TW200413526A - Peptides which accelerate platelet aggregation - Google Patents

Abstract

The present invention relates to the use of the blood platelets aggregation associated factor, gp44, to screen for blood platelets aggregation associated compounds, the methods of measuring the said activities, compounds obtained employed the methods, kits utilizing the methods, mutated polypeptides deprived of the blood platelets aggregation activities, and provides a medical composition for blood platelets aggregation associated diseases, model animals, disgnostic methods, treatment and prevention of the said diseases.

Description

200413526 发明 Description of the invention [Technical field to which the invention belongs] The present invention relates to a screening method for platelet aggregation-related compounds using a factor related to platelet aggregation, a method for measuring activity, a compound obtained by the method, and a set used by the method. Group (Kit), a mutant polypeptide that has lost platelet agglutination activity, and a pharmaceutical composition for a model animal of platelet agglutination-related diseases, a method for diagnosis, treatment, and prevention. [Prior art] Platelet agglutination refers to the exposure of collagen and von Willebrand factor to platelets exposed in blood vessels and blood vessel walls caused by trauma and other injuries. It is also bound by collagen and von Willebrand factor The activated platelet free thromboxane and ADP caused by the destruction of the internal structure cause the activation of new platelets and induce adhesion to the wound site to form a platelet hemostatic plug to stop the blood flow and achieve the important role of preventing blood loss from injury to blood loss. Platelets are known to be involved in many diseases, such as pulmonary thromboembolism (see "Clinical Adult Disease", 1990, 20th, p. 1995-2001), cerebral infarction (see Stroke, 1985, 16th, p. 524-526; Stroke, 1 989, 20th year, P · 1 27 1-1 272), Deficiency heart disease, Venous thrombosis, Peripheral arterial thrombosis, Seeded intravascular coagulation (DIC), Antiphospholipid Antibody syndromes, hyperlipidemia, diabetic chronic cyanosis, glomerulonephritis, liver disease, transplantation, mucocutaneous skin lymphopathy, and platelet agglutination (see Yamagaku Junior High School, Yamazaki Hiroshi, "Platelet" , College of Medicine, January 1991, p.251-253; edited by Hidetoshi Satoshi, "Basics and Clinical Practice of Hyperthrombotic Drugs", Medical Journal, p. 1 23-230; Fujita Lan 200413526 Diseases and Illnesses, "Kanehara Publishing, June 2001). Furthermore, it is known that due to congenital or acquired platelet count reduction and dysfunction, bleeding tendency, blood coagulation disorder, hemophilia, uremia, chronic bone marrow genital disease, etc. are known (see Matsuda, "Clinical of Hemostasis and Thrombosis", Emerging Medical Press, April, 1996, p. 65-84). It is also known that cancer cells release various platelet agglutination substances to promote the formation of thrombus and hypercoagulation in cancer cells, and interact with platelets to form thrombi to promote hematogenous metastasis. Therefore, in cancer metastasis, the interaction between cancer cells and platelets The role plays an important role (see Journal of National Cancer Institute, 1999, 91 巻, p. 22-36). In particular, osteosarcoma (mostly in infants and young children) is known to be caused by platelet aggregation and is known to be related to lung metastasis (see Cancer Research, 1987, 47 第, p. 31 15-31 17 "known mouse peripherals Membrane protein expressing gp38 in type I thymic epithelial cells and interstitial cells in the T-cell-dependent area of the lymph nodes (see The Journal of Experimental Medicine, 1992, p. 176, p. 1 477-1482). Also as gp38 The mouse homolog (Homologue), gp38P was also selected (see Nephrology Dialysis Transplantation, 2002, 17 巻, ρ 97 8-9 84). It has also been reported that the El 1 antigen of the mouse homolog belonging to gP38 is A new marker for the bone bud cell line (see Bone, 1996, 18ρ, ρ · 1 25-1 3 2). The mouse homologue of gp38, TI α, is expressed on lung type I lung cell epithelial cells. Type II pulmonary cell epithelial cell expression (see American Journal of Respiratory cell and 200413526

Molecular Biology, 1990, 19th, p. 143-149) ° It is also known that TI α, which belongs to the mouse homologue of gp38, is expressed as it differentiates from lung cell epithelial stem cells to type I lung cell epithelial cells. In addition, there is a 43 kDa glycoprotein (99% homogeneity with TiAl) that belongs to the mouse homologue of gp3 8 and is expressed on glomerular epithelial cells (podocytes). There are also reports of small changes in humans (kidneys). Pathological tissue type that changes one of the pathological conditions) (see The American Journal of Pathology, 1997, 151151, pl 141-1 152). GP40, which belongs to the gp38 dog homologue, has been revealed to be expressed in type-I φ

In Madin-Darby canine kidney (MDCK) cells (refer to Biochemical Journal, 1997, 326 巻, p.99-108) 5 is a sialoglycoprotein with a molecular weight of 40 kDa, which has many 0-binding sugar chains, but No N-binding sugar chain addition site. gp40 is expressed in the lung, intestine, kidney, brain, and heart. As a human homolog of gp38, gp36 was found (see Biochemical Journal, 1999, 341 第, ρ · 277-284). gp36 is highly expressed in human lung, placenta, and skeletal muscle (low expression in brain, kidney, and liver). · GP38 (or gP38P) and its homologs have been found in many studies, but the mechanism of gp38 (or gp38P) is still unknown. In addition, the mouse colon cancer cell line 26 was subjected to repeated experimental lung metastasis to obtain NL-1 7 cells belonging to the high metastatic strain and NL-14 cells belonging to the low metastatic strain (see Cancer Research, 1983, No. 43 巻, p. 5437-5442). The platelet agglutination factor gp44 expressed on the surface of NL-17 cells aggregates platelets in mice, which results in the suggestion of lung metastasis (200413526 refer to Cancer Research, 1991, 51 (1), ρ · 92] -925). However, the gp44 gene sequence and amino acid sequence of platelet agglutination factor have not been revealed so far. [Summary of the invention] The main object of the present invention is to express a novel gene related to platelet aggregation, to provide a novel substance related to platelet aggregation, and to provide a therapeutic agent and preventive agent for diseases related to platelet aggregation and decreased platelet function. Methods for substances used as preventive agents, methods for measuring platelet aggregation promotion and inhibition of active substances, elucidation of polypeptide functions related to platelet aggregation, and diagnostic methods for diseases related to platelet aggregation or decreased platelet function. The present inventors have made a deliberate effort on the gp44 gene sequence known to have platelet agglutination for the purpose of exploring genes related to platelet aggregation. 'The gene sequence expressing the gp38P protein is encoded by gp44. Moreover, the present inventors' used the protein encoded by the sequence (gp38P) or a substance related to the expression of the protein encoded by the sequence as a platelet aggregation promoter or a platelet aggregation inhibitor, and related to the expression of gp38P homologue protein or homologue protein Substances are used as platelet aggregation promoters or platelet aggregation inhibitors. The protein has specific essential amino acids for platelet aggregation, and the expression of the polynucleotide or peptide encoded by the sequence is determined. The substances related to platelet aggregation are explored and evaluated. And as a platelet aggregation or platelet function to reduce the use of related diseases diagnosis, and thus completed the present invention. The present invention is: (1) a screening method for platelet aggregation-promoting or inhibiting active substances, including the method of: • 10-200413526: (i) culturing cells expressing gp44 in the presence or absence of test substances; ( ii) In the cultured cells obtained in the above (i), detect mRNA expression comprising the nucleotide sequence described in any one of the following (a) to (i): U) Nucleotide numbers 1 to 5 of sequence number 1 in the sequence listing Nucleotide sequence shown in 19; (b) Nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in the Sequence Listing; (c) Nucleotide number 1 to 510 shown in sequence number 5 in the Sequence Listing Nucleotide sequence; (d) Nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7 in the Sequence Listing; (e) Nucleotide sequence shown in nucleotide number 1 to 540 of sequence number 13 in Sequence Listing (F) a polynucleotide made from a nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e) above, hybridizes under severe conditions, and has platelet aggregation to promote active polymerization; Nucleotide sequences encoded by peptides; (g) Amino acid sequences of amino acid numbers 1 to 172 in Sequence Listing Sequence Number 2 The encoded nucleotide sequence; (h) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 166 of sequence number 4 in sequence listing; (i) the amino group of sequence number 6 in sequence listing Nucleotide sequence encoded by amino acid sequence poly-peptide shown in acid numbers 1 to 169; (j) Nucleic acid encoded by peptide poly-amino-peptide sequence shown in sequence number 8 of amino acid number 1 to 162 in sequence listing 8 Nucleotide sequence; (k) Nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of sequence number 14 in the sequence listing; (]) any one of (g) to (k) above In the amino acid sequence described, a nucleotide sequence encoded by a polypeptide having platelet aggregation promotion activity formed by missing, replacing, or adding one or more amino acid sequences formed by amino acids; (iii) The amount of mRNA expression detected in (ii) above is compared with cells cultured in the presence of the test substance and cells cultured in the absence of the test substance; (iv) compared with cells cultured in the absence of the test substance, Choose a substance with a lower mRNA expression as a substance with platelet aggregation inhibitory activity, or choose a substance with a lower mRNA expression High substances are substances with platelet aggregation promotion activity. (2) The screening method described in (1), the method for testing mRNA expression is characterized by the Northern Blot method and the Dot stain method. Blot), Slot Blot, RT-PCR, RNA protection assay or derivative assay, (3) Screening methods with platelet aggregation promoting or inhibiting active substances, including the following engineering methods: (a ) gp44 expressing cells are cultured in the presence of the test substance; (b) in the cultured cells obtained in (a) above, detecting the expression of the polypeptide containing any of the following: (i): (i) the following ( i- 1) to (i-5) the amino acid sequence encoded by the nucleotide sequence described in any one, or a part thereof and a platelet aggregation-promoting polypeptide; -12-200413526 (b. 1) SEQUENCE LISTING SEQUENCE NUMBER 1 SEQUENCE NUMBERS SEQUENCE NUMBERS 1 to 5 1 9; (i-2) SEQUENCE LISTING NUMBER SEQUENCE NUMBERS 3 to 5 1; i-3) the nucleotide sequence shown in nucleotide number 1 to 5 10 of sequence number 5 in sequence listing; (i-4) sequence The nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7; U-5) The nucleotide sequence shown in nucleotide number 1 to 540 of sequence number 13 in sequence listing; (ii) and (i- Ι) to (i-5) the nucleotide sequence complementary to the nucleotide sequence of the nucleotide sequence of the polynucleotide encoded by the polynucleotide hybridized under severe conditions, and Polypeptide with platelet aggregation promoting activity: (iii) amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of sequence number 2 of the sequence listing; (iv) amino acid number 1 of sequence number 4 of the sequence listing Polyaminopeptide of amino acid sequence shown to 1 to 6; (v) Polyaminopeptide of amino acid sequence shown to 1 to 169 in sequence number 6 of the Sequence Listing; (vi) Amine of sequence number 8 to sequence list Amino acid sequence polypeptides represented by amino acid numbers 1 to 162; (vii) amino acid sequence polypeptides represented by amino acid numbers 1 to 172 of Sequence Listing 14;

