polyhedrosis virus (AcNPV) of Autographa californica, expressing it in Sf9 cells of Spodoptera frugiperda or Tn5 cells of Trichoplusia ni, and similarly by purifying it from a culture supernatant by chromatography. Specific examples of the expression vector when the yeast cell is used as the host, include pAM82 [Proc. Nati Acad. Sci., USA., 80: 1 (1983)] which has the promoter for the acid phosphatase gene. Examples of the commercially available expression vector for the yeast cell include pPICZ (Invitrogen) and pPICZa (Invitrogen). The promoter is not particularly limited. When a bacterium belonging to the genus Escherichia is used as the host, it is possible to use preferably the tryptophan (trp) promoter, Ipp promoter, lac promoter, recA promoter and PL / PR promoter. When the host belongs to the genus Bacillus, the SP01 promoter, SP02 promoter, penP promoter, and the like are preferred. When the yeast is used as the host, it is possible to conveniently use the pH05 promoter, PGK promoter, GAP promoter, ADH promoter, and the like. Preferred promoters when the animal cell is used as the host are the SV40-derived promoter, the retrovirus promoter, metallothionein promoter, heat shock promoter, cytomegalovirus promoter, SRa promoter, and the like. When the insect cell is used as the host, it is possible to exemplify the p10 baculovirus promoter, polyhedrin promoter, and the like. As the expression vector of the DNA molecule of SY-001, an expression vector for a protein of fusion. Specific examples of the vector include pGEX (Promega) for expression as the fusion protein with glutathione-S-transferase (GST). As the polynucleotide sequence corresponding to a coding sequence in the mature polypeptide, which facilitates the expression and secretion of the polypeptide from the host cell, it is possible to exemplify a secretory sequence and a leader sequence. These sequences include marker sequences (hexahistidine tag, histidine tag), eg, hemagglutinin tag in the case of the animal cell, used for the purification of the mature polypeptide fused to the host bacterium. The method for introducing the desired recombinant DNA (expression vector) into the host cell, and the method for transformation therewith, are not particularly limited, and various general methods can be used. The resulting transformant can be cultured according to standard methods, and by culture, the target protein (expressed product) encoded by the SY-001 DNA molecule designed as desired, is expressed and produced (accumulated and secreted) intracellularly or extracellularly or on the membrane of a cell in the transformant. As a medium used for culture, several commonly used media can be selected and optionally used, depending on the host cell used. The culture can also be carried out under the condition suitable for the growth of the host cell.

The expressed product (recombinant protein) of the present invention obtained in this way can be separated and purified by several separation manipulations [see, Biochemisty data book II, pgs. 1 175-1259, first edition, first printing, published by Tokyo Kagaku Dojin on June 23, 1980; Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987)], using their physical and chemical natures, as desired. Such methods specifically include customary rearrangement treatment, treatment (salification) by a protein precipitation agent, centrifugation, osmotic shock, sonication, ultrafiltration, various liquid chromatography methods such as molecular sieve chromatography (gel filtration), absorption, ion exchange chromatography, affinity chromatography and high performance liquid chromatography (HPLC), dialysis, and combinations thereof. The particularly preferable method includes affinity chromatography using a column to which the antibody specific for SY-001 has been bound. When designing the polynucleotide encoding SY-001, it is possible to use the DNA sequence of the DNA molecule of SY-001 of SEQ ID NO: 2. In the sequence, it is also possible to select, change and optionally use the codon for each amino acid residue, as desired. In the amino acid sequence of SY-001, when a part of the amino acid residues or the amino acid sequence is modified by substitution, insertion, deletion or addition, various methods such as the site-specific mutagenesis described above can be used. (5) Expressed product of the present invention which is an active component of the pharmaceutical composition of the present invention The expressed product (and the same expressed product used for the identification method of the present invention) of the present invention, which is the Active component of the pharmaceutical composition of the present invention, can be obtained by the genetic engineering techniques shown in subsection (4) above. The inhibitory activity of the expressed product of the present invention on platelet aggregation is the same as that defined for the inhibitory activity of the platelet aggregation of SY-001 described in subsection (1) above. This inhibitory activity can be determined according to the publicly known platelet aggregation test method, for example, by the platelet-rich plasma platelet aggregation (PRP) platelet aggregation determination method, using the turbidimetric platelet aggregometer with light transmittance. More particularly, the inhibitory activity of platelet aggregation of the expressed product of the present invention on platelet aggregation can be determined and evaluated by measuring the level of platelet aggregation by the addition of the platelet aggregation agent in PRP prepared from human blood, in the presence of or absence of SY-001 using the turbidimetric platelet aggregometer with light transmittance, and calculating the inhibitory rate of platelet aggregation of SY-001 from its platelet aggregation curve. The inhibitory activity of the expressed product of the present invention on platelet adhesion is the same as that defined for the inhibitory activity of platelet adhesion (ie, inhibiting platelet adhesion to collagen) of SY-001 described in subsection ( 1) previous. This inhibitory activity can be determined according to the publicly known platelet adhesion test method for collagen, for example, by the method of determining platelet adhesion to collagen using the De protein test kit (BIO-RAD Laboratories ). More particularly, the inhibitory activity of platelet adhesion of the expressed product of the present invention on platelet adhesion to collagen can be determined and evaluated by measuring the level of platelet adhesion to collagen by the addition of the platelet suspension prepared from of human blood in the presence or absence of SY-001, using the De protein test kit, and calculating the inhibitory activity of platelet adhesion of SY-001 from its platelet adhesion curve. The collagen binding capacity exhibited by the expressed product of the present invention on collagen binding is the same as that defined for the collagen binding capacity (ie, SY-001 binds to the collagen) of SY-001 described in subsection (1) above. This ability Collagen binding can be determined according to the publicly known collagen binding test method, for example, by the method of determining collagen binding capacity using a microplate reader with a change in absorbance to 405-410 nm. More particularly, the collagen binding capacity exhibited by the expressed product of the present invention on collagen binding, can be determined and evaluated by measuring the level of binding capacity of SY-00 to collagen by the addition of SY -00 at determined concentrations in the presence or absence of collagen using the microplate reader with a change in absorbance at 405-410 nm, and calculating the binding capacity of SY-001 to the collagen from its collagen binding curve . The composition of the present invention is useful as the therapeutic agent or the preventive agent for the pathological conditions subsequent to the diseases and the complications thereof., for example, acute coronary syndrome, myocardial infarction, cerebral embolism, chronic arterial obstruction, arterial sclerosis, ischemic cerebral infarction, angina, venous thrombosis, hypertension, pulmonary hypertension, cerebral infarction, pulmonary infarction, heart failure, nephritis, renal dysfunction and subarachnoid hemorrhage, caused by the formation of thrombi or emboli, by the inhibitory activity of the expressed product of the present invention on platelet aggregation and / or platelet adhesion, based on which it comprises the expressed product of the present invention as the active component. The composition of the present invention is also useful for the prevention of thrombus formation after PTCA and stent placement, and as the restenosis preventive agent after stent placement by wrapping, applying or including the preventative agent in the stent itself. (6) Chemical synthesis of SY-001, which is the active component of the pharmaceutical composition of the present invention The polypeptide (SY-001) which is the active component of the pharmaceutical composition of the present invention, can also be produced by the method of general chemical synthesis according to the amino acid sequence information of SEQ ID NO: 1 or 3. The method includes peptide synthesis methods by a usual liquid phase method or solid phase method. Peptide synthesis methods include the so-called gradual elongation method, in which each amino acid is synthesized sequentially and linked, one by one, to elongate the chain, and a method of fragment condensation in which fragments comprising several amino acids they are synthesized, and then respective fragments are coupled. SY-001 can be synthesized by any of these methods. A condensation method used for the synthesis of peptides can also be carried out according to standard methods. Examples of standard methods may include an azide method, a mixed acid anhydride method, a DCC method, an active ester method, an oxidation and reduction method, a DPPA (diphenylphosphoryl azide) method, a DCC + additive method (1-hydroxybenzotriazole, N-hydroxysuccinimide, N- hydroxy-5-norbomeno-2,3-dicarboxiimide), and the Woodward method. After the above peptide synthesis reaction, the amino acid not involved in the reaction or the carboxyl group in the peptide can be protected as a lower alkyl ester such as methyl ester, ethyl ester and tert-butyl ester, and ester of aralkyl such as benzyl ester, p-methoxybenzyl ester and p-nitrobenzyl ester, generally by esterification. The hydroxyl group on the amino acid such as tyrosine residue, having a functional group on the side chain, can be protected with acetyl group, benzyl group, benzyloxycarbonyl group or tert-butyl group, but it is not always necessary to be protected. In addition, for example, the guanidino group in the arginine residue can be protected with a suitable protecting group, such as nitro, tosyl, p-methoxybenzenesulfonyl, methylene-2-sulfonyl, benzyloxycarbonyl, isobornyloxycarbonyl or adamantyloxycarbonyl group. A deprotection reaction of these protecting groups on the amino acid, the peptide and the polypeptide which is the active component of the finally obtained pharmaceutical composition of the present invention having the protecting group, can be carried out according to the methods commonly used, such as the contact reduction method, and methods using liquid / sodium ammonia, hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, and the like. SY-001 obtained in this way, which is the active component of the pharmaceutical composition of the present invention, can optionally be purified according to various methods such as methods using an ion exchange resin, partition chromatography and gel chromatography, and a countercurrent distribution method commonly used in the field of peptide chemistry. (7) Pharmaceutical composition of the present invention It is important for the pharmaceutical composition of the present invention, which contains SY-001 or the expressed product of the present invention as the active component. The pharmaceutical composition is useful as the pharmaceutical composition, in particular as an inhibitor of platelet aggregation to inhibit or block the condition in which the substance that induces platelet aggregation is increased in the human blood vessel (in particular in coronary artery, aorta and cerebral artery), or a platelet aggregation capacity is facilitated in the blood vessel, or the condition in which the damage has occurred in the blood vessel and the platelets are added excessively in the damaged site.