(viii) the amino acid sequence of the polypeptide described in any one of (iii) to (Vii) above;} 'missing, substituted or added' in J 200413526 or an amino acid sequence formed by several amino acids Polypeptide with platelet aggregation promoting activity. (C) Detection of the peptide expression level in (b) above, compared with cells cultured in the presence of the test substance and cells cultured in the absence of the test substance; (d) with cells cultured in the absence of the test substance; For cell comparison, choose a substance with a lower peptide expression as a substance with platelet aggregation inhibitory activity, or choose a substance with a higher peptide expression as a substance with platelet aggregation promotion activity, (4) as described in (3) Screening methods, which are specific for the detection of polypeptide expression, include Western blotting, dot blotting, slit blotting, or fixed enzyme immunoassay (ELISA), (5) measuring substances A method for promoting or inhibiting platelet aggregation, including the following methods: (i) gP44 expressing cells are cultured in the presence or absence of the test substance; (ii) in the cultured cells obtained in (i) above, the Contains the expression of the nucleotide sequence mRNA described in any one of the following items (a) to (1): U) The nucleotide sequence shown in nucleotide number 1 to 5 1 9 of sequence number 1 in the sequence listing; (b) SEQUENCE LISTING SEQUENCE NUMBER 3 SEQ ID NOS: 1 to 501 ; (C) the nucleotide sequence shown in nucleotide number 1 to 5 10 of sequence number 5 in the sequence listing; (d) the nucleotide sequence shown in nucleotide number 1 to 48 9 of sequence number 7 in sequence listing; !! J; (e) Nucleotide 200413526 shown in nucleotide number 1 to 540 of sequence number 13 in the Sequence Listing; (f) complementary to the nucleotide sequence described in any one of (a) to (e) above Polynucleotides composed of nucleotide sequences hybridize under severe conditions, and have nucleotide sequences encoded by platelet aggregation-promoting polypeptide; (g) Amino acid numbers 1 to 172 of sequence number 2 in the sequence listing The nucleotide sequence encoded by the amino acid sequence poly-peptide shown; (h) the nucleotide sequence encoded by the amino acid sequence poly-peptide shown by amino acid numbers 1 to 166 in Sequence Listing Sequence Number 4; (i ) Nucleotide sequence encoded by amino acid φ sequence polypeptide shown in amino acid numbers 1 to 169 of sequence number 6 in the Sequence Listing; (j) amino acid numbers 1 to 1 62 of sequence number 8 in Sequence Listing A nucleotide sequence encoded by an amino acid sequence polypeptide; (k) A nucleotide sequence encoded by an amino acid sequence polypeptide shown in amino acid numbers 1 to 17 of Sequence Listing sequence number 14; ( l) In the amino acid sequence described in any one of (g) to (k) above, the amino acid sequence formed by missing, replacing, or adding 1 or more amino acids is formed. Nucleotide sequence encoded by a polypeptide with platelet aggregation promoting activity; (c) The mRNA expression detected in (b) above is compared with the cells cultured in the presence of the test substance and the absence of the test substance. The cultured cells; (d) compared with cells cultured in the absence of the test substance, it is determined that the substance having a platelet aggregation inhibitory activity when the expression of A is low or the mRNA expression level is high as a platelet aggregation promoting activity, (6 ) The screening method as described in (5), whose mRNA expression detection method includes the northern blot method, the dot blot method, the slit blot method, rT-PCr, RNA-15-200413526 protection assay or derivative Assay method, (7) A method for measuring the platelet aggregation promotion or inhibitory activity of a substance, including the following engineering methods: (a) gp44 expressing cells are cultured in the presence of a test substance; (b) in the cultured cells obtained in (a) above To detect the expression of the polypeptide described in any one of the following ⑴ to (viii): ⑴ The amino acid sequence encoded by the nucleotide sequence described in any one of the following (i-1) to (i_5) or a part thereof Platelet aggregation promoting activity Peptide: (i-1) Nucleotide sequence shown in nucleotide number 1 to 5 1 9 of sequence number 1 in sequence listing; (i-2) Nucleotide number 1 to 501 in sequence listing 3 of sequence listing (I-3) Nucleotide sequences shown in nucleotide numbers 1 to 5 10 of sequence number 5 in sequence listing; (i-4) Nucleotide numbers 1 to 7 of sequence listing in sequence number 7 Nucleotide sequence shown as 489; (i-5) Nucleotide sequence shown as nucleotide numbers 1 to 54 in sequence number 13 of sequence listing; (ii) and (i-1) to (i-5) The nucleotide sequence complementary to the nucleotide sequence described in any one of the above) is a polynucleotide encoded by an amino acid sequence that can be hybridized under severe conditions, and has a platelet aggregation promoting activity. Peptides; (iii) amino acid sequence polypeptides shown in amino acid numbers 1 to 172 in sequence number 2 of the sequence listing; -16-200413526 (iv) amino acid numbers in sequence number 4 of the sequence listing] to 166 Shows amino acid sequence polypeptide; (v) Amino acid sequence number 6 to sequence number 169 to 169; (vi) Amino acid number 1 to sequence number 8 of sequence table 162 amino acid Listed polypeptides; (vii) Polypeptides of amino acid sequences shown in amino acid numbers 1 to 172 of SEQ ID NO: 14 in Sequence Listing; (viii) Polymers as described in any one of (iii) to (vii) above The peptide sequence of the peptide is a polypeptide having platelet aggregation promotion activity, which is formed by missing, replacing or adding an amino acid sequence formed by one or several amino acids. (c) Detection of peptide expression in (b) above, comparing cells cultured in the presence of the test substance with cells cultured in the absence of the test substance; (d) cultured in the absence of test substance Cell comparison, determine that the substance with lower peptide expression is a substance with platelet aggregation inhibitory activity, or select the substance with higher peptide expression as a substance with platelet aggregation promotion activity, (8) as described in (7) In the method, the method for measuring the expression level of polypeptide includes the western blotting method, spot blotting method, slot blotting method or fixed enzyme immunoquantitative method. (9) The method according to any one of (1) to (8), wherein the platelet aggregation inhibitory activity is against cancer, cancer metastasis, pulmonary thrombosis, cerebral infarction, myocardial infarction or arteriosclerosis, or platelet aggregation during vascular surgery Therapeutic effect or preventive effect 0 (10) The method according to any one of (1) to (8), wherein platelet aggregation-17-200413526 promotes activity, and has a tendency to bleeding, blood coagulation disorder, hemophilia, uremia Or chronic bone marrow sacral diseases have therapeutic or preventive effects. U 1) In a model animal that promotes platelet aggregation related diseases, a gene containing the nucleotide sequence described in any one of U) to (1) below is introduced into a non-human gene transgenic animal in a highly expressed form of the gene : U) the nucleotide sequence shown in nucleotide number 1 to 5 1 9 of sequence number 1 in the sequence listing; (b) the nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in sequence listing; (c ) The nucleotide sequence shown in nucleotide number 1 to 5 10 of sequence number 5 in the sequence listing; (d) the nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7 in sequence listing; (e ) A nucleotide sequence shown in nucleotide number 1 to 540 of sequence number 13 in the sequence listing; (f) a nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e) above; The resulting polynucleotides hybridize under severe conditions, and have the nucleotide sequence of the polynucleotide encoded by the active peptide that promotes platelet aggregation; (g) the amino acid numbers 1 to 172 of sequence number 2 in the sequence listing The nucleotide sequence encoded by the amino acid sequence poly-peptide shown; (h) the amino acid sequence poly-amino acid shown by the amino acid number 1 to 166 of the sequence listing sequence number 4 Encoded nucleotide sequence: U) Nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 169 of sequence number 6 in the sequence listing; -18-200413526 (j) sequence number 8 The nucleotide sequence encoded by the amino acid sequence polypeptide shown in the amino acid numbers 1 to 16 2; (k) the amino acid shown in the amino acid numbers 1 to 1 2 of the sequence listing 1 4 The nucleotide sequence encoded by the sequence polypeptide; (l) the amine formed by missing, replacing or adding 1 or more amino acids to the amino acid sequence described in any one of (g) to (k) above A nucleotide sequence encoded by a polypeptide derived from a basic acid sequence and having platelet aggregation promotion activity, (1 2) The animal as described in (1 1), wherein the platelet aggregation inhibition activity is effective against cancer, cancer metastasis, pulmonary thrombosis, Cerebral infarction, myocardial infarction or arteriosclerosis or vascular surgery have platelet aggregation effect or preventive effect, (13)-a method to test the substance's platelet aggregation promoting activity or platelet aggregation promoting effect of a therapeutic or preventive agent for related diseases, Contains the following methods: (a) administering test substances to (11) And (12) the animal according to (12); and (b) measuring (a) the platelet aggregation function of the animal. (14) In a model animal that inhibits platelet aggregation-related diseases, a gene containing the nucleotide sequence described in any one of the following (a) to (1) is introduced into a non-human gene transformation in a low expression form of the gene Animals ... (a) Nucleotide numbers shown in SEQ ID NO: 1 in Sequence Listing 1 to 5 and 9; (b) Nuclei shown in nucleotide numbers 1 to 501 in Sequence Listing 3 Nucleotide sequence; (c) Nucleotide sequence shown in nucleotide number 1 to 510 of sequence number 5 in sequence listing; -19-200413526 (d) Nucleotide number 1 to 4 8 9 of sequence number 7 in sequence listing (E) the nucleotide sequence shown in nucleotide number 1 to 540 of sequence number 13 in the sequence listing; (0 as described in any of (a) to (e) above Polynucleotides formed by complementary nucleotide sequences to the nucleotide sequence hybridize under severe conditions, and the nucleotide sequence of the polynucleotide encoded by the active polypeptide with platelet aggregation promotion; (g) Sequence listing sequence Nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of the number 2; φ (h) amino acid numbers 1 to 16 of sequence number 4 in the sequence listing Nucleotide sequence encoded by the amino acid sequence polypeptide shown in 6; (i) Nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 169 of sequence number 6 in the Sequence Listing; ( j) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 162 of sequence number 8 in the sequence listing; (k) the amines shown in amino acid numbers 1 to 172 of sequence number 14 in the sequence listing Nucleic acid sequence encoded by a polypeptide; (1) In the amino acid sequence described in any one of (g) to (k) above, one or more amino acids are missing, substituted or added. A nucleotide sequence encoded by a polypeptide having a platelet aggregation-promoting activity formed from the formed amino acid sequence. (15) Model animals with platelet aggregation-inhibiting diseases, including any of the following (a) to (1) Genes that record nucleotide sequences, knockout non-human animals: U) Nucleotide numbers 1 to 519 of nucleotide numbers 1 to 519 in sequence list sequence number 20-200413526 Sequence number! J; (b) Nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in sequence listing; (c) Nucleotide number 1 to 5 1 in sequence number 5 of sequence listing Nucleotide sequence shown by 0; (d) Nucleotide sequence shown by nucleotide numbers 1 to 489 in sequence number 7 of the Sequence Listing; (e) Nucleotide number 1 through 540 of Sequence number 13 in the Sequence Listing (F) a nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e) above, hybridizes under severe conditions, and has The nucleotide sequence of the polynucleotide encoded by the platelet aggregation-promoting active peptide; (g) The nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of SEQ ID NO: 2 in Sequence Listing (H) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 166 of sequence number 4 in the sequence listing; (shown in the amino acid numbers 1 to 169 of sequence number 6 in the sequence listing Nucleotide sequence encoded by amino acid sequence polypeptide; (j) Nucleotide sequence encoded by amino acid sequence polypeptide shown by amino acid numbers 1 to 162 in sequence number 8 of Sequence Listing; (k) sequence Nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of the sequence number 14; (l) the amine described in any one of (g) to (k) above In the amino acid sequence, a nucleotide sequence encoded by a polypeptide having 200413526 platelet aggregation promotion activity, which is formed by missing, replacing, or adding 1 or more amino acid sequences formed by amino acids, () 6) such as ( [4] or (15) of the animal, wherein the disease associated with the inhibition of platelet aggregation is bleeding tendency, blood coagulation disorder, hemophilia, uremia, or chronic myeloproliferative disease. (17) — A method for testing the platelet aggregation promoting activity of a substance or the effect of a therapeutic or preventive agent for promoting a platelet aggregation promoting related diseases, including the following processes: (a) For animals described in (14) to (16) Administration of test substances; and (b) measurement of (a) platelet aggregation function in animals (18)-a method for diagnosing a disease, which involves subjecting the specimen to the nucleotides described in any one of (a) to (1) below Sequence detection of mRNA expression to diagnose diseases related to platelet agglutination or platelet dysfunction of the specimen provider or the animal provided by the specimen: (a) Nucleotide numbers 1 to 5 1 9 Shows the nucleotide sequence 所示! J; (b) The nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in the sequence listing; (c) The nucleotide number 1 to 5 of sequence number 5 in the sequence listing Nucleotide sequence shown in 10; (d) Nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7 in the Sequence Listing [J; (0 Nucleotide number 1 to 54 of sequence number 13 in Sequence Listing) (F) a nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e) above 200413526 The sequenced polynucleotides hybridize under severe conditions, and have the nucleotide sequence of the polynucleotide encoded by the active peptide that promotes platelet aggregation; (g) Amino acid number 1 of sequence number 2 in the sequence listing To] 72 nucleotide sequence encoded by amino acid sequence polypeptide; (h) nucleotide number encoded by amino acid sequence 1 of 1 to 1 in sequence list 4 Sequence; (i) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 169 of sequence number 6 in the Sequence Listing; (j) the amino acid numbers 1 to 162 of sequence number 8 in the Sequence Listing The nucleotide sequence encoded by the amino acid sequence poly-peptide shown; (k) the nucleotide sequence encoded by the amino acid sequence poly-peptide shown by amino acid numbers 1 to 172 in Sequence Listing sequence number 14; (l ) In the amino acid sequence described in any one of (g) to (k) above, a polymer having platelet aggregation promotion activity formed by missing, replacing, or adding an amino acid sequence formed by one or more amino acids Nucleotide sequences encoded by peptides, (19) A screening method such as (18), a method for examining mRNA expression, including the northern blot method, Ink staining method, slit ink staining method, PT-PCR, RNA protection assay or derivative assay, (20) —A method for diagnosing disease, which detects any one of the following items (i) to (viii) A polypeptide expression level according to one item to diagnose a disease related to platelet aggregation or platelet dysfunction of the specimen provider or the animal provided by the specimen: (i) any one of the following (il) to (i-5) The amino acid sequence encoded by the nucleotide sequence described in the item or a part thereof, a polypeptide having platelet aggregation promoting activity; (i-1) the nucleosides represented by nucleotide numbers 1 to 519 in sequence number 1 in the sequence listing -23-200413526 acid sequence; (i-2) nucleotide sequences 1 to 501 shown in sequence number 3 of the sequence listing; (b 3) nucleotide numbers 1 to 5 of sequence number 5 in the sequence listing 〇 Nucleotide sequence shown; (i. -4) The nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7 in the sequence listing; U-5) The nucleotide sequence shown in sequence number 13 of sequence listing] to 540; 如) such as Polynucleotides formed from the nucleotide sequences complementary to the nucleotide sequences described in any one of the above (i-1) to (i-5) hybridize under severe conditions, and have platelet aggregation to promote active polymerization. Polynucleotide sequence (Hi) of the polynucleotide encoded by the peptide (Hi) SEQ ID NO: 2 of the amino acid sequence shown in SEQ ID NO: 2 to 172; (iv) Amino group of SEQ ID NO: 4 in the Sequence Listing Polyurethane peptides with amino acid sequences shown in acid numbers 1 to 16; (v) Polyurethane peptides with amino acid sequences shown in sequence number 6 in sequence listing 6; (vi) Sequence number in sequence listing The amino acid sequence polypeptide shown in amino acid numbers 1 to 162 of 8; (vii) The amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of sequence list 14; (viii) the above ( iii) The amino acid sequence 200413526 of the polypeptide according to any one of (vii), which is formed by missing, replacing or adding an amino acid sequence formed by one or more amino acids Promoting activity of platelet aggregation with poly-peptide. (2 1) The screening method according to (20), the method for measuring the expression level of polypeptide, using Western blotting method, dot blotting method, slit blotting method or fixed enzyme immunoquantitative method is special, (2 2 ) The screening method according to any one of (18) to (21), wherein the platelet aggregation-associated disease is cancer, cancer metastasis, pulmonary thrombosis, cerebral infarction, myocardial infarction or arteriosclerosis or platelet aggregation during vascular surgery. (2 3) The screening method according to any one of (1 8) to (2 1), wherein the platelet agglutination-associated disease is colon cancer, rectal cancer, small intestine cancer, seminal carcinoma, seminoma ), A special feature of flat epithelial cancer, (24) The screening method according to any one of (18) to (21), wherein the platelet aggregation-related disease is bleeding tendency, blood coagulation disorder, hemophilia, uremia or chronic bone marrow Special features of colonic diseases, (25) — A method for separating platelet aggregation promoting or inhibiting active substances, which comprises a polypeptide of the amino acid sequence described in any one of the following (viii), and a test peptide containing The reagent of the substance is contacted, and then separated from the polymer Peptide-binding reference substance. (i) The amino acid sequence encoded by the nucleotide sequence described in any one of (i-1) to (i-5) below, or a polypeptide (i-1) having platelet aggregation promotion activity in part thereof ) Nucleotide sequence shown in nucleotide number 1 to 519 of sequence number 1 in sequence listing; (i-2) Nucleoside 25-200413526 acid sequence shown in nucleotide number 1 to 501 of sequence number 3 in sequence listing; (i-3) Nucleotide numbers shown in Sequence Listing Sequence Number 5] to 5 10; (i-4) Nucleotides shown in Sequence Listing Sequence Number 7, Nucleotide Numbers 1 to 4 89 Acid sequence; (i_ 5) the nucleotide sequence shown in nucleotide number 1 to 540 of sequence number 13 in the sequence listing; (ii) the core described in any one of (i-1) to (i-5) above Polynucleotides composed of nucleotide sequences complementary to the nucleotide sequence hybridize under severe conditions, and have a nucleotide sequence encoded by a platelet aggregation-promoting active peptide; (iii) the amine group of sequence number 2 in the sequence listing Polyaminopeptides of amino acid sequences shown in acid numbers 1 to 172; (iv) Polyaminopeptides of amino acid sequences shown in amino acid numbers 1 to 166 in Sequence Listing; (v) Sequence No. 6 of sequence listing Amino acid number 1 Amino acid sequence polypeptide shown in 169; (vi) Amino acid sequence polypeptide shown in sequence number 8 of sequence listing 1 to 162; (vii) Amino acid number of sequence number 14 in sequence listing Polypeptide of amino acid sequence shown in 1 to 172; (viii) The amino acid sequence of polypeptide as described in any one of (iii) to (vii) above is missing, substituted or added with one or more amines A polypeptide with amino acid sequence formed by amino acid and having platelet aggregation promoting activity. (26) —A method for isolating platelet aggregation-promoting or inhibiting active substances-26-200413526, which involves contacting the polypeptide described in any one of the following (1) to (xiv) with a reagent containing a test substance, and then , Isolate the substance binding to the polypeptide: (i) the amino acid sequence encoded by the nucleotide sequence described in any one of (il) to (i-5) below, or a part thereof having platelet aggregation promotion activity (I-2) Nucleotide sequences shown in nucleotide numbers 67 to 132 of sequence number 1 in sequence listing 1; (i-2) Nuclei shown in nucleotide numbers 67 to 132 of sequence number 3 in sequence listing Nucleotide sequence; (i-3) Nucleotide sequence numbered from nucleotide sequence number 103 to 198 in sequence number 5 in sequence listing; (i -4) Nucleotide number sequence 8 in sequence listing 7 (I-5) the nucleotide sequence shown in nucleotide number 82 to 186 of sequence number 13 in the sequence listing; (ii) the same as (i-1) to (i- 5) The nucleotide sequence of any of the nucleotide sequences described in any one of the nucleotide sequences, the polynucleotide can hybridize under the severe conditions of the polynucleotide-encoded amino acid sequence, and has platelet aggregation Into the active polypeptide; ◦ ii) as in the amino acid sequence of sequence number 2 in the sequence listing, the polypeptide containing the amino acid number 34 amino acid, more than 6 consecutive amino acid sequences; (iv) such as In the amino acid sequence of SEQ ID NO: 4 in the Sequence Listing, a polypeptide containing an amino acid number 34 of the amino acid and six or more consecutive amino acid sequences; (v) As in the amino acid sequence of SEQ ID NO: 6 in the Sequence Listing ， Polypeptide containing amino acid number 41 1 amino acid, 6 or more consecutive amino acid sequences; -27-200413526 (Vi) As in the amino acid sequence of sequence number 6 in the sequence listing, the amino acid number 50 Amino acid, a polypeptide with more than 6 consecutive amino acid sequences; (vii) As in the amino acid sequence of SEQ ID NO: 8 in the Sequence Listing, containing an amino acid number 5 amino acid, 6 or more consecutive amino acids Polypeptide of sequence; (viii) Polyaminopeptide containing amino acid number 34 amino acid sequence in the amino acid sequence of sequence number 14 in the sequence listing, and more than 6 consecutive amino acid sequences; (IX) as ordered In the amino acid sequence of the sequence number 2, the polypeptide containing the amino acid sequence of 39 to 44 amino acid sequence; (X) The amino acid sequence of the sequence number 4 In the sequence, the polypeptide containing the amino acid number of the amino acid number 3 9 to 44; (XI) As in the amino acid sequence of the sequence number 6 of the sequence listing, the amino acid number is 46 to 5 1 Polypeptide of amino acid sequence; (XII) As in the amino acid sequence of SEQ ID NO: 8 in the Sequence Listing, a polypeptide containing amino acid sequences 57 to 62; (XIII) as the sequence listing sequence number In the amino acid sequence of 14, a polypeptide containing an amino acid number of 39 to 44; (XIV) The amino acid of the polypeptide according to any one of (iii) to (XIII) above In the sequence, a polypeptide lacking, substituting, or adding an amino acid sequence formed by one or more amino acids, (27) —A method for screening active substances having platelet aggregation promotion activity or platelet aggregation inhibition activity, including the following projects Method: (a) contact gp44-expressing cells with platelets in the presence of the test substance, (b) measure platelet agglutination activity, -28-200413526 (C) the results of (b) can determine whether the test substance has Platelet aggregation promoting or inhibiting activity, (2 8) —A screening for platelet aggregation promoting or inhibiting activity The method of the active substance includes the following engineering methods: U) contact gp44 with platelets in the presence of the test substance, (b) measure the platelet agglutination activity, (c) determine the result of the test substance from (b) With platelet aggregation promoting activity or inhibitory activity, (29) —A method for screening platelets with platelet aggregation promoting activity or inhibiting active substances' includes the following methods: U) Gp44-expressing cells are administered to non- Human mammals, (b) determine the number of tissue nodules of non-human mammals in (a), (c) determine from the results of (b) whether the test substance has platelet aggregation promotion activity or platelet aggregation inhibition activity, (3 0 ) ~ A method for selecting a cancer metastasis-inhibiting activity or cancer metastasis-promoting active substance by a seed shop 'including the following engineering methods: (a) administering gp44 expressing cells to a non-human mammal in the presence of a test substance, Measure the number of tissue nodules in non-human mammals in (a), (0) Determine whether the test substance has cancer metastasis-inhibiting activity or cancer metastasis-promoting activity from the results of (b), (3 1)-a test A set of platelet aggregation-promoting or inhibiting active substances comprising one or more of the following: [J (a) to (c): 200413526 (a) Able to specifically amplify sequence listing sequence numbers 1, 3, Partial or all of the nucleotide sequence of the nucleotide sequence described in 5, 7, or 13 of 15 to 30 bases long continuous oligonucleotide primers; (b) the sequence number 1, 3, 5 The 7 'or 13' polynucleotides described in the nucleotide sequence hybridize under severe conditions, and can detect more than 15 consecutive nucleotide probes of the polynucleotide; (c) ( The substance described in any one of a) or (b) is an immobilized immobilizing agent. 0 (3 2) —a kit for detecting platelet aggregation promoting or inhibiting active substances, which contains more than one as follows (a) to (C) Any of the substances described in: (a) An antibody that can specifically bind to the polypeptide of the amino acid sequence described in sequence number 2, 4, 6, 8 or 14 of the sequence listing; (b) and (a ) A secondary antibody that describes antibody binding; (c) an immobilizing agent that fixes the substance described in any one of (a) or (b). (3 3) A test set for detecting platelet aggregation-associated diseases, which includes one or more of the substances described in any one of the following (a) to (c) ... (a) Amplification of sequence number 1 in the sequence table specifically, 3 to 5, 5 or 7 or 13 of the nucleotide sequence of the nucleotide sequence of a part of or all of the 15 to 30 base long continuous oligonucleotide primers; (b) and sequence number 1, 3 , 5, 7, or 13 Polynucleotides of the nucleotide sequence described in the hybridization under severe conditions, and can detect consecutive polynucleotide probes of more than 15 nucleotides; (c ) An immobilizing agent for immobilizing a substance described in any one of (a) or (b). · (3 4) —A set for detecting platelet aggregation-associated diseases, which includes a substance described in any one of (a) to (0) above -30-200413526: (a) can be specifically sequenced with the sequence table Polypeptide-binding antibody of the amino acid sequence described in No. 2, 4, 6, 8 or 4; (b) Secondary antibody that binds to the antibody described in (a); (c) Immobilized (a) or (b (35) The kit according to any one of (33) or (34), wherein the platelet aggregation-related disease is cancer, cancer metastasis, pulmonary thrombosis, cerebral infarction, myocardial infarction, or Arteriosclerosis 5 (3 6) The set described in (3 3) or (3 4), wherein the platelet aggregation-associated diseases are colon cancer, rectal cancer, small intestine cancer, seminal carcinoma, seminoma, flat epithelial cancer, (37) An antibody having platelet aggregation inhibitory activity, which can specifically bind to the polypeptide described in any one of the following (XIV): (i) the following (i-1) to (i-5) ) Any one of the amino acid sequences encoded by the nucleotide sequence, or part of the polypeptide: (i-1) Nucleotide number 6 of sequence number 1 in the sequence listing Nucleotide sequences shown in 7 to 132; U-2) Nucleotide sequences shown in sequence number 3 of nucleotide number 67 to 132; (i_3) Nucleotide number 10 of sequence number 5 in sequence list Nucleotide sequences shown to 198; (i_4) Nucleotide sequences shown to nucleotide numbers 88 to 186 in sequence number 7 of the Sequence Listing; (i-5) Nucleotide numbers 82 to 186 to Sequence number 13 in the Sequence Listing Nuclei 200413526 shown in the nucleotide sequence; (ii) a nucleotide sequence complementary to the nucleotide sequence described in any of (1 · 1) to (i-5) above, the polynucleotide may be stringent A polypeptide of an amino acid sequence encoded by a hybridized polynucleotide under conditions; (Hi) As in the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing, it contains an amino acid number 34 of an amino acid, and more than 6 consecutive amines Polypeptide of amino acid sequence; (iv) Polyaminopeptide containing amino acid number 34 amino acid in the amino acid sequence of sequence number 4 in the Sequence Listing, and more than 6 consecutive amino acid sequences; (v) For example, in the amino acid sequence of sequence number 6 in the Sequence Listing, a polypeptide containing an amino acid number 41 of an amino acid and more than six consecutive amino acid sequences; (vi) As the amino acid sequence of sequence number 6 in the Sequence Listing In the column, a polypeptide containing an amino acid number 50 of an amino acid and six or more consecutive amino acid sequences; (vi i) As in the amino acid sequence of sequence number 8 in the sequence listing, an amino acid number 52 is contained in the amino acid sequence. Amino acid, a polypeptide of 6 or more consecutive amino acid sequences; (viii) the amino acid sequence of sequence number 14 in the sequence listing, containing amino acid number 34 amino acid sequence, and 6 or more consecutive amino acid sequences Polypeptide; (IX) As in the amino acid sequence of SEQ ID NO: 2 of the Sequence Listing, a polypeptide containing the amino acid sequence of 39 to 44 amino acid; (X) As the amino group of SEQ ID NO: 4 of the Sequence Listing In the acid sequence, a polypeptide containing an amino acid number of 39 to 44 amino acid sequence; (XI) As in the amino acid sequence of sequence number 6 of the Sequence Listing, an amino acid number of 46 to 5 1 Polypeptide of amino acid sequence; (XII) As in the amino acid sequence of sequence number 8 in the Sequence Listing, polypeptide containing amino acid numbers 57 to 62; -32-200413526 (XIII) such as In the amino acid sequence of SEQ ID NO: I4 in the Sequence Listing, a polypeptide containing an amino acid sequence of 39 to 44 amino acids; (XIV) As in any one of (iii) to (XIII) above Poly amino acid sequence disclosed peptides of the lack of, or poly-substituted amine acid peptide sequences formed of several amino acids or additional〗. (38) Substances selected as described in (1) to (4), (27) to (30), or substances separated as described in (25) to (26) (39) The polypeptide described in any one of the following (a) to (f): (a) A variant of the amino acid sequence polypeptide shown in SEQ ID NO: 2 in the Sequence Listing, and its amino acid number 34 The amino acid can be transformed into other amino acids to obtain a mutant polypeptide; (b) a variant of the amino acid sequence polypeptide shown in sequence number 4 in the sequence listing, and the amino group of the amino acid number 34 The acid can be converted into other amino acids to obtain mutant polypeptides; (0 A variant of the amino acid sequence polypeptide shown in Sequence Number 6 of the Sequence Listing, and the amino acid of the amino acid number 41 can be changed to (D) A variant of the amino acid sequence polypeptide shown in SEQ ID NO: 6 in the Sequence Listing. The amino acid of the amino acid number 50 can be converted into other amines. (E) A variant of the amino acid sequence polypeptide shown in SEQ ID NO: 8 in Sequence Listing, the amino acid of amino acid number 52 can be converted to other amino acids change (F) a variant of the amino acid sequence polypeptide shown in sequence number 14 in the sequence listing, -33-200413526 the amino acid of the amino acid number 34 can be changed to other amino acids to obtain mutations (40) the variant polypeptide according to (3), wherein the other amino acid is alanine, (4 1) a DNA encoding the variant according to (39) or (40) Polypeptide, (42) —a pharmaceutical composition containing a polynucleotide containing a nucleotide sequence as described in any one of the following (a) to (1) as an active ingredient: (a) sequence number 1 The nucleotide sequence shown in nucleotide numbers 1 to 519; (b) The nucleotide sequence shown in nucleotide number 1 to 501 in sequence number 3 in the sequence listing; (c) The nucleotide number shown in sequence number 5 in the sequence listing Nucleotide sequences shown from 1 to 5 10; (d) Nucleotide sequences shown from nucleotide numbers 1 to 489 in sequence number 7 of the Sequence Listing; (e) Nucleotide numbers 1 to 13 of Sequence Listing 13 Nucleotide sequence shown in 540; (0) A nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e) above hybridizes under severe conditions, A nucleotide sequence encoded by a polypeptide having platelet aggregation-promoting activity; (g) A nucleotide sequence encoded by an amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of Sequence Listing Sequence Number 2; (h) Nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 166 of sequence number 4 in the sequence listing; -34-200413526 (i) shown in sequence number 6 of the amino acid sequence number 1 to 169 Nucleotide sequence encoded by amino acid sequence peptide; (j) Nucleotide sequence encoded by amino acid sequence peptide sequence shown in SEQ ID NO: 8 amino acid number of sequence table to] 62; (k) sequence The nucleotide sequence encoded by the amino acid sequence polypeptide shown in SEQ ID NO: 14 amino acid numbers 1 to 17 in the list; (l) in the amino acid sequence described in any one of (g) to (k) above, Nucleotide sequence encoded by a polypeptide that is formed by the absence, substitution, or addition of one or more amino acids and has an amino acid sequence and has platelet aggregation promotion activity. Lu (43), a pharmaceutical composition, internal Polynucleotide containing the antisense sequence of the nucleotide sequence according to any one of the following (a) to (1): U) Sequence Listing Sequence Number 1 Nucleotide sequence shown in nucleotide numbers 1 to 519; (b) Nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in the Sequence Listing! J; (c) Nucleus of sequence number 5 in Sequence Listing Nucleotide sequences shown in nucleotide numbers 1 to 5 10; (d) Nucleotide sequences shown in nucleotide numbers 1 to 489 in sequence number 7 in the Sequence Listing; (e) Nucleotides shown in sequence number 13 in the Sequence Listing The nucleotide sequences shown in acid numbers 1 to 540; (f) Polynucleotides made of nucleotide sequences complementary to the nucleotide sequence described in any one of (a) to (e) above are severe Under the conditions of hybridization, and has a platelet aggregation-promoting active peptide-encoded nucleotide sequence; -35-200413526 (g) SEQ ID NO: 2 of the amino acid sequence of SEQ ID NO: 1 to 172 (H) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 166 of sequence number 4 in the sequence listing; (i) sequence number 6 amino acid number of the sequence listing The nucleotide sequence encoded by the amino acid sequence polypeptide shown in 1 to 169; (j) SEQ ID NO: 8 in the sequence listing. Nucleotide sequence; (k) Nucleotide sequence encoded by the amino acid sequence polypeptide shown in SEQ ID NO: 14 amino acid number 1 to 172 in the Sequence Listing; (l) any one of (g) to (k) above In the amino acid sequence described, a nucleotide sequence encoded by a polypeptide having platelet aggregation promotion activity formed by missing, replacing, or adding one or more amino acid sequences formed by amino acids, (44) A pharmaceutical composition containing a polynucleotide combination carrier according to any one of the following (a) to (1): (a) sequence table sequence number 1 nucleotide number 1 to 5 1 Nucleotide sequence shown in 9; (b) Nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in the Sequence Listing; (c) Nucleotide number 1 to 5 10 of sequence number 5 in Sequence Listing (D) the nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7 in the sequence listing; (e) the nucleotide sequence shown in nucleotide number 1 to 540 of sequence listing 13 in sequence listing 200413526 Acid sequence; (0) a polynucleotide composed of a nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e) above, which hybridizes under severe conditions, Nucleotide sequence of a polynucleotide encoded by a polypeptide with platelet aggregation promoting activity; (g) Nucleotide encoded by an amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of SEQ ID NO: 2 in Sequence Listing Sequence; (h) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 166 of sequence number 4 in the sequence listing; (i) sequence number 6 amino acid numbers 1 to 169 The nucleotide sequence encoded by the amino acid sequence poly-peptide; (j) the nucleotide sequence encoded by the amino acid sequence poly-peptide shown in Sequence Listing Sequence Number 8 amino acid number 1 to 162; (k) the sequence The nucleotide sequence encoded by the amino acid sequence polypeptide shown in SEQ ID NO: 14 amino acid numbers 1 to 172; (l) in the amino acid sequence described in any one of (g) to (k) above, A nucleotide sequence encoded by a polypeptide having platelet aggregation promoting activity that is missing, substituted, or added with one or more amino acid sequences formed by amino acids, (45) —a pharmaceutical composition containing Polynucleotide combination vector of antisense sequence of the nucleotide sequence according to any one of the following (a) to (1): U) Sequence Listing Sequence Code 1 Nucleotide Sequences 1 to 5] 9; (b) Nucleotide Sequences 1 to 501 in Sequence Listing Sequence Number 3; U) Nuclei in Sequence Listing 5 Nucleotides 200413526 shown in nucleotide numbers 1 to 510; (d) Nucleotide sequences shown in nucleotide numbers 1 to 4 8 in sequence number 7 in the Sequence Listing; (e) Sequence numbers 13 in sequence list Nucleotide sequences represented by nucleotide numbers 1 to 540; (f) Polynucleotides formed from nucleotide sequences complementary to the nucleotide sequences described in any one of (a) to (e) above Nucleotide sequence encoded by a polypeptide that hybridizes under severe conditions and has platelet aggregation-promoting activity; (g) Nucleotide coded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of sequence listing sequence number 2 Nucleotide sequence; (h) Nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 166 of Sequence Listing No. 4; (i) Sequence number 6 of amino acid sequence No. 1 to Nucleotide sequence encoded by the amino acid sequence polypeptide shown in 169; (j) Nucleus coded by the amino acid sequence polypeptide shown in the sequence listing sequence number 8 amino acid number 1 to 162 Acid sequence; (k) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of sequence number 14 in the sequence listing; (l) any of the above (g) to (k) In the recorded amino acid sequence, a nucleotide sequence encoded by a polypeptide having platelet aggregation promotion activity formed by missing, replacing, or adding an amino acid sequence formed by one or several amino acids is added. (46) A pharmaceutical composition containing a polypeptide as described in any one of the following (i) to (viii): (i) as in any one of the following (i-1) to (i-5) The recorded nucleotide sequence encodes -38-200413526 amino acid sequence, or a part thereof, a polypeptide with platelet aggregation promotion activity: (il) Sequence Listing Sequence Number 1 Nucleotide Numbers 1 to 51 9 Nucleotide sequence 歹 0; U-2) Nucleotide sequences shown in nucleotide numbers 1 to 501 in sequence number 3 in the sequence listing; (i-3) Nucleotide numbers 1 to 5 in sequence number 5 in the sequence listing Nucleotide sequence shown in 10; (i-4) Nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7 in sequence listing; (Bu 5) Nucleotide number 1 to sequence number 13 in sequence listing Nucleotide sequence shown in 540; (ii) Polynucleotide formed by the nucleotide sequence complementary to the nucleotide sequence described in any one of (i-1) to (i-5) above Nucleotide sequence of a polynucleotide encoded by a polypeptide having platelet aggregation promoting activity under severe conditions > (iii) the amino acids shown in SEQ ID NO: 2 in the Sequence Listing 2 Sequence peptide; (iv) sequence Amino acid sequence polypeptides shown in amino acid numbers 1 to 166 of the sequence number 4 in the list; (v) Sequence number 6 amino acid sequence polypeptides shown in amino acid numbers 1 to 169 in sequence list; (vi) SEQUENCE LISTING SEQUENCE NUMBER 8 AMINO ACID SEQUENCE POLYPeptides shown in amino acid numbers 1 to 162; -39-200413526 (vii) SEQUENCE LISTING SEQUENCE NUMBER 14 AMINO ACID SEQUENCE POLYPEPTIDES IN SEQUENCE NUMBER 14 ; (Viii) the amino acid sequence of the polypeptide described in any of (in) to (vii) above is' missing, substituted, or added with an amino acid sequence formed by one or more amino acids and has Platelet aggregation-promoting polypeptide, (47) a pharmaceutical composition containing an antibody that can specifically bind to the polypeptide described in any one of the following (i) to (viii): (0 or less (i-I The amino acid sequence encoded by the nucleotide sequence described in any one of (i) to (i-5), or a part thereof, having a platelet aggregation promoting activity j U-1) SEQ ID NO: 1 nucleoside The nucleotide sequence shown in the acid number 丨 to 519; U-2) The nucleotide sequence shown in nucleotide number 1 to 501 of the sequence number 3 of the sequence listing; (i-3) sequence listing order Nucleotide sequence shown in column number 5 from nucleotide numbers 1 to 510; (i_4) Nucleotide sequence shown by nucleotide number 1 to 489 in sequence list sequence number 7; (i-5) Sequence number in sequence listing Nucleotide sequences shown by nucleotide numbers 1 to 5 40 of 1 3; (ii) the nucleotide sequence 歹 IJ complementary nucleus as described in any one of (i-1) to (i-5) above The nucleotide sequence of the nucleotide sequence of the nucleotide sequence hybridizes under severe conditions, and the platelet aggregation promotes the nucleotide sequence of the polynucleotide encoded by the active peptide; (Hi) the amino acid of sequence number 2 in the sequence listing Polyaminopeptide of amino acid sequence of 1 to 172 shown in 2004 to 200413526; (iv) Polyaminopeptide of amino acid sequence of 1 to 166 of amino acid sequence number 4 of Sequence Listing; (v) Sequence Listing sequence Number 6 amino acid number] to 169 amino acid sequence polypeptide; (vi) Sequence Listing sequence number 8 amino acid number 1 to 162 amino acid sequence polypeptide; (vii) Sequence Listing sequence No. 14 amino acid sequence polypeptide shown in amino acid numbers 1 to 172; (vii i) The amino acid sequence of the polypeptide described in any one of (iii) to (vii) above, lacking, Replace Or a polypeptide with 1 or more amino acid sequences formed from amino acids and having platelet aggregation promoting activity; (48) — a pharmaceutical composition containing from (1) to (4), and (27) to (30) The substance selected by the method described in any one of the methods, or the substance isolated according to the method described in (25) or (26), (49) selected from any of (42), (44) and (46) A pharmaceutical composition according to one item, which can promote platelet aggregation, (50) A pharmaceutical composition selected from any one of (42), (44) and (46), which can inhibit platelet aggregation, (51) selected from the group consisting of (43) ), The pharmaceutical composition of any one of (45), (47), and (48), which can treat or prevent colon cancer, rectal cancer, small intestine cancer, seminal carcinoma, seminoma, squamous cell carcinoma, cancer metastasis , Pulmonary thrombosis, cerebral infarction, myocardial infarction, arteriosclerosis, bleeding tendency, blood clotting disorders, hemophilia, uremia or chronic myeloproliferative disease or inhibition of platelet coagulation during vascular surgery -41-200413526 episode. In the present specification, "disease related to platelet aggregation" refers to the above-mentioned diseases including diseases that are caused or indirectly related to platelet aggregation. In the present specification, "a disease related to a decrease in platelet function" refers to a disease that includes a decrease in the number of platelets or a decrease in platelet function as a cause or an indirect related disease. The "substance with platelet aggregation inhibitory activity" of the present invention refers to platelet aggregation as a cause or indirectly related diseases, for example, cancer (specifically, cancer metastasis) 'pulmonary thrombosis, cerebral infarction, and virtual heart disease (myocardial infarction) (Specifically refers to the suppression of pulmonary thrombosis during severe symptoms of trauma such as traffic accidents), arteriosclerosis, venous thrombosis, peripheral arterial thrombosis, seeded intravascular coagulation (DIC), antiphospholipid antibody syndrome, hyperlipidemia, diabetes Chronic Burst, Glomerulonephritis, Liver Disease, Transplantation, Mucosal Skin Lymphatic Syndrome and Platelet Agglutination, etc. Treatment or Prevention of By-Pass or Vascular Surgery Narrow prevention of useful substances. In addition, "substances with platelet aggregation promoting activity" refer to diseases that are caused by or decrease in platelet count or platelet function, such as bleeding tendency, blood coagulation disorder, hemophilia, uremia, chronic myelogenous diseases, etc. A substance useful in the treatment or prevention. In the present specification, "gp44" means a polypeptide selected from any one of (i) to (viii) below. (i) An amino acid sequence encoded by the nucleotide sequence described in any one of (i-1) to (i-5) below, or a polypeptide formed from platelet aggregation promotion activity or a part thereof: -42-200413526 (i-1) Nucleotide sequences shown in nucleotide numbers 1 to 5 1 of sequence number 1 in sequence listing; (i-2) Nucleotide numbers 1 to 5 of sequence listing 3 in sequence listing. Nucleotide sequence shown in 1; (i-3) Nucleotide sequence shown in nucleotide number 1 to 510 of sequence number 5 in sequence listing; (i-4) Nucleotide number 1 to sequence number 7 in sequence listing The nucleotide sequence shown in 489; (i-5) The nucleotide sequence shown in nucleotide number 1 to 540 of sequence number 13 in the sequence listing; (ii) any of the above (i-1) to (i-5) A nucleotide sequence complementary to the nucleotide sequence described in the present invention, a polynucleotide capable of hybridizing under stringent conditions, an amino acid sequence encoded by a polynucleotide, and a platelet aggregation-promoting polypeptide; (iii) an amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of sequence number 2 in the Sequence Listing;