The pharmaceutical composition is also useful, in particular as an inhibitor of platelet adhesion to inhibit or block platelet adhesion to substances as an aggregating agent, such as collagen, in which the substance that induces adhesion with platelets in the human blood vessel (in particular in the coronary artery, aorta and cerebral artery) or a platelet adhesive capacity is facilitated in the blood vessel, or the condition in which the damage has occurred in the blood vessel and the platelets are added excessively in the damaged site. The pharmaceutical composition of the present invention is useful as the therapeutic agent or preventive agent for the pathological condition subsequent to the diseases and complications thereof, for example, acute coronary syndrome, myocardial infarction, cerebral embolism, chronic arterial obstruction, arterial sclerosis, ischemic cerebral infarction, angina, venous thrombosis, hypertension, pulmonary hypertension, cerebral infarction, pulmonary infarction, heart failure, nephritis, renal dysfunction and subarachnoid hemorrhage, caused by the formation of thrombi or emboli, taking advantage of their inhibitory activity on platelet aggregation and / or platelet adhesion. The pharmaceutical composition is useful for the prevention of thrombus formation after PTCA and stent placement, and prevention of restenosis after stent placement, by wrapping, applying or including the pharmaceutical composition in the stent itself, waiting for the inhibitory activity of SY-001 on platelet aggregation and / or platelet adhesion. SY-001 or the expressed product of the present invention which is the active component in the pharmaceutical composition of the present invention, has the inhibitory activity on platelet aggregation and / or platelet adhesion, and can be used for the treatment procedure of the disease associated with platelet aggregation and / or platelet adhesion in the target cell or tissue, taking advantage of this action or activity. Examples of the target cell affected by said platelet aggregation and / or platelet adhesion may include platelets and blood cells. Examples of tissues comprising these cells may include coronary artery vessel, cerebral arterial vessel, cervical arterial vessel, arterial vessel, venous vessel, peripheral arterial vessel, peripheral venous vessel, renal arterial vessel and hepatic arterial vessel. According to the inhibitory activity of platelet aggregation and / or the platelet adhesion inhibitory activity or the action to inhibit platelet aggregation and / or the action to inhibit platelet adhesion in the target cell, that the pharmaceutical composition of the present invention has, it is possible to treat or prevent the pathological conditions subsequent to the diseases and the complications thereof, for example, myocardial infarction, cerebral embolism, chronic arterial obstruction, arterial sclerosis, cerebral ischemic infarction, angina, venous thrombosis, hypertension, pulmonary hypertension, cerebral infarction, pulmonary infarction, insufficiency cardiac, nephritis, renal dysfunction and subarachnoid hemorrhage, caused by the formation of thrombi or emboli. According to the inhibitory activity of platelet aggregation and / or the inhibitory activity of platelet adhesion of SY-001 on platelet aggregation and / or platelet adhesion, it is possible to prevent the formation of thrombi after PTCA and stent placement, and preventing restenosis after stent placement, by wrapping the pharmaceutical composition of the present invention, applying it or including it, in the stent itself. The pharmaceutical composition of the present invention for the prevention of restenosis after stent placement can be used as a cyclodextrin clathrate. The pharmaceutical composition of the present invention can also be used by applying it (applying or spraying it thickly) onto a biodegradable resin that is a stent material, or including it in the stent itself. SY-001 or the expressed product which is the active component in the pharmaceutical composition of the present invention, also includes pharmaceutically acceptable salts thereof. Examples of said salt include non-toxic alkali metal salts such as sodium, potassium, lithium, calcium, magnesium, barium and ammonium salts, alkaline earth metal salts and ammonium salts. These salts can be produced according to standard methods. The above salts further include non-toxic acid addition salts obtained by the reaction of SY-001 or the expressed product of the present invention, with a suitable organic or inorganic acid. Examples of the representative non-toxic acid addition salts include hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, malate, p-toluenesulfonate (tosylate), citrate, fumarate salts, succinate, tartrate, sulfonate, glycolate, ascorbate, benzenesulfonate and napsylate. The pharmaceutical composition of the present invention is prepared in a pharmaceutical formulation form by obtaining SY-001, the expressed product of the present invention or the salt thereof which is the active component and which contains a pharmaceutically effective amount thereof, together with a suitable pharmaceutical vehicle or diluent. As the pharmaceutical carrier used to prepare the pharmaceutical formulation, it is possible to exemplify excipients and diluents such as fillers, thickeners, binders, wetting agents, disintegrators, surface activators and lubricants. These are optionally selected and used, depending on a unit dosage form of the resulting formulation. The particularly preferable pharmaceutical formulation is prepared by optionally using various ingredients, for example, a stabilizer, a germicidal agent, a pH regulator, an isotonization agent, a chelating agent, a pH adjuster, a surface active agent, and the like, used for usual protein formulations. Examples of the stabilizer in the above include human serum albumin, common L-amino acids, saccharides and cellulose derivatives.