Uv) Amino acid sequence polypeptides shown in amino acid numbers 1 to 166 in sequence number 4 of the Sequence Listing; (v) Amino acid sequence poly shown in amino acid numbers 1 to 169 of sequence number 6 in Sequence Listing Peptides; (vi) amino acid sequence poly-peptides shown in amino acid numbers 1 to 16 of sequence number 8 in sequence listing; (vii) amino acid numbers 1 to 1 7 2 of sequence listing in sequence number 1 4 Polyaminopeptide of the amino acid sequence shown; (viii) the amino acid sequence of the polypeptide described in any of (iii) to (vli) above 200413526 column 'missing, replacing, or adding 1 or several amino acids A polypeptide formed from the formed amino acid sequence and having platelet aggregation promoting activity. Furthermore, "the gP44 of the present invention contains modified peptides such as sugar chains. In addition, "" gp44-expressing cell "means a cell expressing a gp44-encoding gene introduced by its own gp44 or expressing an artificial operation. Cells can be used, animal cells, insect cells, etc., preferably animal cells, more preferably mouse, mouse 'canine or human cells, particularly mouse bone bud cell line MC3T3-E1 cells, mouse lymph node matrix CA -12 cells, mouse brain capillary MBEC-4 cells, CHO cells or human 293T cells. Autologous gp44 expression cells can be animal cells, osteoblasts, or cells derived from lung, brain, skin, bone, intestine, kidney, heart, placenta, and skeletal muscle. The artificially introduced cell may be, for example, a transient gene-transformed expression cell or a stable gene-transformed expression cell. "Substance" refers to a mixture other than a single compound, such as a synthetic organic compound, a naturally occurring organic compound, an antibody, a peptide, or a gene. "Specimen" means blood, living tissue or fecal matter obtained from a subject (patient, including a subject in a clinical trial) or a test animal, and is preferably a bone bud cell, lung, brain, The tissue of skin, bone, intestine, kidney, heart, placenta or skeletal muscle is preferably the tissue of lung or skeletal muscle. In the present specification, "polypeptide" also includes those modified with a sugar chain or the like. "Hybridization under severe conditions" refers to a commercially available hybridization solution ExpressHyb Hybridization Solution (manufactured by Clonetech), which is hybridized at 68 ° C. When using DNA-fixed filter paper, perform hybridization at 68 ° C in the presence of 0.7 ~ 1.0-44-200413526 M NaCl, and then use 0.1 to 2 times the concentration of SSC solution (1 times the concentration of SSC containing 150 m). Μ N a C 1,15 μM sodium citrate) was washed at 6 8 ° C under the same conditions or under the same conditions. The present invention has platelet aggregation inhibitory activity or platelet aggregation promotion active substance which can be used for medicine. Its administration form is, for example, lozenges, capsules, granules, powders or syrups and the like, and injections or suppositories. Oral administration. 1. Screening method using cultured cells The method of the present invention is, in particular, expressing the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, or 13 in the Sequence Listing, or having the same peptide as the polypeptide encoded by it An active polycapeptide-encoded mRNA sequence is measured for specific detection of the mRNA, and a method for selecting a platelet aggregation inhibitor or a test substance for which the mRNA expression level is decreased or increased is determined. Furthermore, the sequence contained in the nucleotide sequence shown in sequence number 1 of the sequence listing is: mouse glycoprotein 38 mRNA, complete coding regions (complete cds), which is listed in GenBank accession number M96645, and sequence sequence number 3 Shows the D aw 1 ey (T 1-a) m RNA contained in the nucleotide sequence, all coding regions, published in accession number U07 7 97 in Ge η Bank, and the nucleoside shown in sequence number 5 of the sequence listing The acid sequence contains the sequence of 斤 J 丨 J is, C. familiaris mucin-type membrane proteing P40, published in GenBank accession number Z81018, and the nucleotide sequence shown in sequence number 7 in the sequence listing is' H 0 m 0 sa P 丨 ens 1 ung type-I cell niembrane-associated protein hTla-2 (h T 1 a -2) m RNA, full 咅 M flat code region, access number in G en Bank AF 0 3 0 4 2 Listed in 8-45-200413526. The nucleotide sequence shown in sequence number 1 in the Sequence Listing 1 3 is Μ υ smuscu 1 us mRN A for glycoprotein 3 8 podoplanin homologue (Gp38P gene), access number AJ297944 in GenBank Posted. And, the method of the present invention is, specifically, expressing the amino acid sequence of sequence number 2, 4, 6, 8 or 14 in the sequence list, or the polyamino acid sequence encoded by a polypeptide having the same activity as the polyamino acid sequence Peptide, a measurement of specific detection of the polypeptide, and a method for selecting a platelet aggregation inhibitor / promoter for a test substance whose expression level of the polypeptide is decreased / increased. φ In addition, the amino acid sequence shown in the sequence number 2, 4, 6, 8 or 14 of the sequence listing is listed in J, GenBank accession number M96645, U07797, Z81018, AF030428 or AJ297944. (1) Production of gp44-expressing cells According to conventional methods such as selective colonization and hybridization, a cDNA library obtained from mouse, mouse, dog, or human tissues is used to obtain a full-length cDNA, and the cDNA is introduced into the cells to obtain gp44-expressing cells. The probe used to obtain the full-length cDNA from the cDNA library is a part of the nucleotide sequence described in sequence number 1, 3, 5, 7, or 13 of the beta list, 15 to 30 bases Long continuous oligonucleotide fragments were used as probes. The length of the probe is preferably 15 to 20 bases. The introduced cells can be animal cells, insect cells, etc., preferably animal cells, more preferably primates, sun-toothed or dogs, and particularly preferably mouse, mouse, dog or human cells. Cell introduction method, method using viral vector, direct expression plastid administration method, liposome method (liposome), lipid infection > 46-200413526 (L ip fecti ο η) method 'microinjection (Micr 〇injecti ο) method, phosphoric acid fishing method or electroporation method. When a viral vector is used, the virus used is a retrovirus, adenovirus, adenovirus, herpes virus, vaccinia virus, pox virus, polio virus, or a DNA virus, or RN A virus. Among them, it is preferably a retrovirus' adenovirus, an adenovirus or a vaccinia virus, and particularly preferably a retrovirus or an adenovirus. (2) Cultivation of gp44-expressing cells and addition of test substances Cells expressing gp44 can be cultured in a common culture method under conditions where gp44 may be expressed. For example, ‘Cultivation according to the method described in the Freshman Chemistry Experiment Lecture (Edited by the Tokyo Biochemical Society, Tokyo Chemical Dojin 1 990). The test substance, culture medium, phosphate buffer, etc. are prepared at a desired concentration, and the medium is added and cultured for a fixed period. The test substance is not limited, and extracts of microbial cultures and animal and plant tissues, extracts of cultured cell cultures, artificially synthesized inorganic or organic compounds, recombinant proteins, antibodies or fragments thereof, and antisense (Anti-sense ) Nucleic acids such as nucleic acids, siRNA, their salts, their derivatives, and combinations of multiple types of substances. The amount and concentration of the test substance can be set to a suitable amount or to create a set of dilution series. The culture time after the test substance is added can be set appropriately, usually 30 minutes to 24 hours. According to the above cultured cells, if necessary, use the following method (3) to detect its mRNA expression, or use the following (4) method to detect its polypeptide expression -47-200413526 ′ to screen the test substance. (3) Screening method using mRNA (3-l) Preparation of mRNA reagents (including mRNA, the same applies hereinafter), the cells after culture are directly extracted with an RNA extraction solvent (for example, 'containing phenol and other inert components with RNA Substance) should be dissolved directly. In addition, RNA is extracted after the cells are recovered with a spatula with a spatula, as far as possible without damaging the cells, or by using a proteolytic enzyme such as trypsin to stably separate the cells from the culture dish. _ RN A extraction method 'can use guanidinium thiocyanate · chlorinated high-speed centrifugation method, guanidinium thiocyanate · hot phenol method, guanidine-HC1, acidic guanidinium thiocyanate · phenol · chloroform method (Chomczynski, P. and Sacchi (N ,, (1987) Anal. Bichem ('162, 156-159)) and the like, preferably the acid guanidine thiocyanate · phenol · chloroform method. In addition, commercially available RNA extraction reagents (for example, ISOGEN (manufactured by Japan Gene Corporation) TRIZOL reagent (manufactured by Gibco-BRL), etc.) are used in accordance with an operation template (p r 〇 t 〇 C ο 1) attached to the reagent.