These may be used alone or in combination with the surfactant. In particular, by this combination, the stability of the active component is further improved in some cases. The L-amino acid is not particularly limited, and any of glycine, cysteine, glutamic acid, and the like can be used. The saccharides are not particularly limited. For example, monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohol such as mannitol, inositol and xylitol, disaccharides such as sucrose, maltose and lactose, polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate and hyaluronic acid can be used. , and derivatives thereof. The surfactant is not particularly limited, and any of ionic surfactants and nonionic surfactants can be used. Specific examples thereof include surfactants based on polyoxyethylene glycol sorbitan alkyl ester, based on polyoxyethylene alkyl ether, based on monoacyl sorbitan ester and based on fatty acid glyceride. The cellulose derivative is not particularly limited. Methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and sodium carboxymethylcellulose, and the like can be used. An amount of the above saccharides to be added is about 0.0001 mg or more, and preferably about 0.01 to 10 mg relative to 1 pg of the active component. The amount of the agent surfactant to be added is approximately 0.00001 mg or more, and preferably approximately 0.0001 to 0.01 mg relative to 1 pg of the active component. The amount of human serum albumin to be added is approximately 0.0001 mg or more, and preferably approximately 0.001 to 0.1 mg relative to 1 pg of the active component. The amount of the L-amino acid to be added is conveniently from about 0.001 to 10 mg relative to 1 pg of the active component. The amount of the cellulose derivative to be added is about 0.0001 mg or more, and preferably about 0.001 to 0.1 mg relative to 1 pg of the active component. The amount of the active component contained in the pharmaceutical formulation of the present invention is optionally selected from the broad scale. It is desirable that the amount of the active component is typically from about 0.00001 to 70% by weight, and preferably from about 0.0001 to 5% by weight in the formulation. Various additives such as pH regulators, isotonization agents and chelating agents can be added in the pharmaceutical formulation. Examples of the pH regulator include boric acid, phosphoric acid, acetic acid, citric acid, e-aminocaproic acid, glutamic acid, and / or salts thereof (e.g., alkali metal salts such as sodium, potassium, calcium salts and magnesium, and alkaline earth metal salts). Examples of the isotonization agent include sodium chloride, chloride potassium, saccharides and glycerin. Examples of the chelating agent include sodium edetate and citric acid. The pharmaceutical formulation can be prepared as a solution formulation, and can additionally be obtained in a lyophilized dosage form obtained by lyophilizing the pharmaceutical formulation, which is prepared at a suitable concentration in use by dissolving in the pH regulating solution containing saline. The unit dosage form of the pharmaceutical formulation can optionally be selected, depending on a therapeutic purpose. Representative examples thereof include solid dosage forms such as tablets, pills, powdered drugs, powders, granules and capsules, and liquid dosage forms such as solutions, suspensions, emulsions, syrups and elixirs. These are further classified into oral agents, parenteral agents, nasal agents, vaginal agents, suppositories, sublingual agents and ointment agents, depending on the routes of administration, and can be combined, molded and prepared according to common methods. If necessary, coloring agents, preservatives, perfumes, flavoring and sweetening agents and other pharmaceutical formulations can also be contained in the pharmaceutical formulation of the present invention. A method of administering the pharmaceutical formulation is not particularly limited, and is determined depending on various formulation forms, an age, a gender, other conditions and the severity of the disease of a patient. For example, the tablet, the pill, the liquid, the suspension, the emulsion, the granule and the capsule are administered orally. An injectable agent is administered alone or in a mixture with a common fluid replacement such as glucose and amino acid intravenously, if necessary intramuscularly, intracutaneously, subcutaneously or intraperitoneally alone. The suppository is administered intrarectally. The vaginal agent is administered in the vagina, the nasal agent is administered in the nose, the sublingual agent is administered in the oral cavity, and the ointment agent is administered topically percutaneously. A dose of the pharmaceutical formulation is not particularly limited, and is optionally selected from the broad scale, depending on the desired therapeutic effect, the method of administration, a period of treatment, age, gender and other conditions of the patient. In general, it is preferable that the dose be determined, so that the amount of the active component is typically from about 0.01 pg to 10 mg, preferably from about 0.1 pg to 1 mg per kg of body weight per day. The formulation can be administered by dividing it once or several times per day or intermittently. (8) Identification of substances (agonist) that facilitate the inhibitory activity on platelet aggregation v / o platelet adhesion The present invention also provides the method for identifying candidate compounds that facilitate the inhibitory activity on platelet aggregation and / or platelet adhesion . The method is characterized by measuring the level of inhibitory activity of platelet aggregation and / or the platelet adhesion inhibitory activity of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3, or the polypeptide comprising the sequence of amino acids having one or more deletions, insertions, substitutions or additions of amino acid in the amino acid sequence of SEQ ID NO: 1 or 3, and having the inhibitory activity of platelet aggregation and / or the inhibitory activity of platelet adhesion in presence or absence of a subject substance, and selecting the subject substance that affects the inhibitory activity of platelet aggregation and / or the platelet adhesion inhibitory activity as the agonist, comparing the measurement value in the presence of the subject substance with the value of measurement in the absence of the subject substance. The level of inhibitory action on platelet aggregation can be obtained by the inhibition rate of platelet aggregation calculated from the measurement values obtained using the turbidimetric platelet aggregometer with light transmittance. Plus particularly, the human blood sample is collected using the syringe with the anticoagulant, and the human platelet-rich plasma (PRP) is prepared from the whole blood collected by centrifugation. A solution of the purified SY-001 previously dissolved in PBS is added to the PRP, which is then preincubated at 37 ° C. Next, a solution of collagen (provided by NYCOMED) as the platelet aggregant agent is added thereto to aggregate the platelets, and the incubation is continued for a determined period. Then, a transmittance of the solution is measured using the turbidimetric platelet aggregometer with light transmittance (MCM HEMA TRACER 313M: provided by MC Medical), to obtain the platelet aggregation curve. The rate of inhibition of platelet aggregation can be calculated from this curve in each case of SY-001 or SY-001 + subject substance. As the above aggregating agent, it is possible to use ADP, CRP, convulxin, TRAP, epinephrine, arachidonic acid, U-46619, A23817, and the like, in addition to collagen. The level of inhibitory activity of platelet adhesion can also be obtained by the rate of inhibition of platelet adhesion calculated from the measurement values obtained using the De protein test kit (Lowry method [Lowry, O. et al. ., J. Biol. Chem., 193, 265 (1951)] BIO-RAD Laboratories). More particularly, the human blood sample is collected using the syringe with the anticoagulant, and the human platelet suspension is prepared from the PRP obtained by centrifugation of whole blood. A solution of SY-001 Purified protein previously dissolved in PBS is added to a 96-well plate coated with collagen, and incubated for 30 minutes at room temperature. After incubation, the platelet suspension is added to the well, and incubated for 45 minutes at room temperature. After incubation, the incubation solution is removed with a pipette from the well, and washed with PBS. PBS solution containing 1% SDS is added to the cavity, and the cavity is air-dried after agitation. Then, distilled water is added to the cavity, the amount of protein from each cavity is measured using a De-protein test kit (BIO-RAD Laboratories), and a rate of platelet adhesion inhibiting activity of SY-001 it is calculated from a measurement value obtained based on the value in the control that does not contain SY-001, and calculating the inhibitory activity of the platelet adhesion of SY-001 from its platelet adhesion curve. The rate of inhibition of platelet adhesion can be calculated from this curve in each case of SY-001 or SY-001 + subject substance. As the inducer of anterior platelet adhesion, it is possible to use collagen. The present invention also provides the method for identifying the candidate substance that increases the platelet aggregation inhibitory activity of SY-001, comprising the steps (1) to (4) below: (1) a step for preparing the culture medium , which comprises the cell transformed with the expression vector of SY-001 and the platelet-rich plasma (PRP); (2) a step to induce platelet aggregation by adding the platelet aggregation substance to the culture medium of the preceding (1) in the presence or absence of the subject substance; (3) a step to measure the levels of inhibition of platelet aggregation in the presence of the subject substance and in the absence of the substance subject in subsection (2) above; and (4) a step for selecting the subject substance as the candidate substance when the measurement value in the presence of the subject substance is greater than the measurement value in the absence of the subject substance. The present invention also provides the method for identifying the candidate substance which increases the platelet aggregation inhibiting action of the expressed product of the present invention, comprising the steps (1) to (4) below: (1) a step for preparing the culture medium comprising the cell containing the expressed product of the present invention-- and the platelet-rich plasma (PRP); (2) a step to induce platelet aggregation by adding the platelet aggregation substance to the culture medium of the preceding (1) in the presence or absence of the subject substance; (3) a step to measure the levels of inhibition of platelet aggregation in the presence of the subject substance and in the absence of the substance subject in subsection (2) above; Y (4) a step for selecting the subject substance as the candidate substance when the measurement value in the presence of the subject substance is greater than the measurement value in the absence of the subject substance. The present invention also provides the method for identifying the candidate substance that determines the platelet adhesion inhibiting activity of SY-001, comprising the following steps (1) to (4): (1) a step for preparing the culture medium (in the cavity) comprising the cell transformed with the expression vector of SY-001 and the coating of the cavity plate with platelet-adhering substance (i.e., collagen); (2) a step to induce platelet adhesion by adding the platelet suspension in the culture medium of the preceding (1) in the presence or absence of the subject substance; (3) a step to measure levels of inhibition of platelet adhesion in the presence of the subject substance and in the absence of the substance subject in subsection (2) above; and (4) a step for selecting the subject substance as the candidate substance when the measurement value in the presence of the subject substance is greater than the measurement value in the absence of the subject substance. The present invention further provides the method for identifying the candidate substance that determines the inhibitory action of the platelet adhesion of the expressed product of the present invention, comprising the steps (1) to (4) below; (1) a step for preparing the culture medium (in the cavity) comprising the cell containing the expressed product of the present invention and coating the cavity plate with platelet-adhering substance (i.e., collagen); (2) a step to induce platelet adhesion by adding the platelet suspension in the culture medium of the preceding (1) in the presence or absence of the subject substance; (3) a step to measure levels of inhibition of platelet adhesion in the presence of the subject substance and in the absence of the substance subject in subsection (2) above; and (4) a step for selecting the subject substance as the candidate substance when the measurement value in the presence of the subject substance is greater than the measurement value in the absence of the subject substance. The identification method of the present invention can be practically carried out by applying high performance identification technology. According to the practical application of this technology, for example, in the identification of a catalyst of platelet aggregation inhibitory activity and / or platelet adhesion inhibitory activity, a preparation (including SY-001 and labeled ligand of the polypeptide ) with either a synthesis reaction mixture, a cell fraction, a blood fraction, e.g., platelet-rich plasma or platelet suspension, is incubated in the presence or absence of the subject substance to be identified. If the subject substance serves as an agonist for the action of SY-001 or antagonizes the action of SY-001, is detected by decreasing the bound labeled ligand. The level of activity of SY-001 can be detected by the use of a reporter system or colorimetric mark (it is not limited to this), the incorporation of a reporter gene responsible for the change in activity of the polynucleotide or the polypeptide, or a test of Union known publicly in the art. A competitive test in which SY-001 or the other mutant [eg, SY-001 (151-269), SY-001 (21-269)] and the other inhibitor of platelet aggregation (eg, acetylsalicylic acid, cilostazol) are combined with the compound that is bound thereto, can be used to identify the candidate compound as the catalyst of the platelet aggregation inhibitory activity. A competitive test in which SY-001 or the other mutant [for example, SY-001 (151-269), SY-001 (21-269)] and the other inhibitor of platelet adhesion (for example, triclopidine) are also combined with the compound that is bound thereto, can be used to identify the candidate compound as the catalyst of the platelet adhesion inhibiting activity. The subject substance (candidate compound) which is identified by the identification method of the present invention, is most likely the agonist of SY-001 as the catalyst of the platelet aggregation inhibitory activity and / or the platelet adhesion inhibitory activity. .

The agonist may be the substance that increases the inhibitory activity of platelet aggregation and / or the inhibitory activity of platelet adhesion measured by the presence thereof in the identification system of the present invention. Specific examples of these subject substances include oligopeptides, proteins, antibodies, RNA molecules, siRNA, non-peptide compounds (synthetic compounds), fermented products, cell extracts (plant extracts, animal extracts) and plasma. These substances can be those newly developed or known substances. Examples of the platelet aggregation inhibiting substance and / or the platelet adhesion inhibiting substance include low molecular weight organic or inorganic compounds, synthetic or naturally occurring peptides and polypeptides, or peptides and polypeptides prepared by the recombinant technology of genes, which increase the activity of SY-001 by binding to it. The sense DNA molecule for the DNA molecule encoding SY-001, administered directly in vivo or administered in the form inserted into the recombinant vector, is also included in the platelet aggregation inhibitory substance and / or the inhibitory substance of previous platelet adhesion. The candidate substance having the activity to increase the inhibitory activity of platelet aggregation and / or platelet adhesion inhibitory activity identified in accordance with the identification method of the present invention is evaluated as follows. That is, the candidate substance that increases or facilitates platelet aggregation inhibiting activity and / or platelet adhesion inhibiting activity by about 20% or more, preferably about 30% or more, and more preferably about 50% or more compared to the control (absence of the subject substance), can be evaluated as the compound that facilitates the inhibitory activity of platelet aggregation and / or the inhibitory activity of platelet adhesion. It is thought that the compound that facilitates platelet aggregation inhibitory activity and / or platelet adhesion inhibitory activity is useful as the therapeutic agent or the preventive agent for the pathological condition subsequent to the diseases and complications thereof, for example, myocardial infarction, cerebral embolism, chronic arterial obstruction, arterial sclerosis, ischemic cerebral infarction, angina, venous thrombosis, hypertension, pulmonary hypertension, cerebral infarction, pulmonary infarction, heart failure, nephritis, renal dysfunction and subarachnoid hemorrhage, caused by the formation of thrombi or emboli. Among the substances that facilitate the inhibitory activity of platelet aggregation and / or the inhibitory activity of platelet adhesion, obtained by the identification method of the present invention, it is thought that there is also the same substance that has the activity to inhibit aggregation platelet-induced platelet aggregation in vivo, and / or to inhibit platelet adhesion induced by the platelet-binding substance in vivo, and it is thought that the substance is useful as the inhibitor of platelet aggregation and / or platelet adhesion inhibitor in several fields including the pharmaceutical field. (9) Equipment for identification The present invention can provide a kit for identifying the agonist for SY-001, characterized in that it comprises the platelet-rich plasma, SY-001 (including the expressed product of the present invention) and the platelet aggregant agent as the constituents. The kit for identifying the present invention comprises (1) SY-001 (e.g., the polypeptide having the amino acid sequence of SEQ ID NO: 1 or 3) or the expressed product of the present invention (e.g., the expressed product of the DNA molecule that codes for SY-001, or the DNA sequence of SEQ ID NO: 2 or 4), (2) the cell culture medium comprising the platelet-rich plasma, and (3) the platelet aggregant agent as the essential ingredients. Similar to the equipment for identification of this type, the equipment may comprise several reagents such as cell culture media, reaction diluents, staining agents, pH regulators, fixing solutions and washing solutions as other optional ingredients. Specific examples of the kit of the present invention include those comprising the following constituents 1 to 3: constituent 1: cells (cells expressing SY-001, cultured cells comprising Escherichia coli cells or transformed insect cells with the polynucleotide (DNA molecule) coding for SY-001) cultured on a 60 mm plate at 0.5 to 1 x 105 cells / well at 37 ° C under 5% CO2, using MOPS pH regulator containing EGTA- Na; constituent 2: the substance (eg, collagen, ADP) that induces platelet aggregation; and constituent 3: platelet-rich plasma. In the identification method used by the team to identify the present invention, the rate of inhibition of platelet aggregation is detected in the platelet-rich plasma by unit visual field of the cavity, from the cavity in which the substance has been added. of test (subject substance, candidate drug substance). Then, the rate of inhibition of platelet aggregation is detected in the cavity in which the test substance has not been added. Then, the significant difference is tested between the first speed and the last speed. The measurement and evaluation can be carried out according to standard methods. The present invention can also provide a kit for identifying the agonist for SY-001, characterized in that it contains the platelet-adhering agent (ie, collagen), SY-001 (including the expressed product of the present invention) and the platelet suspension. as the constituents. The kit for identifying the present invention comprises (1) SY-001 (eg, the polypeptide having the amino acid sequence of SEQ ID NO: 1 or 3) or the expressed product of the present invention (eg, example, the expressed product of the DNA molecule encoding SY-001, or the DNA sequence of SEQ ID NO: 2 or 4), (2) the substance that adheres platelets (ie, collagen), and (3) ) the suspension of platelets as the essential ingredients. Similar to the equipment for identification of this type, the equipment may comprise several reagents such as cell culture media, reaction diluents, staining agents, pH regulators, fixing solutions and washing solutions as other optional ingredients. Specific examples of the kit of the present invention include those comprising the following constituents 1 to 3: constituent 1: cells (cells expressing SY-001, cultured cells comprising Escherichia coli cells or insect cells transformed with the polynucleotide (molecule DNA) coding for SY-001) cultured on a 60 mm plate at 0.5 to 1 x 105 cells / well at 37 ° C under 5% CO2, using PBS pH regulator; constituent 2: the substance that adheres platelets (for example, collagen, ADP) that induces platelet adhesion; and constituent 3: platelet suspension. In the identification method that the equipment uses to identify the present invention, the rate of inhibition of platelet adhesion is detected as the level of platelet adhesion to the collagen by unit visual field of the cavity, of the cavity in which it is has added the test substance (subject substance, drug candidate substance). Then, the rate of inhibition of platelet adhesion is detected in the cavity in which the test substance has not been added. Then, the significant difference is tested between the first speed and the last speed. The measurement and evaluation can be carried out according to standard methods.