The mRNA can be obtained by refining the RNA extract obtained by the above method. Most of the mRNA in eukaryotic cell cytoplasm has a poly (A) sequence at the 3 terminus, and mRNA can be refined using this feature. For example, an mRNA is adsorbed on a biotinylated oligo (dT) probe to form a complex 'and then this complex uses a biotin / streptavidin binding' to capture the streptavidin in the The magnetic particles were 'washed' and eluted to purify the mRNA. It is also possible to adsorb the mRNA on an oligo (dT) cellulose column and then dissolve it to purify the mRNA -48-200413526 However, in the method of the present invention, the mRNA refining step is not necessary, as long as the detection of the above (a) to (1) is satisfied ) MRNA expression of any one of the recorded nucleotide sequences, the RNA extract can be used in this detection step. (3-2) Detection and evaluation of mRNA expression The extracted mRNA will be protected by RT-PCR method, Northern hybridization method, ribonuclease (R ib ο nuc 1 ease), derivative assay, DNA chip analysis' DNA Macro array (Micr Array) analysis, membrane filtration, paper analysis or other analytical detection. (3-2- 1) RT-PCR method to perform reverse transcription enzyme reaction on the mRNA of the nucleotide sequence described in any one of (a) to (1) above as a template, and then perform specificity by PCR DNA fragment amplification. The primer used in the reverse transcription enzyme reaction is an antisense primer with a length of 15 to 40 bases which is complementary to the sequence of a specific part of the target mRNA. The length of the primer is preferably 18 to 30 bases, and more preferably 21 to 30 bases. The primers used in the PCR amplification of the cDNA purified by the reverse transcription enzyme reaction are synonymous primers that are complementary to a specific partial sequence of the cDNA and have a continuous length of 15 to 40 ^ bases. The length of the primer is preferably 18 to 30 bases, and more preferably 21 to 30 bases. RT-PCR reactions are performed by conventional methods. Rebeijing, the reagent used for RT-PCR is preferably a purified mRNA grade.