EXAMPLES The present invention will be described in more detail with reference to the following examples. These examples are for exemplification only, and do not limit the present invention.

EXAMPLE 1 1 . Preparation of a cDNA collection from the salivary glands of Anopheles stephensi The Anopheles stephensi female mosquito (strain SDA500) from 3 to 7 days after hatching was made to suckle blood in a mouse (strain BALB / c acquired from CLEA Japan Inc .). The wings, legs, a portion of the head and a portion of the abdomen were removed from the mosquito 6 hours after blood suction, and only a portion of the thorax that included the salivary glands was stored in liquid nitrogen. At the time the portions of the thorax of 300 mosquitoes were collected, RNA was extracted using the RNeasy Midi (QIAGEN) equipment. A collection collecting RNA was prepared added with poly A, obtaining cDNA using reverse transcriptase, adding restriction enzyme sites (EcoRI and Hind I II sites) at both ends, and incorporating into cloning equipment? SCREEN-1 (Novagen). 2. Immunoidentification of the cDNA collection from the salivary glands Escherichia coli (ER-1647, Takara) was infected with each phage from the previous collection, and was identified using an antibody from the salivary glands anti-Anopheles stephensi as a probe. Salivary gland antibody an \\ - Anopheles stephensi was obtained by immunizing a rabbit with a homogenate of the salivary glands of Anopheles stephensi together with Freund's adjuvant. A positive clone obtained by selection was infected with Escherichia coli ER-1647 and amplified. Then, the phage was removed and an inserted portion was cloned into a plasmid pSCREEN-1 b (+) (Novagen) using a plasmid excision kit mediated by Cre (Novagen). A base sequence of the inserted portion was sequenced using portions of DNA (SP6 promoter primer: SEQ ID NO: 10, product code No. TKR3867, Takara Bio, and terminator primer T7: SEQ ID NO: 1 1, code of product No. NV432, Novagen) added at both ends of pSCREEN using the genetic analyzer 310 provided by ABI. As a result of the analysis of the base sequence, it was found that a cloned gene fragment contained a stop codon, but spelled a start codon. To obtain the 5 'region deleted from the SY-001 cDNA, the RACE 5' method was carried out [Frohman, M.A., et al., PNASC, 8, 8998-9002 (1988)] using phage DNA? screen extracted from the DNA collection of the salivary glands as template and using primers, SP6 promoter primer (SEQ ID NO: 10) and primer pAnS-1 of SEQ ID NO: 12, to clone a DNA fragment of approximately 470 bp . This DNA fragment was overlapped with the DNA fragment obtained in the above identification, and also contained the start codon. The two DNA fragments were ligated to determine the entire base sequence of the DNA molecule encoding SY-001. An open reading frame (ORF) found in the cDNA encoded 269 amino acid residues of a putative 28.5 kDa protein and contained 807 bp. It was predicted that this protein is the secreted protein like saliva, because it was predicted from the deduced amino acid sequence, that this protein was an acidic protein with pl = 3.8, 21 amino acid residues at the N-terminus were hydrophobic and exhibited a signal peptide sequence, and the C-terminal end did not have a membrane anchoring region. This deduced amino acid sequence is shown in SEQ ID NO: 5. The base sequence of the DNA molecule encoding the amino acid sequence is shown in SEQ ID NO: 6. 3. Expression of SY-001 by recombinant DNA, and purification thereof (1) Production of SY-001 (22-269) A DNA fragment encoding the amino acid residues at positions 22 to 269 of SY-001 (SEQ ID NO: 5), was amplified by PCR. The cDNA of the salivary glands shown in point 1 above was used as the template. The primer pAnSG-F7 (SEQ ID NO: 3) used had the Ncol site (CCATGG) at the third to eighth sites starting at end 5, and the primer pAnSG-R1 (SEQ ID NO: 14) used had the Notl site (GCGGCCGC) in the second to ninth sites from the 5 'end. Then, the DNA fragment was cloned into pENTR / D-TOPO (Invitrogen) to construct the plasmid pENTR-SY-001 - ???? 1 -4. Next, a DNA fragment of SY-001 (approximately 760 bp) obtained by digesting pENTR-SY-001 - ???? 1-4 with Ncol and Notl, was inserted into the Ncol / Notl site of pET32-b (+) (Novagen), to construct the plasmid pET32-SY-001-Exon 1 -4. The BL21 strain of Escherichia coli (DE3) (Novagen) was transformed with the plasmid pET32-SY-001-Exon 1 -4, and the resulting transformant was cultured with agitation in 6 ml_ of LB medium (BAL) containing 50 pg / mL of ampicillin for 15 hours at 37 ° C. Then, the cultivated medium was added in 600 ml_ of LBA, which was then cultured with agitation for 4 hours at 37 ° C. Then, 6 mL of 100 mM IPTG was added in the culture medium, which was further cultivated with agitation for 4 hours at 37 ° C. The resulting culture medium was centrifuged at 6,000 xg for 15 minutes, and a supernatant was removed. A resulting pellet was lysed by adding 40 mL of 6 M guanidine hydrochloride thereto. A resulting bacterial solution was centrifuged at 30,000 xg for 25 minutes, and the supernatant was collected. To this, 1.8 mL of Ni-NTA (QIAGEN) was added, which was then stirred for 15 minutes at 4 ° C. The mixture was centrifuged at 3,000 xg for 3 minutes, the supernatant was removed, and Ni-NTA was collected. A solution of 6 M TBS-urea (6M urea, 150 mM NaCl, 50 mM Tris-HCl [pH 7.5]) containing 10 mM imidazole was added, and Ni-NTA was collected. The resulting mixture was centrifuged at 3,000 xg for 3 minutes, and the supernatant was removed. The resulting Ni-NTA was packed into a column. The SY-001 Exon 1 -4 protein was eluted from the column, as follows. That is, 2 mL of 6 M TBS-urea solutions containing 20 mM, 50 mM, 100 mM and 200 mM imidazole, respectively, were run sequentially through the column, and respective fractions were collected. A portion of each fraction was electrophoresed on 2% SDS-PAGE, and then stained with Coomassie stain to determine the fraction that contained the SY-001 Exon 1 -4 protein. This fraction was dialyzed against PBS for 48 hours. The amount of the protein was quantified using the BCA protein test kit (PIERCE), and the yield was 5.0 mg. The amino acid sequence of the recombinant protein obtained in this manner was as shown in SEQ ID NO: 7. sequence of amino acids at positions 1 to 162, and the amino acid sequence at positions 410 to 420, are the amino acid sequences derived from a thioredoxin protein and pET32-b (+) containing a His tag sequence, respectively. The amino acid sequence of SY-001 (22 to 269) of the present invention is located at positions 162 to 409. (2) Production of SY-001 (148-269) PCR was carried out with the plasmid pET32-SY-001 Exon1 -4 prepared in part (1) above as the template, using the 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-TOPO (Invitrogen), to construct plasmid pENTR-SY-001 Exon 3-4. Plasmid pENTR-SY-001 Exon 3-4 was digested with Ncol / Notl to obtain the 372 bp DNA fragment. This was inserted into the Ncol / Notl sites of pET32 (b) + (Novagen), to construct the plasmid pET32-SY-001 Exon 3-4. The BL21 strain of Escherichia coli (DE3) (Novagen) was transformed with the plasmid pET32-SY-001-Exon 1 -4 obtained above, and the resulting transformant was cultivated with agitation in 6 mL of LB medium (BAL) containing 50 pg / mL of ampicillin for 15 hours at 37 ° C. Then, the cultivated medium was added in 600 mL of LBA, which was then cultured with agitation for 4 hours at 37 ° C. Then, 6 mL of 100 mM IPTG was added to the culture medium, which was further cultivated with stirring for 4 hours at 37 ° C. The resulting culture medium was centrifuged at 6,000 xg for 15 minutes, and the supernatant was removed. The resulting pellet was lysed by adding 40 mL of 6 M guanidine hydrochloride thereto. A resulting bacterial solution was centrifuged at 30,000 xg for 25 minutes, and the supernatant was collected. To this, 1.8 mL of Ni-NTA (QIAGEN) was added, which was then stirred for 15 minutes at 4 ° C. The mixture was centrifuged at 3,000 xg for 3 minutes, the supernatant was removed, and Ni-NTA was collected. The 6 M TBS-Urea solution (6 M urea, 150 mM NaCl, 50 mM Tris-HCl [pH 7.5]) containing 10 mM imidazole was added, and Ni-NTA was collected. The resulting mixture was centrifuged at 3,000 xg for 3 minutes, and the supernatant was removed. The resulting Ni-NTA was packed into the column. The SY-001 Exon 3-4 protein was eluted from the column, as follows. That is, 2 mL of 6 M TBS-urea solutions containing 20 mM imidazole, 50 mM, 100 mM and 200 mM, respectively, were run sequentially through the column, and respective fractions were collected. A portion of each fraction was electrophoresed on 12% SDS-PAGE, and then stained with Coomassie stain to determine the fraction containing the SY-001 Exon 3-4 protein. This fraction was dialyzed against PBS for 48 hours. The amount of the protein was quantified using the BCA protein test kit (PIERCE), and the yield was 1.8 mg. The amino acid sequence of the recombinant protein obtained in this manner was as shown in SEQ ID NO: 8, and had the amino acid sequence that contained 293 amino acid residues. In this amino acid sequence, the amino acid sequence at positions 1 to 160, and the amino acid sequence at positions 283 to 293, are the amino acid sequences derived from a thioredoxin protein and pET32-b (+) that contained a marker sequence of His, respectively. The amino acid sequence of SY-001 (148 to 269) is located at positions 161 to 282.

EXAMPLE 2 Production of recombinant SY-001 (21-269) using a baculovirus expression system (1) Production of Baculovirus SY-001 (21-269) A DNA fragment encoding the polypeptide at positions 21 to 269 of SY-001 (SEQ ID NO: 1) was amplified by PCR using the cDNA of the salivary glands shown in Example 1, 1 as the mold. The primer pAnSG-F10 (SEQ ID NO: 16) used had the BamHI site (GGATCC) in the fifth to tenth sites of the 5 'end, and a FLAG sequence in the twelfth G to the forty-first A. The initiator pAnSG-R1 (SEQ ID NO: 14) used had the Notl site (GCGGCCGC) in the second to ninth sites of the 5 'end. The DNA fragment was cloned into pENTR / D-TOPO (Invitrogen), to construct the plasmid pENTR-SY-001 - ???? 1 -4. Next, a fragment of SY-001 (780 bp) obtained by digesting pENTR-SY-001 - ???? 1-4 with BamHI / Notl was inserted into the sites BamHI / Notl from pBACgus-1 (Novagen), to construct a baculovirus transfer vector plasmid pBACgus-SY-001 - ???? 1 -4. Recombinant baculovirus was obtained using a recombinant baculovirus production kit (BacVector-2000 transfection kit, Novagen), co-transfecting Sf9 cells with the above baculovirus transfer vector plasmid pBACgus-SY-001 - ???? 1-4 and BacVector-2000 DNA. The recombinant baculovirus prepared was designated as AcNPV-SY-001 - ???? 1 -4. That is, the Sf9 cells were cultured at 1 x 10 7 cells per 150 mm Petri dish, and were infected with AcNPV-SY-001 - ???? 1-4 at a multiplicity of infection of approximately 5. After 3 to 4 days, approximately 250 mL of the culture supernatant was collected from Petri dishes of 10 to 150 mm, and 1.5 mL of Ni were added thereto. -NTA (QIAGEN), which was then stirred for 15 hours at 4 ° C. The mixture was centrifuged at 3,000 xg for 3 minutes, and the supernatant was discarded. Ni-NTA was collected, and 50 mL of a TBS solution (150 mM NaCl, 50 mM Tris-HCl [pH 7.5]) containing 10 mM imidazole was added. The resulting mixture was centrifuged at 3,000 xg for 3 minutes, and the supernatant was discarded. The Ni-NTA obtained was packed in the column. The SY-001 Exon 1 -4 protein was eluted from the column, as follows. That is, 2 mL of the TBS solutions containing 20 mM, 50 mM, 100 mM and 200 mM imidazole, respectively, were run sequentially through the column, and fractions were collected. respective. A portion of each fraction was electrophoresed on 12% SDS-PAGE, and then stained with Coomassie stain to determine the fraction containing the SY-001 Exon 1 -4 protein. This fraction was dialyzed against PBS for 48 hours. The amount of the protein was quantified using the BCA protein test kit (PIERCE), and the yield was 0.8 mg. The amino acid sequence of the recombinant protein SY-001 (21-269) obtained in this way is shown in SEQ ID NO: 9. In the sequence, the sequence in positions 1 to 10 is the FLAG sequence, the sequence of amino acids at positions 1 1 to 259 is the sequence of SY-001 (21 -269), and the sequence at positions 260 to 270 is the sequence derived from pBACgus-1 containing the His marker sequence.