After PCR of the amplified mRNA, the reaction solution is detected by electrophoresis to determine the nucleic acid band showing the target length. In order to carry out quantitative detection, using the cDNA clones diluted in previous stages as standard template DNA under the same conditions was expanded with PCR -49-200413526 to determine the possible temperature and cycle number for quantitative detection, or for example, 5 cycles will A part of the reaction solution was sampled and subjected to electrophoresis. Then, for example, a radiolabeled dCTP is used in the PCR reaction to obtain the amount of radiation in the band as an index for quantification. In addition, methods with high reliability of polynucleotide quantification can also be used, such as the modified competitive RT-PCR (competitive RT-PCR) method of RT-PCR described above (Souaze et al. (1996) Bio. Techniques 21 ^ 280 -285), and Taqman PCR (Heid et al. (1996) Genom. Res. 6, 986-994) and the like. Compare the amount of mRNA extracted from cells cultured in the presence of test substance with the amount of mRNA extracted from cells cultured in the absence of test substance. It is a platelet aggregation inhibitory active substance. (3-2-2) Northern hybridization method This method can be implemented by a conventional method in the technology used in the present invention (for example, 'biotechnology experiment i 11 u s t r a t e d 4, p 1 4 8, Xiu Run Company). The choice of material is implemented in the same way as above (3-2 -1). (3-2-3) Detection of ribonucleic acid protection The target mRNA in the RNA reagent is hybridized with a complementary labeled probe to form a double-stranded polynucleotide, and then ribonuclease is added for culture. Whereas the double-stranded polynucleotide is not degraded by ribonuclease, the RNA that has not formed double-stranded polynucleotide can be degraded by ribonuclease. Therefore, quantification of double-stranded polynucleic acid after the above-mentioned culture can determine the expression level of the target m R N A. Furthermore, double-stranded labeled probes should be separated from double-stranded polynucleotides for quantification -50-200413526

In this method, DNA or RNA can be used as the labeled probe, preferably RNA. The length of wooden needles is preferably 50 to 500 bases. Marking probes should be used in an amount equivalent to 5 X 1 05 p m ο 1. The RNA probe can be prepared, for example, by the following method. Apply the template 〇να to a plastid carrier (? 1 * 〇111〇 [6 1.) (D7, 3-6, D3 promoter, etc.) in a plastid vector (eg, pGEM-T (Promega Made by the company), etc.). The plastid carrier is cut to limit enzyme degradation and is cut down (down stream). The obtained linear DNA template was subjected to an in vitro translation reaction using a polymerase corresponding to a promoter in the presence of a radiolabeled ribonucleotide. This reaction is performed using, for example, ribose probe system_T7, ribose probe system-SP6, or ribose probe system_T3 (both manufactured by proinega). The use of ribonucleic acid can be selected as required. When the labeled probe is DNA, single-stranded DNA should be degraded. The RNA reagent is preferably a purified mRNA grade. Furthermore, it is preferable to use an equivalent amount of 10 to 20 g of the RNA reagent. Furthermore, this method is performed using a commercially available kit (HybSpeed RPA Kit, manufactured by Ambion). The obtained ribonuclease-degraded reagent was electrophoresed on a 4 to 12% polyacrylamide gel containing 8 M urea, the gel was dried, and X-ray film was used for autoradiography detection. The quantification of the detection zone is performed according to the usual method. Moreover, the difference in the amount of RNA between the reagents can be corrected based on the expression of actin genes measured simultaneously with the reagents. Substance selection is performed in the same way as above (3-2-1.). (3-2-4) Derivative assay (R u η-ο n a s s a y, Greenberg, M.E. and -51-200413526

Ziff 'EBB (1984) Natu re 311, 433-438, Groudine, M. e al. (198 1) Mol. Cel] Bi 01, 281-288) This method is to directly detect the amount of mRNA in cells. A method for measuring the transcriptional activity of a target gene from a nucleus isolated from a cell. The "method for measuring the expression amount of a polynucleotide" in the present invention also includes this method. Isolate the nucleus and carry out the transcription reaction in the test tube. If the transcription is started before the nuclear separation, the mRN A chain can be extended in the middle of the generation. In this transcription reaction, radiolabeled ribonucleotides are added, and the extended m R N A is radiolabeled. The m R N A of unlabeled probe hybridization can be measured to detect the transcription activity of the target polynucleotide when isolated in the nucleus. Comparing the nuclear transcription activity of cells cultured in the presence of the test substance with the nuclear transcription activity of cells cultured in the absence of the test substance, a substance having platelet aggregation promotion activity can be selected when it is increased, and Platelet aggregation inhibitory substance. (3-2-5) D N A wafer analysis' D N A macro array analysis, membrane filter paper analysis (labeling of probes) As described above, (3 -1) the obtained mRN A is labeled with a probe reagent. The labeling reagent is preferably purified mRNA.

For the analysis of DNA wafers made by Affymetrix, the probes used are labeled with biotin according to the operation template attached to the wafer. For DNA microarray analysis, the probes used are used for poly (A) + RNA to cDNA production in the reverse transcription enzyme reaction. Fluorescent pigments (for example, Cy3 'Cy5, etc.) are added to d-UTP, etc. Fluorescently labeled cDNA. At the same time, the reagents obtained from culturing cells in the presence of the test substance and the cells cultured in the absence of the test substance are labeled with different pigments. In the subsequent hybridization operation, the two are mixed and used together. When using Wu filter paper for analysis, the poly (A) + R N A to c D N A was prepared in the reverse transcription reaction, and radioactive isotopes (for example, 3 2 p, 3 3 p, etc.) labeled d-CTP labeled probes were added. (Preparation of Immobilization Reagents) ① Immobilized Type I reagents for DNA wafer analysis can be used, for example, EST (expressed sequence tag) sequences in a database or immobilized antisense oligonucleotides based on mRNA sequences. Gene chip (for example, manufactured by Affymetrix (Lipshuts, RJ. Et al. (1 999) Nature genet. 21,

Supplement, 20-24), etc.) The source of the EST sequence or mRNA sequence of the immobilized oligonucleotide, but there is no particular limitation on the person who can detect the polypeptide of the present invention. Obtained from animals used for probe preparation. ② DNA microarray analysis and membrane filter paper analysis. 'Immobilization reagents use the same or similar animal organs and tissues as those used in the preparation of the probe to isolate or isolate mRNA obtained from cells to make cDNA or RT-PCR. The product is immobilized with a DNA microarray or membrane filter paper. The immobilized cDNA or RT-PCR product is used. For example, based on sequence information such as the EST database of mRNA material animals, make primers to perform a reverse enzyme reaction to perform PCR化 产品。 Chemical products. For the preparation of reagents, use -53-200413526 cells with the nucleotide sequence genes described in any one of the prefaces (a) to (]). The cells are preferably the same type as the cells used in the preparation of the probe or Organs and tissues of related animals to isolated or singulated cells. The CDNA or RT-PCR product 'due to different RNA expression levels, a selective self-subtraction method (diatchenk 〇, L. et al. (1996) Proc. Natl. Acad. Sci. USA 93 j 6025 -6030) was used to compare the expression method ( Kato, K. (1 995) Nucleic Acids Res. 23, 36 85-3 690). DNA microarray or membrane filter paper can be fixed by using a commercially available gene containing the detection target, or using a commercially available Spoter (for example, GMS417 arrayer: manufactured by Takara Shuzo Co., Ltd.) Made by. (Hybridization analysis) The probes obtained above were hybridized using an immobilized reagent. (Brown, P.O. eta 1. (1 999) Nature Genet. 21, Supplement, 3 3-37) ① DNA chip analysis For example, according to Affymetrix manufacturing gene chip operation template (expression analysis technology manual), use eight 〖£ ¥ 11 ^ 1 ^ \ An analytical device (〇6116 (: 1 * ^ F1 uidics Tati ο η 4 0 0) manufactured by the company) was used for hybridization and analysis, and avidin fluorescence was detected for analysis. ® ② DNA macroarray analysis hybridization in accordance with the usual method, after washing, use a fluorescence detector to detect the fluorescence intensity. For example, if a matrix is used, for example, a commercially available matrix manufactured by Takara Shuzo will be operated by the company The template was hybridized and washed, and the fluorescence signal was detected by a fluorescent signal detector (such as GMS418 matrix scanner 49 (manufactured by Takara Shuzo), etc.), and then analyzed. ③ Analysis of membrane filter paper -54-200413526 For hybridization, for example, a commercially available filter paper is made into a macro array, and after hybridization and washing with an AUas system (C) made by Ontech, detection is performed with an analysis device (for example, A11 as I mage: C 1 ο ntech). Preface ① to ③ Medium The method described is to use the same batch of (] 0t) immobilized fixative to hybridize the probes obtained from the cells cultured in the presence of the test substance with the probes obtained from the cells cultured in the absence of the test substance. Hybridization under the same conditions except for needles. When fluorescently labeled probes are used, each probe is labeled with a different fluorescent pigment on an immobilized reagent, and a mixture of two probes is used at the same time to hybridize the parent. § Buy all light intensity (Brown, P. 〇. 〇. etal. (1999;) Natui * e_genet, 21, Supplement, 3 3 -37) (Judgment) Material selection was performed in the same way as (3-2 -1) above. (3-2-6) Other analysis methods Inventive method 'A method other than the above can be used to detect mRNA obtained from cells cultured in the presence or absence of the test substance, and there is no particular limitation. The detection method other than the above can be, for example, Substration Cloning Method, Differential Display method, the "D & 9111 & 11" known technology (1 ^ 〇11 & 11 ( },?.] \ 4.6131. (1991)? 1'〇 (:.

Natl · Acad. Sci. USA 88, 7276-7280), a method of combining PCR and hybridization exploration in a single reaction (Higuchietal. Biotechnology, 10, 413-417 (1992)) '' LightCycler system (see Roche Diagnostic, Japan Patent Publication 2000-3 1 2600) and so on. (4) Screening method using polypeptides -55-200413526 (4-1) Preparation of polypeptide reagents ① Western ink stain method Reagents are used in western ink stain method, for example, the culture supernatant liquid is treated with trichloroacetic acid The protein was precipitated, and the centrifugal precipitate was recovered. The recovered precipitate was washed with ice-cold acetone, air-dried, and dissolved in a sample buffer (Bio Rad, etc.) containing 2-thiohydrin ethanol by s DS polyacrylamide electrophoresis. ② Spot ink stain method, slit ink stain method, fixed enzyme immunoquantification method Spot ink stain method, slit ink stain method or fixed enzyme immunoquantification method uses reagents, for example, it can be prepared by appropriately diluting the culture palate or buffer solution. . Chun (4-2) Polypeptide Reagent Immobilization Polypeptide was specifically detected from the reagents obtained above, and the reagent was immobilized. ① Western ink stain method, dot ink stain method, slit ink stain method In western ink stain method, dot ink stain method, or slit ink stain method, after electrophoresis of the reagent, the polypeptide is moved from the glue to the membrane to fix Chemicals: used membranes, such as nitrocellulose membranes (Bio Rad, etc.), nylon membranes (Hibond-ECL (Amersham · Pharmacia, etc.), etc.), cotton membranes (Blot Adsorbent membranes (Bio Rad, etc.), etc.) or PVDF membrane (Bio Rad, etc.). The ink stain method is a wet ink stain method, a semi-dry ink stain method (see CURRENT PROTOCOLS IN IMMUNOLOGY volume2 ed. By J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.