EXAMPLE 3 The inhibitory action of SY-001 on platelet aggregation was evaluated by measuring platelet-rich platelet aggregation (PLP) using a turbidimetric platelet aggregometer with light transmittance. An outline of the method is as follows. That is, first, a blood sample was collected from a healthy donor using a syringe with anticoagulant, and the platelet-rich plasma (PRP) was prepared by centrifuging the collected whole blood. The prepared PRP was mixed and preincubated with a solution of SY-001 [PBS solution in which SY-001 had been dissolved having the amino acid sequence of SEQ ID NO: 7 prepared in Example 1, 3- (1)] or PBS as the control, and then the platelets were added by adding a platelet aggregant agent. As the platelet aggregant agent was used, respectively, ADP (provided by Sigma), collagen (provided by NYCOMED), CRP (provided by Peptide Institute Inc.), convulxin (provided by Alexis), TRAP (provided by Sawaday Technology Co.) , epinephrine (provided by Daiichi Pharmaceutical Co., Ltd.), arachidonic acid (provided by Sigma), U-46619 (provided by Cayman) or A23187 (provided by Sigma). Then, the rate of platelet aggregation was measured for 5 minutes using the turbidimetric platelet aggregometer with light transmittance (MCM HEMA TRACER 313M: provided by MC Medical), and the maximum aggregation rate was obtained in the measurement by 5 minutes. The rate of inhibition of platelet aggregation (%) of SY-001 was calculated according to the following formula. Rate of inhibition of platelet aggregation (%) = (1 - As / Ac) x 00, where: As: Maximum platelet aggregation rate in PRP with SY-001 Ac: Maximum platelet aggregation rate in PRP only as control. The details of the previous manipulation are as shown in subsections (a) to (c) below. (a) First, 60 mL of blood was collected from the healthy donor using the syringe with 6 mL of 3.8% sodium citrate as the coagulant. Then, the blood was centrifuged at 1, 100 rpm for 10 minutes, and a layer of human platelet-rich plasma (PRP) as an upper layer was transferred to another test tube. A portion of the remaining lower layer was centrifuged at 3,000 rpm for 10 minutes, and a resulting upper bright yellow layer (platelet deficient plasma, PPP) was transferred to another test tube. The mixture in which the number of platelets had been adjusted to 3 x 108 / mL was obtained by mixing the PRP and the PPP obtained above, and was used in the next measurement. This mixture is referred to as a "PRP measurement sample" in the next measurement. The platelet aggregometer platelet aggregation velocity was adjusted so that the light transmittance in the PPP was 100% of the platelet aggregation rate, and the light transmittance in the PRP was 0% of the rate of platelet aggregation. (b) Determination of the collagen concentration and measurement of the platelet aggregation inhibitory activity. First, an aggregation tube in which 200 pL of the PRP measurement sample not containing SY-001 had been added was adjusted in the platelet aggregometer, and incubated for 2 minutes at 37 ° C. Then, 22.2 pL of a collagen solution (provided by NYCOMED GmBH, Moriya Sangyo) was added to the miso, and the rate of platelet aggregation was continuously measured for 5 minutes at 37 ° C. The highest platelet aggregation rate in 5 minutes it was considered as the maximum platelet aggregation velocity. In the collagen solution, concentrations at 5 to 20 pg / mL (final concentrations in the PRP measurement sample were 0.5 to 2 pg / mL) were used as submaximal concentrations at which 70% of the speed was induced of maximum platelet aggregation. c) Then, (i) SY-001 (22-269) [having the amino acid sequence of SEQ ID NO: 7 prepared in Example 1, 3- (1)] prepared at 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 100 mM, 300 nM and 100 nM, respectively, or (iii) PBS (137 mM NaCl, 2.7 mM KCI, 8.1 mM Na2HPO4, 1.5 mM KH2PO4) as the control were added in each tube in which 200 pl_ of the PRP measurement sample had been added. Each mixture was adjusted in the platelet aggregometer, and incubated for 2 minutes at 37 ° C. Then, 22.2 μl of the collagen solution was added thereto at a given concentration determined above, and. the platelet aggregation rate was measured during 5 minutes from the addition of the same. The result of the inhibitory activity of the platelet aggregation of SY-001 (22-269) induced by the stimulation with collagen, is shown in figure 1. The result of the inhibitory activity of the platelet aggregation of SY-001 (148-269) induced by the stimulation with collagen, is also shown in figure 2.

In each figure, a horizontal axis represents a time course (-0.5 to 5 minutes) of 30 seconds before the addition of collagen, and a vertical axis represents the rate of platelet aggregation (%). In figure 1, curve 1 shows the result in the case of the addition of SY-001 (22-269) at a concentration of 300 nM, curve (2) shows the result of SY-001 (22-269) at 100 nM, curve (3) shows the result of SY-001 (22-269) at 30 nM, and curve (4) shows the result of the control. In figure 2, curve 1 shows the result in the case of the addition of SY-001 (148-269) at a concentration of 1000 nM, curve (2) shows the result of SY-001 (148-269) at 300 nM, curve (3) shows the result of SY-001 (148-269) at 100 nM, and curve (4) shows the result of the control. As is evident from the results shown in these figures, it has been shown that SY-001 of the present invention reduces platelet aggregation induced by collagen stimulation, since its addition amount is increased, i.e., SY-001 has the inhibitory activity of platelet aggregation. The same experiments as the previous ones were carried out using the recombinant protein SY-001 (21-269) produced in the baculovirus expression system in example 2. As a result, substantially the same results as shown in the figure were obtained 1 . In Consequently, it is obvious that the SY-001 of the present invention has the inhibitory activity of platelet aggregation. In experiments using SY-001 above (21-269), platelet aggregation rates were obtained by changing the concentrations of SY-001 (21 -269) from 3 nM to 1000 nM. Then, log-logit analysis was performed for the inhibitory activity of platelet aggregation (IC50) of SY-001 (21 -269), using the SAS software (SAS Institute, Japan, Reléase 8.1). As a result, it was calculated that the IC50 of SY-001 (21 -269) is 25 nM. From the above results, it has been speculated that a portion of epitope present on the C-terminal side between positions 148 and 269 in the amino acid sequence of SY-001 of SEQ ID NO: 5, could contribute to the inhibitory activity of the platelet aggregation of SY-001 of the present invention.

EXAMPLE 4 Synthesis of SY-001 mutant SY-001 is then synthesized by the solid phase synthesis method using the Fmoc (9-fluorenylmethyloxycarbonyl) method. A synthetic polypeptide having the desired number of amino acid residues from the N-terminal end to the C-terminal end of the amino acid sequence of SEQ ID NO: 5, can be obtained by reaction in a continuous flow mode using TBTU [2- (1 H-benzotriazole-, 1, 3,3-tetramethyluronium] tetrafluoroborate and HOBt [1-hydroxybenzotriazole hydrate] as active reagents, if necessary, the resulting synthetic polypeptide can be dissolved in dimethyl sulfoxide, and can be further diluted with acetonitrile.

EXAMPLE OF FORMULATION 1 (1) The pharmaceutical composition of the present invention in the injectable formulation was prepared by adding and mixing 100 pg / mL of SY-001 (having the amino acid sequence of SEQ ID NO: 8), 0.01 mg / mL of Tween 80 (monooleate of polyoxyethylene (20) sorbitan, polysorbate 80), 15 mg / mL of dextran 40, 0.1 mg / mL of cysteine and 1.0 mg / mL of HSA (human serum albumin) in pH regulator of citric acid 0.01 M- sodium citrate (pH 6.0), filtering the mixture (using a 0.22 pm membrane filter), then sterilizing the filtrate by 1 mL in a vial, and lyophilizing it. The formulation can be used by dissolving in 1 mL of saline in use. (2) The pharmaceutical composition of the present invention in the injectable formulation was prepared by adding 10 pg / 0.1 mL of SY-001 (having the amino acid sequence of SEQ ID NO: 8), 5 mg of cysteic acid and 1 mg of human serum albumin (HSA) per vial in water distilled for injection, filling the resulting solution in a vial for 1 mL, and lyophilizing it.

Action of AAPP on platelet adhesion to collagen A solution of SY-001 (50 μ? _) At 3, 10, 30, 100, 300, 1000 or 3000 nM was added in a 96-well plate coated with 40 pg / mL of collagen (NYCOMED GmBH), and incubated for 30 minutes at room temperature. After incubation, 50 pl_ of a platelet suspension (6 x 108 / ml_ of cells) was added to each well, and incubated for 45 minutes at room temperature. After incubation, the solution in each cavity was removed using a pipette, and the wells were washed with 200 μl of PBS. Then, 20 μl of PBS containing 1% SDS was added to each well, which was then stirred with stirring, and dried in air at 45 ° C. Then, 5 μl of distilled water was added to each cavity, and the concentration of protein in the cavity was measured, using the De (BIO-RAD) protein test kit. The results are shown in Figure 3. As shown in Figure 3, it was identified that SY-001 of the present invention inhibited platelet adhesion to collagen in a dose-dependent manner, and that SY-001 exhibited the action potent inhibitor on platelet adhesion, particularly at doses of 300 pg / mL or more.