Shevach, W. Strober) and others. Commercially available kits (eg, BioRad, etc.) using Western blotting, spot blotting or slot blotting. -56-200413526 ② Immobilized enzyme immunoassay method (EIA method, ELIS A 丨 J) In the fixed enzyme immunoassay method, add a reagent or its diluent to a 96-well plate (for example, an immunoplate (manufactured by NUNC), etc.), Allow to stand overnight at 40 ° C to room temperature or at 37 ° C] to 3 hours, and immobilize the polypeptide on the bottom surface of the acupoint to immobilize it. (4_3) Antibody preparation The antibodies used in this method can be used exclusively Identification of gp 4 4 or a portion thereof of the present invention. Specifically, the antibody used in this method can specifically identify the polypeptide or a part thereof described in (i) to (viii) below. _ (I) the following ( The amino acid sequence encoded by the nucleotide sequence described in any one of (i-1) to (i-5) or a part thereof and a platelet aggregation promoting activity (i-1) sequence listing sequence Nucleotide sequence shown in nucleotide number 1 to 5 1 9 of number 1; (i-2) Nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in sequence listing; (b) Sequence list Nucleotide® acid sequence shown in SEQ ID NO: 5 to 510; (i-4) Nucleotide number 1 to 489 in SEQ ID NO: 7 in Sequence Listing 5) The nucleotide sequence shown in nucleotide numbers 1 to 540 of sequence number 13 in the sequence listing; (ii) any of the items (i-1) to (i-5) described above Polynucleotide composed of nucleotide sequences complementary to the nucleotide sequence, polynucleotide-57-200413526 which hybridizes under severe conditions, and a polypeptide with platelet aggregation promoting activity; (iii) Amino acid sequence poly-peptide shown in amino acid numbers 1 to 172 of sequence number 2 in the Sequence Listing; (iv) Amino acid sequence poly-peptide shown in amino acid numbers 1 to 166 of sequence number 4 in sequence listing Peptides; (v) the amino acid sequence polyamino acid peptides shown in the sequence listing sequence number 6 amino acid numbers 1 to 169; (vi) the amino acid sequence polyamino acid peptides shown in the sequence listing sequence number 8 amino acid numbers 1 to 162 Peptide; _ (vii) a polypeptide of the amino acid sequence shown in the sequence listing of the amino acid number 1 to 1 72 of the sequence listing; (viii) the polyvicin according to any one of (iii) to (vii) A polypeptide in which the amino acid sequence of the peptide is reduced, substituted or added with one or more amino acids, and still has platelet aggregation promoting activity. The antibody used in the method of the present invention is preferably an antibody capable of binding to the polypeptide of any one of (i) to (viii) above, and not binding to any other protein from mouse or human. · The antibody used in the method of the present invention, according to the usual method (for example, freshman chemistry experiment lecture 1 'Protein i, p3 89_397, (1 992)), using the antigen polypeptide or a part thereof to immunize the animal and collect live The antibodies produced in the body are refined and guarded. Moreover, according to a conventional method (for example, K 〇 h 1 e r a n d M i 1 s t e i η,

Nature 256 '495-497 (1 975), Kennet, R. ed., Monoclonal Antibody p. 3 65 -3 67 (1 980), Prenum press, N.Y.), will be able to produce a polypeptide for the present invention The antibody-producing cells of the antibody were fused with fibroid cells-58-200413526 to establish a hybridoma, and a single antibody was obtained. As the antigen, the polypeptide described in any one of (i) to (viii) above, or a polypeptide containing at least 6 consecutive partial amino acid sequences, or an arbitrary amino acid sequence can be added to the polypeptide Derivatives with monomers. Polypeptides of the antigen can be synthesized in vitro or produced using genetically modified host cells. Specifically, a polynucleotide encoded by a peptide that will become an antigen is combined in an expression vector and reacted in a solution containing an enzyme, a substrate, and an energy substance necessary for transcription and translation of the vector. Furthermore, the form is transformed into a prokaryotic or eukaryotic host cell, and the nucleotide is expressed to obtain the desired polynucleotide. A method for in vitro synthesis of a polypeptide can be, for example, a Rapid Translation System (RTS) (Roche Diagnotics). The method is to clone the target gene into an expression vector controlled by the T7 promoter, add the reaction system of the vector and T7 RNA polymerase, transcribe mRNA according to the template DNA, and then 'add the liposomes in the E. coli lysate, The method of translating mRNA into the target polypeptide reaction solution (Biochemica, 1, 20-23 (2001), Biochemica, 2, 28-29 (2001)). Polypeptide can be used as a host when it is obtained from genetically modified host cells. As long as the prokaryotic cells that can be used in this method are not limited, such as E. coli (E sch er ic hi ac ο 1 i ), Bacillus subtilis (b ac i 1 lu ssubti 1 is) and the like. For E. coli, for example, K 1 2 strain can be used. Bacillus subtilis can be used, for example, strain 207-25. The vector is not limited as long as it can be used in this method. For example, when -59-200413526 uses E. coli as the host, pBR322 and pUC can be used. When using B. subtilis as the host, pTUB 2 8 can be used. 8 (Ohmura, K. et a]. (1984) J. Biochem. 95, 87-9 3). When using Bacillus subtilis as a host, it binds to the DNA sequence encoded by the SigIia peptide sequence of the Bacillus subtilis and secretes the expressed polypeptide outside the bacteria. When using a prokaryotic cell as a host, the promoter is not limited as long as it can be used to produce the promoter of the target polypeptide. For example, in E. coli, the tryptophan (Up) promoter 'lactose (1 ac) promoter, Amino acid, lactose (tac) promoter, lipoprotein (] PP) promoter, poly (peptide bond chain extension factor Tu (tufB) promoter, etc.). Furthermore, when using eukaryotic cells as the host, as long as this method The eukaryotic cells that can be used are not limited, such as vertebrate cells, insect cells, yeasts, and the like. Vertebrate cells are, for example, monkey cells COS cells (Gluzman, Y. (1981) Cell 23, 175-182, ATCCCRL-1 650), dihydrogen of Chinese golden rat ovary cells (CHO cells) (ATCC CCL-61) Deletion mutant of folic acid reductase (Urlaub, G. and Chasin, LA (1 980) Proc. Natl. Acad. Sci · USA 77, 4126-4220) ° When eukaryotic cells are used as hosts, promoters can be used Those with promoters, RNA splice sites, poly (A) sites, and translation termination sequences located upstream of the gene to be expressed can also use promoters with origins of replication when necessary. The promoter with the promoter can be, for example, pCR3.1 with an early promoter of CMV virus (manufactured by InviUogen), and pSV2dhfr with an early promoter of SV40 (Subramani, S. et al. (1981) Mol. -60- 200413526

Cell. Biol. 1,854-864) and the like. For example, when cos cells are used as host cells, the expression vector can be used to prepare SV40 replication origins, which can be replicated in COS cells by themselves, translation promoters, translation termination signals, and RNA splice sites. The expression vector can be used by the diethylaminoethyl (DEAE) -glucose method (Luthman, H. and Magnusson j G. (1983) Nucleic Acids Res, 11, 1295-1 308), phosphate fishing-DNA co-precipitation method ( Graham, FL and van der Eb, AJ (1 973) Virology 52, 456-457) or electroporation (Neumann, E. et al. (1 982) EMBO J. 1, 84, 845), etc. , And have to transform cells of hope. Furthermore, when CHO cells are used as host cells, the expression vector functions as an antibiotoxin G4 18 marker marker to express a neo gene, such as pRSVneo (Sambrook, J. eta 1. (1 9 8 9) · " Molecular Cloning A Laboratory Manual "Cold Spring Harbor Laboratory, NY) or pSV2neo (Southern, PJ and Berg j P. (1 982) J.