Binding capacity of SY-001 to collagen A blocking solution (300 μ? _) Was added to each well of a 96-well plate (NUNC, 152038) coated with collagen or a 96-well plate (NUNC, 260895) uncoated, and incubated for 1 hour at room temperature. The solution in each well was removed, and 100 μl of a protein solution SY-001 at 3, 10, 30, 100 or 300 nM was added to each well, and incubated for 1 hour at room temperature. The solution in each cavity was removed, 200 pL of sucrose at 2% was added to the cavity, and they were incubated for 5 minutes at room temperature. The solution in each cavity was removed, and the cavity dried. Then, 100 pL of a reconstituted Ni-HRP solution (ExpressDetector Nickel-HRP, provided by KPL) was added to each well and incubated for 30 minutes at room temperature. After washing with a washing pH regulator, 100 μ? of ABTS peroxidase substrate, and mixed gently with agitation. After the reaction was terminated, 100 pL of SDS at 1% was added, and an absorbance at a wavelength of 405 to 410 nm was measured using a microplate reader. The results are shown in Figure 4. The triangles represent a reaction curve of the empty vector as a control. As shown in Figure 4, it was identified that SY-001 had the ability to bind collagen. As in the above, it could be identified that SY-001 of the present invention not only had the inhibitory action on aggregation platelet, but also the inhibitory action on platelet adhesion to collagen. It could also be identified that SY-001 of the present invention had the ability to bind collagen. Of these results, the pharmaceutical composition comprising SY-001 of the present invention as the active component, may be the pharmaceutical composition useful as the therapeutic agent and the preventive agents for myocardial infarction, pulmonary embolism, cerebral infarction, and the like.

Inhibitory action of platelet adhesion and collagen binding capacity of recombinant AAPP Figure 3: AAPP inhibits platelet adhesion to collagen. Figure 4: AAPP has the ability to bind collagen.

Sequence listing free text SEQ ID NO: 10 represents the sequence of the SP6 promoter primer, SEQ ID NO: 1 represents the initiator sequence of the T7 terminator, SEQ ID NO: 12 represents the sequence of the primer pAnS-1, SEQ ID NO: 13 represents the sequence of the primer pAnSG-F7, SEQ ID NO: 14 represents the sequence of the primer pAnSG-R1, SEQ ID NO: 15 represents the sequence of the primer pAnSG-F8, and SEQ ID NO: 16 represents the sequence of the primer pAnSG-F10.

Field of industrial application In accordance with the present invention, it is possible to provide the pharmaceutical composition containing SY-001 or the expressed product of the present invention as the active component. It is thought that the composition of the present invention will be useful as a therapeutic agent or a preventive agent for subsequent pathological conditions to various diseases, for example, diseases and complications caused by thrombi or emboli, for example, acute coronary syndrome, myocardial infarction. , cerebral embolism, chronic arterial obstruction, arterial sclerosis, ischemic cerebral infarction, angina, venous thrombosis, hypertension, pulmonary hypertension, cerebral infarction, pulmonary infarction, heart failure, nephritis, renal dysfunction and subarachnoid hemorrhage, in which SY-001 are involved and the polynucleotide (DNA molecule) encoding the SY-001 polypeptide. Furthermore, it is thought that the composition of the present invention will be useful for the prevention of thrombus formation after PTCA and stent placement, and for the prevention of restenosis after stent placement, including SY-001, by applying SY-001 or including SY-001, in the same stent. By using SY-001 and the DNA molecule encoding SY-001 provided by the present invention, it is possible to identify the agonist for the polypeptide or the product expressed by the DNA molecule (expressed product of the present invention), that is, identify the substance having the activity to facilitate the inherent inhibitory activity for these proteins on platelet aggregation and / or platelet adhesion, such as the candidate compound.

Claims (4)

1. NOVELTY OF THE INVENTION CLAIMS 1 .- A pharmaceutical composition comprising at least one polypeptide of the following items (a) to (d): (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1; (b) a polypeptide comprising an amino acid sequence comprising one or more deletions, insertions, substitutions or additions of amino acid in the amino acid sequence of subsection (a) above, and having an inhibitory activity on platelet aggregation and / or an activity inhibiting platelet adhesion; (c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3; and (d) a polypeptide comprising an amino acid sequence comprising one or more deletions, insertions, substitutions or additions of amino acid in the amino acid sequence of subsection (c) above, and having an inhibitory activity of platelet aggregation and / or an inhibitory activity of platelet adhesion as an active component.
2. 2 - A pharmaceutical composition comprising an expressed product, which is expressed by at least one polynucleotide of the following (e) to (j): (e) a polynucleotide comprising the DNA sequence of SEQ ID NO: 2, or a complement to it; (f) a polynucleotide that hybridizes with the polynucleotide of part (e) above under a severe condition, and is capable of expressing a polypeptide having an inhibitory activity on platelet aggregation and / or an inhibitory activity of platelet adhesion; (g) a polynucleotide comprising the DNA sequence having 80% or more homology to the polynucleotide of part (e) above, and which is capable of expressing the polypeptide having a platelet aggregation inhibitory activity and / or a inhibitory activity of platelet adhesion; (h) a polynucleotide comprising the DNA sequence of SEQ ID NO: 4, or a complement thereof; (i) a polynucleotide that hybridizes with the polynucleotide of part (h) above under the severe condition, and that is capable of expressing the polypeptide having the platelet aggregation inhibitory activity and / or a platelet adhesion inhibiting activity; and (j) a polynucleotide comprising the DNA sequence having 80% or more homology to the polynucleotide of part (h) above, and which is capable of expressing the polypeptide having the platelet aggregation inhibitory activity and / or an activity inhibiting platelet adhesion, as an active component.
3. 3. The pharmaceutical composition according to claim 1 or 2, further characterized in that said inhibitory activity of platelet aggregation and / or platelet adhesion inhibiting activity, is the platelet aggregation inhibitory activity induced by collagen and / or the inhibitory activity on platelet adhesion to collagen.
4. 4. - The pharmaceutical composition according to claim 1 or 2, further characterized in that said pharmaceutical composition has the ability to bind collagen. 5. - A platelet aggregation inhibitor and / or an inhibitor of platelet adhesion, comprising the polypeptide described in claim 1 or the expressed product, which is expressed by the polynucleotide described in claim 2. 6. - A method for identifying an agonist for platelet aggregation inhibitory activity and / or a platelet adhesion inhibiting activity, wherein a level of platelet aggregation inhibitory activity and / or a platelet adhesion inhibitory activity of the polypeptide described in claim 1 is measured in the presence or absence of a subject substance, and a measurement value in the presence of the subject substance is compared to a measurement value in the absence of the subject substance to select the subject substance which increases the inhibitory action as the agonist 7. - The method for identifying an agonist for platelet aggregation inhibitory activity and / or platelet adhesion inhibitory activity, further characterized by a level of the inhibitory activity of the expressed product, which is expressed by the polynucleotide described in claim 2, is measured in the presence or absence of a subject substance, and a measurement value in the presence of the subject substance is compared to a measurement value in the absence of the substance subject to selecting the subject substance which increases the inhibitory activity of platelet aggregation and / or an inhibitory activity of platelet adhesion such as the agonist. 8. A method for identifying a candidate substance that serves as an agonist for platelet aggregation inhibitory activity and / or a platelet adhesion inhibiting activity of the polypeptide described in claim 1 or the expressed product described in claim 2, which comprises the following steps (1) to (4): (1) a step for preparing a culture medium comprising a cell transformed with an expression vector, which expresses the polypeptide described in claim 1 or a cell comprising the product expressed as described in claim 2 and platelet-rich plasma; (2) a step for adding a platelet aggregant agent to the culture medium of subsection (1) above, to induce platelet aggregation in the presence or absence of a subject substance; (3) a step to measure a level of platelet aggregation in the presence or absence of the substance subject in subsection (2) above; and (4) a step for selecting the subject substance as the candidate substance when a measurement value in the presence of the subject substance is greater than a measurement value in the absence of the subject substance. 9. A kit for identifying an agonist for the polypeptide described in claim 1 or the product expressed by the polynucleotide described in claim 2, characterized in that it contains platelet-rich plasma, either one of the polypeptide described in claim 1 and the the expressed product described in claim 2, and a platelet aggregant agent as components. 10. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1. 1 1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 3. 12. - An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 5. 3. - A polynucleotide comprising the DNA sequence of SEQ ID NO: 2, or a complement thereof. 14. - A polynucleotide comprising the DNA sequence of SEQ ID NO: 4, or a complement thereof. 15. - A polynucleotide comprising the DNA sequence of SEQ ID NO: 6, or a complement thereof.