Mol. Appl. Genet. 1,327-3 4 1) and other transformations, and screened G41 8-resistant cell lines, to obtain transformed cells that can stabilize and produce the desired polypeptide. When insect cells are used as host cells, the insect cells use, for example, cells derived from Spodoptera frugi perda ovary cells of the family Lepidoptera (Sf-9 or Sf-2 1) and High FWe cells (Wickham from Trichoplusiani egg cells) , T. J. et al '(1992) Biotechnol. Prog. I: 39 Bu 3 9 6) and the like. When insect cells are used as host cells, the vector used is a baculovirus transfer vector, specifically pVL using the polyhedrin protein promoter of the Polygonum californicum-61-200413526 polyhedrovirus (AcNPV). 1 3 92/1 3 93 (Kidd, IM and V. C, Emery (1 993) The use of baculovi ruses as expression vectors. Applied Biochemistry and Biotechnology 42, 137-159), or use baculovirus p 1 〇 or Vectors of promoters of isobasic proteins. Furthermore, the GP 67 secretion signal sequence 歹 ij of the AcNPV outer sheath surface protein was fused to the N-terminus of the target protein, and the recombinant protein was expressed as a secreted protein. When yeast is used as the host cell, the yeast is preferably a yeast of the genus Saccharomyces (for example, Saccharomyces cerevisiae, Pichia pastoris, etc.). When yeast is used as the host cell, the vector is preferably a promoter of an alcohol dehydrogenase gene (Bennetzen, JL and Hall, BD (1982) J Biol. Chem. 257, 30 1 8-3025) or an acid phosphatase gene. Promoter (Miyanohara, A. eta 1. (1 9 8 3) Proc. Natl. Acad. Sci. USA 80, 1-5) and so on. Furthermore, the secretion signal sequence and the intrinsic protease or known protease cleaving site contained in the host cell are fused with the N-terminal side of the target protein, and the recombinant protein is expressed as a secreted protein. For example, if human mast cell tryptase belonging to trypsin-type serine protease of petroleum yeast is to be expressed, at the N-terminal side of the target protein, the secretory signal sequence of α factor of yeast and the KE2 protease of petroleum yeast The site connection is cut to express the protein of interest and the active tryptase is secreted in the culture medium (Andrew, L. Niles, eta 1. (1998) Biotechnol. App 1. Biochem. 28, 125-131). The target polypeptide can be cultured according to the conventional method and produced in -62-200413526 intracellular or extracellular. The medium used for the culture is selected from various conventional mediums suitable for the host cells used. For example, in the above-mentioned cos cell culture, RPMI] 640 medium was used, Dibaco modified the necessary culture medium (hereinafter referred to as "DMEM"), etc., and a medium containing bovine fetal blood pupa and other blood pupa components should be used if necessary. According to the above-mentioned culture, the recombinant protein produced inside or outside the transformed cell is separated and purified by a conventional separation operation method by utilizing the physical and chemical properties of the protein. The method is, for example, treatment with a conventional protein precipitant; ultrafiltration; molecular chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography or high-speed liquid chromatography (HPLC) and other liquid layers Analysis; dialysis; or a combination thereof. A 6-residue histidine is linked to the recombinant protein to be expressed, and the nickel affinity column is used for efficient purification. It is desirable to separate and purify the above methods in combination. The antibody that can be used in the method of the present invention is an antibody obtained according to the above method, and a known antibody that specifically binds to gp44 can also be used. The antibody is, for example, a monoclonal antibody 8F11 (M. Watanabe et al, Cancer Res., 1988, 48 • 6 4 1 1-6 4 1 6 (4-4). The antibody is labeled with the obtained polypeptide as an antigen. The obtained antibody can be used as a direct label in the following detection projects, or the antibody is used as a primary antibody and used in conjunction with a labeled secondary antibody that uniquely identifies the antibody. There is no specific labeling method as long as it is a known method of use Limit, it should be enzyme (for example, alkaline dephosphatase or wasabi peroxidase, etc.) or biotin (used in conjunction with streptavidin). Label a secondary antibody (or label avidin chain-63-200413526 bacteriocin ) A variety of commercially available products can be used. For RIA, for example, radioactive isotope labeling such as I125 can be used. (4_5) Detection of polypeptide The use of enzyme activity in antibody labeling can be used to determine the amount of polypeptide containing antigen. For example, when alkaline dephosphatase or wasabi peroxidase is used for labeling, the enzyme is reacted with a commercially available substrate, and then the coloration or fluorescence is measured to determine the amount of polypeptide. Furthermore, when the antibody is labeled with a radioisotope Using liquid Use a scintillation counter (Liquid scitilation counter), etc. to measure the amount of polypeptide. Φ When using a color-producing substrate, Western blotting, spot blotting, or slit blotting can be detected visually. In the ELISA method, a commercially available microplate reader should be used to measure the absorbance of each cavity (the measurement wavelength is different from the matrix) for quantification. Furthermore, the antigen dilution series used for the production of the antibody in (4-3) above should be used. It is a standard antigen reagent and a target reagent to perform a detection operation at the same time. According to the prepared standard antigen concentration and measurement 値 standard curve chart, the original concentration of target reagent in L / L is quantified. When using a fluorescent substrate, the western ink stain method The dot stain method or slit * In the stain method, 'use an X-ray film or an imaging (Imagine) flat plate autoradiography or take a picture to detect the image. Then, use densit 〇me try or m ο 1 ec u 1 ar Iminger-Fx system (Bio Rad), etc. Quantitative ELISA method, using a luminous microplate reader (B i 〇R ad, etc.). (4_6) Measurement operation ① Western blot method, spot blot method Or slit ink method to prevent Non-specific adsorption, the membrane is in a buffer containing specific adsorption inhibitor -64-200413526 (skimmed milk, casein, bovine blood albumin, gelatin, polyvinylpyrrolidone, etc.) in a buffer solution (hereinafter referred to as "blocking (Blocking solution ") for a certain period of time (referred to as" blocking (B 1 ocking) "for F). The composition of the blocking solution may be, for example, containing 5% skim milk and 0.05% to 0.]% T wi Phosphate buffered saline (pb S), or phosphate buffered saline (TBS) at η 2 0. Skimmed milk can be replaced by Block Ace (Dai Nihon Pharmaceutical), 1 to 10% bovine blood prion protein, 0.5 to 3% gelatin or 1% polyvinylpyrrolidone. Blocking temperature and time are 16 to 24 hours at 4 ° C or 1 to 3 hours at room temperature. φ The membrane is then filled with 0.05% to 0.1% D? 83 or D63 (hereinafter referred to as "washing solution"), after removing the blocking solution, and the antibody prepared in the method described in (4-3) and (4-4) above, appropriately diluted in the blocking solution. Immerse for a certain period of time to allow the antibody to bind to the antigen on the membrane. The dilution ratio of the antibody is determined according to, for example, the western blotting experiment performed in the preparation of the recombinant antigen stage dilution reagent described in (4-3) above. The antibody reaction operation is preferably performed at room temperature for 2 hours. After the antibody reaction operation is completed, wash with a membrane washing solution to remove antibodies. ® When using a labeled antibody, detect it with an appropriate labeling method. When an unlabeled antibody is used, a secondary antibody reaction is performed. The labeled secondary antibody can be used, for example, by a commercial person, and then diluted with a blocking solution at 2000 to 20000 times (the appropriate dilution ratio described in the manual attached to the commercially available secondary antibody is used as described). The membrane after the primary antibody was washed and removed was immersed in a secondary antibody solution at room temperature for 45 minutes to 1 hour, and washed with a washing solution. The washing method can be, for example, 'the membrane is first shaken in the washing solution for 15 minutes -65-200413526 is replaced with a new washing solution, and after shaking for 15 minutes, the washing solution is replaced again and shaken for 5 minutes. Detection is then performed in a manner suitable for the marking method. ② Immobilized enzyme immunoassay (ELIS A method / RIA method) In order to prevent the non-specific adsorption of the antibody on the bottom surface of the plate hole immobilized by the reagent in (4-2) above, the same method as in ① above is performed. Wash the points with washing solution for several minutes. After removing the blocking solution, add the antibody prepared according to the method described in (4-3) and (4-4) above, and add the appropriate diluted solution of the blocking solution to the culture for a certain period of time. To bind the antibody to the antigen. The dilution ratio of the antibody is determined according to, for example, the ELISA experiment in which the recombinant antigen is prepared with a stepwise dilution reagent as described in (4-3) above. The antibody reaction operation is preferably performed at room temperature for 1 hour. After the antibody reaction operation is completed, the cells are washed with a washing solution to remove antibodies. When using a labeled antibody, detect it with an appropriate labeling method. When an unlabeled antibody is used, a secondary antibody reaction is performed. The labeled secondary antibody can be, for example, a commercially available one, and is then diluted with a blocking solution at a ratio of 2000 to 20000 times (when a suitable dilution ratio is described in the specification attached to the commercially available secondary antibody, the method is performed as described). The secondary antibody solution was added to the holes after the primary antibody was washed and removed, and the cells were incubated at room temperature for 1 to 3 hours, and then washed with a washing solution. The washing method can be, for example, firstly adding the washing solution into the hole, shaking it for 5 minutes, and then changing to a new washing solution, shaking it for 5 minutes, changing the washing solution again, and shaking it for 5 minutes. Detection is then performed in a manner suitable for the marking method. Furthermore, the so-called sandwich E LIS A method (I n d i r e c t E LIS A) can be implemented, for example, by the method described below. In the amino acid sequence of any polypeptide of the present invention, 'select two highly hydrophilic fields, and synthesize a partial peptide containing 6 residues in the field from -66-200413526, and use this partial peptide Two antibodies were made for the antigen. One of the antibodies is labeled by the method described in (4-4) above. The unlabeled antibody was fixed on the bottom surface of the cavity with a 96-hole ELIS A plate according to the method described in (4-2) above. After blocking, the reagent solution was added to the cavity and incubated at room temperature for 1 hour. After washing the wells, add the labeled antibody dilution to each well. After washing the acupuncture points, the detection of appropriate marking methods is performed. (4-7) Ligand In addition to antibodies, ligands such as substrates, complementary enzymes, and regulators that can specifically bind to the polypeptide of the present invention can also be used to quantify the polypeptide of the present invention. (4-8) It is determined that the amount of the polypeptide of the present invention extracted from cells cultured in the presence of the test substance is compared with the amount of the polypeptide of the present invention extracted from cells cultured in the absence of the test substance, If you increase, you can choose to have platelet aggregation-promoting active substances, if you reduce it, you can choose to have platelet aggregation-inhibiting active substances. Furthermore, in addition to the methods described above, a protein wafer method or a protein matrix method can also be used as the method of the present invention. 2. Screening method using an individual animal The screening method of the present invention also includes, after administering a test substance to a mammalian individual, 'detecting the amount of the mRNA of the present invention or the amount of the polypeptide of the present invention in organs or tissue cells removed from the animal Method. The test substance can be a synthetic compound, a microbial metabolite, a tissue extract of a plant or an animal, a salt or derivative thereof, or a mixture thereof. Administration of test substances to individual animals can be performed by oral administration, intravenous injection, intraperitoneal injection, percutaneous administration -67-200413526 or subcutaneous injection, depending on the material characteristics of the test substance. The test substance dosage and concentration can be appropriately set or made into various dilutions and other dosages. The feeding period after test substance administration can be appropriately set, usually between one week and one year. After the test substance is administered, an organ or tissue cell expressing gp44 is taken from an animal individual who has been bred for a certain period of time, and the amount of the mRNA or the polypeptide of the present invention in the organ or tissue cell is determined in the same way as the above-mentioned cultured cell assay Determination. The organ or tissue cell expressing gp44 may be, for example, a bone bud cell ' or lung, brain, skin, bone, intestine, kidney, heart, placenta, skeletal muscle, and the like. The amount of the mRNA or peptide of the present invention extracted from the organ or tissue of an animal individual to which the test substance is administered and the amount of the mRNA or peptide of the present invention extracted from the organ or tissue of an animal individual to which the test substance is not administered Compared with the amount, if it is increased, the platelet aggregation-promoting active substance is selected; if it is decreased, the platelet aggregation-promoting active substance is selected. 3 · Method for measuring the platelet aggregation promotion or inhibitory activity of the test substance The platelet aggregation promotion or inhibition activity of the test substance can be detected by the method described in 1. or 2. above, and the expression of mRN A or polypeptide can be compared. the amount. For example, when compared with a non-administered person, it is judged that it has platelet aggregation promotion activity when it is high, and platelet aggregation inhibition activity when it is low. It should be measured simultaneously with a known control group of platelet aggregation-promoting or inhibitory substances, and compared with the control group to determine the strength of the platelet aggregation-promoting or inhibitory activity of the test substance -68-200413526 4 · Use of transgenic animals / gene knockout animals The test method is based on genetic manipulation to create a gene transgenic animal with high or low expression of the gene of the present invention in a normal animal or a gene knockout animal that destroys the gene of the present invention to provide a model animal with abnormal platelet aggregation regulation. Animals of this model can be used for evaluation of medicines against disorders of platelet aggregation regulation. Transgenic animals are obtained from fertilized eggs of animals. After the genes have been introduced, they are transferred to pregnant animals for production. This step can be based on established methods (Manual Engineering Experiment Manual, Supervised by Nomura. Also edited by Katsuki Moto. (1 987), Mouse Embryo Operation Manual (refer to "Manipulating the Mouse Embryo, A Laboratory Manual") B. Hogan, F. Costantini and E. Lacy, Yamauchi Ichi, Sumida Yu, Mori Yosuke, Iwakura Yoichiro, (1 989) 'Niu inch fair 5-4 8 0 9 3 5 Tiger bullet temple). Specifically, for example, in mice, after ovulation inducers are administered to female mice, they are mated with male mice of the same system, and then prokaryotic fertilized eggs are collected from the oviducts of female mice. Then, the D N A fragment-introduced solution was added to the fertilized egg pronucleus through a micro glass tube. In order to express the introduced gene in animal cells, it can be used together with regulatory genes such as promoters and enhancers for those who have function in the introduced animal cells. Furthermore, according to the expected expression level of the introduced gene, regulators such as promoters and promoters can be selected. Fertilized eggs added with DNA were transplanted into the oviducts of pseudo-mother female mice (Sic: ICR, etc.) that gave birth to pregnant women, and they were produced by natural delivery or by incision about 20 days later. The method for confirming the maintenance of the introduced gene of the obtained animal includes: extracting DNA from the tail of the animal, using the DNA as a template, and performing a PCR method using synonymous and antisense primers specific to the introduced gene; and using the DNA with several restriction enzymes After the degradation, -69-200413526 gel electrophoresis was performed. The DNA in the gel was labeled with nitrocellulose membranes and nylon membranes, and all or part of the introduced genes were labeled. The probes were used for southern blot analysis. Furthermore, whether or not the introduced gene is actually expressed in the animal is determined by measuring the platelet aggregation function according to the method of τ column 7. Introduced Genes 丨 Observation When there is high expression in animals, it has a high platelet aggregation function compared with animals without gene introduction, and when it is low expression, it has a low platelet aggregation function compared with animals without gene introduction. Furthermore, transgenic animals can be obtained by methods other than the above, such as the method of introducing DNA into human embryonic stem cells and then forming a heterochimera (chimera) method, the introduction of embryos at the initial stage of retroviral vector infection, etc. (see Kanegae γ. et al., Nucleic Acids Res. 23: 3816-3821 (1995) »Kanegae Y. et al., Gene. 1 8 1: 207-2 1 2, Wakita T. et al., J.

Biol. Chem. 273: 9001-9006). After the knockout animal has obtained the same recombinants into which the gene has been introduced, the embryos of the recombinant at the blastoderm stage are added to form a heteromer chimera (chimera) animal to obtain the heterogeneous chimera (c h i m e r a) animal combination. Specifically, for example, a gene of interest is selected from a gene bank (g e η om 1 m b a r y), and an exon and its surroundings are deleted to create a gene map. According to the map, the DNA of the area required for the same recombination will be sub-selected, and the part to be deleted will be replaced with a neomycin (neommy η) resistance gene or another drug resistance gene to create a gene targeting site. ) Recombinant DNA used. In order to improve the efficiency of shuffling, the expressed gene is linked to a negative selection gene that causes cell death. Then, the embryonic stem cells are introduced into the gene by means of electroporation, etc., and a transformant of the embryonic stem cell is prepared. The resulting stem cell -70-200413526 can be obtained from the gene system (g e η om s s s s t em) or Lexicongenetic. After ovulation inducers were administered to female mice, they were mated with male mice of the same system, and pronuclear fertilized eggs were collected from the oviducts of female mice 4 days later. After that, 15 to 20 introduced embryonic stem cell transformants are brought into contact with the inner cell mass of the blastoderm via a micro glass tube. The fertilized eggs added with DNA were transplanted to the fallopian tubes of females (mice, Sic: ICR, etc.) of pseudo-mothers at the age of 4 days, and they were born by natural delivery or king incision about 20 days later. The obtained heterochimeric animal is bred with a normal animal to breed a heterozygous animal, and then the heterozygous animal is bred to a homozygous animal. Furthermore, the Cre-loxP system can also be used to eliminate unwanted drug resistance genes or to produce knockout animals specific for the expression period. The method for confirming the disappearance of the introduced gene in the obtained animal includes: extracting DNA from the tail of the animal, etc., using the DNA as a template, and performing PCR using synonymous and antisense primers specific to the introduced gene; and limiting the DNA to several types After the enzyme was degraded, it was analyzed by gel electrophoresis. The DNA in the gel was labeled with nitrocellulose membrane and nylon membrane, and all or part of the introduced genes were labeled. As a probe, southern ink stain analysis was performed. Whether the disappeared gene is actually expressed in the animal body is determined by measuring the platelet agglutination ability according to the following 7. Method. If the introduced gene disappears in the body of an animal, the platelet aggregation ability is lower than that of an animal that has not lost the gene. The obtained gene is introduced into the animal and the test substance is administered to measure the platelet aggregation promotion or inhibitory activity. (It is advisable to compare the results with the gene introduction animal to which the test substance is also administered to determine the significant platelet aggregation promotion or inhibition activity. 200413526 sex). Furthermore, a test substance can be administered to a gene-transduced animal to test the therapeutic or preventive effect of the subject's disease. According to this method, not only substances that can inhibit or hinder the expression of the introduced gene can be found, but also the function of the polypeptide itself that can hinder the gene encoding, and interact with the polypeptide to participate in the polypeptide. Peptide function-expressing factors (such as 'receptors') are inhibitors of in vivo reactions related to platelet aggregation due to the effect of the polypeptide. The substance may also be a therapeutic or preventive agent for promoting or inhibiting platelet aggregation. 5. Use of platelet agglutination activity to enter screening methods with platelet aggregation-promoting activity or platelet aggregation-inhibiting active substances. Platelet aggregation-promoting activity or platelet aggregation-inhibiting active substances can be determined according to the platelet aggregation activity of the test substance. Learned. Specifically, a substance having platelet aggregation promoting activity or platelet aggregation inhibiting activity is selected according to the following A or B screening methods. A • Screening method with platelet aggregation promoting or inhibiting active substances, including the following engineering methods: (1) contacting gp44 expressing cells with platelets in the presence of the test substance: · (2) measuring platelet aggregation activity: (3) According to the measurement result of (2), determine whether the test substance has platelet aggregation promotion activity or inhibitory activity. • Screening methods with platelet aggregation promoting or inhibiting active substances, 3 methods of the following projects: (1) contacting gP44 with platelets in the presence of the test substance: (2) determination of platelet aggregation activity -72-200413526 (3) Press (2) The measurement results determine whether the test substance has platelet aggregation promotion activity or inhibitory activity. The above A and B screening methods are described below in order. A • Screening method with platelet aggregation promotion activity or inhibitory active substance, including the following projects Method: In the presence of the test substance, g p4 4 expressing cells are contacted with platelets. The "test substance", which is shown in the above item "1 ·", can be a synthetic compound, a microbial metabolite, plant or animal tissue extraction. Substances, their salts or derivatives or mixtures thereof, etc. The amount and concentration of the test substance can be appropriately set or various addition amounts can be set based on dilution series, etc. ^ gp44 expressing cells "may be the cells described in the above item" 1 · " "Platelet" is not particularly limited as long as it can be used to measure platelet agglutination activity, for example, using a solution containing platelets. Solution The platelet concentration is also not limited as long as it can measure the activity of platelet aggregation. The solution containing platelets can be blood itself, plasma except blood cells, and a solution in which the plasma is diluted with an appropriate amount (for example, 3 times) of PBS, etc. As long as it can be used to determine platelet agglutination activity, platelets can be derived from that organism and use platelets derived from appropriate organisms. For example, when using cells expressing human-derived gp44, use Human-derived platelets can be used when mouse-derived gp44-expressing cells are used. When mouse-derived gp44-derived cells are used, mouse-derived platelets can be used. For gp44 cells, platelets derived from dogs can be used. The method for contacting gp44 expressing cells with platelets can be, for example, adding -73-200413526 gp44 expressing cell solution to a solution containing platelets. (2) Measurement of platelet aggregation activity

Platelet agglutination activity is not limited as long as it can measure platelet agglutination activity. For example, a platelet agglutination assay using increased light permeability accompanying platelet aggregation (Watanabe M, Okochi E, Sugimoto Y, Tsuruo T

Cancer Res. 1 988; 48: 64 1 bu 6 and Kato Y, Fujita N, Yano H, Tsuruo T. Cancer Res. 1 997; 57: 3040-5), using calcium-sensitive fluorescent substances (Quin2, Fura2), Platelet Agglutination Assay Using Raised Calcium Concentrations in Platelets (Sag e SO, Merritt JE, Ha 11 am TJ, Rink TJ. Biochem J. 1 989; 25 8: 923-6, Rink TJ, Sage SO J Physiol. 1987; 393: 513-24 and Soslau G, Class R,

Morgan DA, Foster C, Lord ST, Marchese P, Ruggeri ZM. J Biol Chem. 2001; 276: 21173-83), using a calcium sensitive photoprotein (Aequorin), using a platelet activation assay for increasing calcium concentration in platelets ( Johnson PC, Ware JA, Cliveden PB, Smith M, Dvorak AM, Salzman EW. J Biol Chem. 1 985; 260: 2069-76), or 45Ca, a platelet activation assay using elevated calcium concentrations in platelets (Feins tei n MB, Fraser C. J Gen Physiol. 1 975; 66: 56 1 -8 1) and the like. (3) Based on the measurement result of (2), the project to determine whether the test substance has platelet aggregation promotion activity or inhibitory activity Based on the above measurement results, it is compared with the control group to show platelet aggregation promotion activity or platelet aggregation inhibition activity. The substance shows high platelet agglutination activity, which can be judged to have platelet aggregation promoting activity. Conversely-74-200413526, if compared with the control group, it shows a lower platelet aggregation activity, or a test substance that does not show platelet aggregation activity at all. It was determined to have a platelet aggregation inhibitory active substance. The platelet aggregation inhibitory active substance was obtained according to the above screening method, and a single antibody 8F11 against gp44 was obtained. B. Screening method with platelet aggregation promoting activity or inhibiting active substance, including the following engineering methods: (1) The project of contacting gp44 with platelets in the presence of the test substance "S-type substance" and "platelet" as described above " A. " "Gp44" is purified from biological cells expressing gp44 and can be prepared according to the method described in "1." above. "(2) Project for measuring platelet aggregation activity" and "(3) Project for determining whether a test substance has platelet aggregation promoting activity or inhibiting activity based on the measurement result of (2)" are defined as "A." above. 6. Identification of antibody binding sites The binding sites of gp44 and the corresponding anti-gP44 antibodies can be determined according to the methods used in this art (Fujita N, Sato S, Katayama K, Tsui · uo TJ Biol. Chem. 2002; 277: 28706- 13) For example, combining various gene fragments of gP44cDNA with expression vectors such as pET-21a (manufactured by Novagen) and expressing them in E.coli, the reactivity of the expressed polypeptide and anti-gp44 antibody will be investigated, and the fragments showing reactivity will Don't show clips for comparison. In addition, g p 4 4 c DNA-specific mutated genes were combined with expression vectors such as pcDNA vectors (manufactured by in vitro gen) to express them in mammalian cells, and the expressed mutated proteins were tested for reactivity with anti-gp44 antibodies. According to the specific site that does not show reactive mutations, -75-200413526 can also determine the binding site of gp44 and the corresponding anti-gp44 antibody. For example, in the anti-gp44 monoclonal antibody 8F1], the antibody binding site was investigated according to the above method, and the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4 of the amino acid numbers 39 to 44 in the sequence listing were clearly recognized. That is, an antibody capable of recognizing the amino acid sequence shown in amino acid numbers 39 to 44 of sequence number 2 or sequence number 14 in the sequence table has an inhibitory activity against gp44 platelet aggregation. Similarly, for example, in the amino acid sequence of sequence number 4 in the sequence listing, the polypeptide from amino acid number 39 to amino acid number 44 amino acid sequence, such as the amino acid sequence of sequence number 6 in the sequence listing The polypeptide from the amino acid sequence of 46 to 51 amino acid sequence, such as the amino acid sequence of the sequence number 8 in the sequence listing, the polypeptide from the amino acid sequence of the amino acid number 57 to 62 The specifically recognizable antibody is also an antibody that inhibits gp44 platelet aggregation activity. 7. Identification of the site required for platelet aggregation in gp44. The site required for platelet aggregation in the gp44 amino acid sequence can be determined according to the methods used in this art. For example, the gp44 cDNA encoding a specific position of the amino acid is replaced with another amino acid. Various variant genes were introduced into the pcDNA3 vector and expressed on the surface of CHO cells. Platelet-rich plasma fraction (P 1 ate 1 et -richp 1 asma: PRP) was added, and platelet aggregation activity was observed to determine. In this experiment, for example, the amino acid sequence of amino acid number 34 in the protein of the amino acid sequence shown in sequence number 2 in the sequence listing, and the amino acid sequence of amino acid number 34 in the protein of the amino acid sequence shown in sequence number 4 Glycine, amino acid number 4 1 and amino acid number 50 in the amino acid sequence of protein shown in SEQ ID NO: 6 -200413526 Glycine with acid number 5 2 and sequence number] 4 Glycerine with amino acid number 34 in the amino acid sequence of protein shown in 4 is necessary for platelet aggregation activity. Therefore, the above platelet agglutination activity must replace the amino acid sequence mutant protein obtained with amino acids with other amino acids and the mutant protein without platelet agglutination activity. For example, compared with the natural protein with platelet agglutination activity, analyze the platelet aggregation mechanism and add It can be used to investigate platelet aggregation promoting substances and platelet aggregation inhibiting substances. 8. Search for Direct Interaction Substances Modification Another aspect of the present invention includes a drug design method based on the three-dimensional structure of the protein for the purpose of obtaining a gp44 activity inhibitory substance. This type of method is known by inferential drug design method, used to explore the functions of enzyme activity, etc., to search for ligands, cofactors, or compounds that are effectively inhibited or activated by DNA binding. For example, known protease inhibitors of marketed anti-HIV agents. For gp44 three-dimensional structure analysis, X-ray crystallographic analysis and general known methods of nuclear magnetic resonance method can be considered. Moreover, in the search for gp44 function inhibiting substances, computer drug design (CADD) can also be used for design. Furthermore, ^ gp44 activity must have an amino acid, as known in the amino acid sequence of sequence number 2 in the sequence listing, amino acid of amino acid number 34, as in the amino acid sequence of sequence number 4 in the sequence listing, Amino acid of amino acid number 34, such as the amino acid sequence of sequence number 6 in the sequence listing, amino acid of amino acid number 41 or amino acid of amino acid number 50, such as the sequence number of the sequence table In the amino acid sequence of 8, the amino acid of amino acid number 52 is the amino acid shown by the amino acid number 34 in the amino acid sequence of sequence number 14 in the sequence listing; and the anti-gP44 -77-200413526 single The 8F Π of the antibody can recognize the amino acid sequence of the amino acid numbers 39 to 44. It is known that it has the activity of inhibiting platelet aggregation, so it can use the three-dimensional structure near the relevant field. For example, new novel genomic drugs for treating chronic joint rheumatism are known to inhibit low molecular weight compounds of AP-1 (International Patent Application Publication No. W099 / 585 1 5). In these methods, a substance that inhibits gP44 function can be obtained by directly binding to gP44 or inhibiting the interaction of gp44 and other factors. Furthermore, another form is a polypeptide that is polymerized with gp44, that is, a partnership protein of gP44 or a compound that binds to gp44. That is, the present invention is a screening method for partnership proteins and binding compounds related to the regulation of gp44 activity. One form of this screening method is a process that includes gp44 in contact with a test substance and selects a test substance that is combined with gp44. These methods may be, for example, using a purified gp44 and performing an affinity purification method with a protein bound thereto. Furthermore, a partial polypeptide containing the amino acid sequence of an amino acid that is required for the aggregation of g P 4 4 platelets was used. This part of the polypeptide is, for example, in the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing, a polypeptide containing an amino acid number 34 of an amino acid with 6 or more consecutive amino acid sequences, such as the sequence number of a sequence listing In the amino acid sequence of 4, a polypeptide containing an amino acid number 34 of the amino acid sequence of more than 6 consecutive amino acid sequences, the amino acid sequence shown in sequence number 6 in the sequence listing 'containing the amino acid number 4 1 ~ Amino acid number 50 Polyamic acid peptide with 6 or more consecutive amino acid sequences, such as the amino acid sequence of sequence number 8 in the sequence listing, containing amino acid number 5 2 consecutive 6 Polypeptides with more than amino acid sequences, such as the amino acid sequence of sequence number 14 in the sequence listing, -78-200413526 containing 6 consecutive amino acid sequences of amino acid number 34 amino acid Peptides, such as the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing, a polypeptide containing an amino acid sequence of 39 to 44 amino acids. The specific method can be exemplified as follows: gp44 or some of the above-mentioned polypeptides are made to be fused with affinity tags of 6 histidine sequences, and they are fused with the cell extract (preliminarily passed into a nickel-agar sugar column and divided by this column) ), Incubate at 4 t for 12 hours, and then add another nickel-agar sugar carrier to the mixture and incubate for 1 hour. After the nickel-agar sugar carrier was washed with a washing buffer, 100 mM imidazole was added, and the protein in the cell extract specifically binding to gp44 or a part of the polypeptide was dissolved and purified to determine its structure. Based on this, proteins that directly bind to gP44 or some of its peptides can be purified, and proteins that do not have binding activity to gp44 or some of its peptides, but subunits and gp44 directly bind to proteins to form complexes that bind to gp44. : Biotechnology Handbook Series 5 "Research on Transcription Factors" PP2 15-219 (Yangtusha). Other methods can be FarWestern Blot [Special Volume of Experimental Medicine, "New Genetic Engineering Handbook" pp76-81 (Yangtusha)] and a two-hybrid system using yeast and mammalian cells [Experiment Separate volume of medicine, "Handbook of New Genetic Engineering" ρρ66-75 (Journal of Yangtusha), "Chechmate · Mammal · Two-Hybrid System" (manufactured by Promega)] are selected, but are not limited to such methods. When a test substance compound or the like is used, the cell extract may be replaced with a solution containing the test substance. According to this, in order to obtain a partnership protein -79-200413526 that directly or indirectly interacts with gp44, functional screening can be performed using an inhibitory substance that interacts with gp44 and the partnership protein. Specifically, for example, gp44 and glutathione transferase (GST) are made into a fusion protein, and after binding to an anti-glutathione transferase antibody-coated microplate, the biotinylated partner protein is fused to the fusion 'Protein contact' is detected using streptavidinated alkaline dephosphatase bound to the fusion protein. When the biotinylated partner protein is added, the test substance is added, and the fusion protein is selected to bind to the partner protein to promote or inhibit the substance. In this method, a substance directly interacting with the fusion protein or a substance directly interacting with the partner protein can be obtained. When the fusion protein is indirectly bound to the partner protein, and any other factor is involved, such as in the presence of a cell extract containing the factor, the same measurement method is used. In this case, it is also possible to choose a substance that acts on the factor. Furthermore, when the obtained partnership protein has the function of inhibiting gP44 function, it can be screened as a candidate substance having platelet aggregation inhibitory activity according to the documented test method using the gp44 gene expression vector. In addition, when the obtained partner protein has the function of inhibiting gp44 function, the polynucleotide of the nucleotide sequence encoded by these inhibitory factors can be used as gene therapy for diseases caused by platelet aggregation. These polynucleotides, such as amino acid sequence analysis to identify inhibitors, can be obtained from oligonucleotide probes synthesized from nucleotide sequences encoded by the amino acid sequences to screen cDNA libraries and genomic libraries. Furthermore, if a peptide having gp44 function inhibitory activity is selected from an artificial peptide library synthesized at a natural opportunity, the nucleotide sequence encoded by the peptide amino acid sequence can be synthesized by DNA chemistry. -80-200413526 Furthermore, the test substance bound to gp44 or a part of its polypeptide can be used to determine whether it has platelet aggregation promotion activity or inhibitory activity according to the screening method shown in the item "5 ·" above. Cancer-specific expression of 9-gP44 gene Whether the gp44 gene has cancer-specific expression can be determined by comparing the expression of gp44 gene in cancer tissues and normal tissues, or in cancerous and non-cancerous parts of cancer patients. For example, comparing Cancer site and non-cancer site mRNAs of various cancer patients with Cancer Profiling Array II (manufactured by BD Bioscience), comparing the expression levels of gp 36 human homologs, Colon (Rectum), Rectum (Rectum), Smal 1 intestine (small intestine) and cancerous parts of the testis compared with non-cancerous parts to observe the increase in gp36 expression. That is, the gp44 and gP44 genes can be used as diagnostic markers for intestinal and testicular cancer (for example, S e mi i η 0 m a). Furthermore, when the expression of gp44 in cancerous and non-cancerous or metastatic sites of the same cancer patients was compared, the expression of colorectal cancer was increased compared with non-cancerous sites, and the tendency of increased expression was also observed in metastatic sites. That is to say, the expression of gp44 gene is related to the promotion of colorectal cancer metastasis. Taking gP44 expression as an index, specifically, using at least one of the following items (1) to (6) 'as a diagnostic kit for colon cancer and / or testicular cancer. In addition, the kit can detect not only the presence or absence of cancer from tissues, but also cancerous and non-cancer sites in tissues, as well as metastatic sites. (1) A part of the nucleotide sequence of the nucleotide sequence described in the sequence number 1, 3, 5, 7, or 13 of the sequence list to the specificity of all nucleotides] 5 to 30 base lengths Consecutive oligonucleotide primers; -81-200413526 (2) hybridize with a polynucleotide of the nucleotide sequence described in sequence number i, 3, 5, 7 or [3] of the sequence listing under severe conditions, Continuous polynucleotide probes capable of detecting more than 15 nucleotides of the polynucleotide; (3) Immobilized reagents in which the substance described in any one of (1) or (2) has been immobilized. (4) An antibody capable of specifically binding to the amino acid sequence polypeptide described in the sequence number 2, 4, 6, 8 or M of the sequence listing; (5) Obtained by binding to the antibody described in (4) Secondary antibody; (6) (4) or (5) the immobilization reagent of the substance described in any one of the substances. 10. Differences in gp44 gene and gp44 cancer tissue-type expression levels (1) gp44 gene expression levels The method described above was the same as described in "9. gp44 gene cancer-specific expression", using Cancer Profiling Array II (manufactured by BD Biosciences) ) And Cancer Profiling

Array (manufactured by BD Biosciences) membrane, investigated the relationship between lung cancer tissue type and gp36 human homolog gene expression level, and found that the expression of gp36 was mostly increased in patients with squamous cell carcinoma and adenocarcinoma. That is, the gp44 gene has a different expression level in cancer tissues, and its expression level increases in flat epithelial cancer, but it does not increase in adenocarcinoma. Therefore, by detecting the expression level of gp44 gene, when the expression level increases, it can be judged that there is a squamous epithelial cancer, and when the expression level does not increase, it can be judged that there is no squamous epithelial cancer. That is, the gP44 gene can be used as a target gene for squamous cell carcinoma. (2) gp44 expression level In human lung cancer tissues and normal lung tissues, gp36 human homologs were expressed by immunohistochemical staining. It was found that gp36 expression was -82-200413526 in flat epithelial cancer, and there was no gp36 expression in adenocarcinoma and normal lung. Therefore, it is shown that the expression level of gp36 increases in squamous cell carcinoma. That is, gp44 has a different expression level in cancer tissues, an increase in expression levels in flat epithelial cancers, and no increase in expression levels in adenocarcinomas. Therefore, by detecting the expression level of gp44, when the expression level increases, it can be judged that there is a squamous epithelial cancer, and when the expression level does not increase, it can be judged that there is no squamous epithelial cancer. That is, gp44 can be used as a standard for epithelial squamous cell carcinoma. (3) The gP44 and gp44 genes were detected in the squamous cell carcinoma type, and the expression level of the squamous cell carcinoma was increased. Therefore, when gp 4 4 and / or g p 4 4 gene expression levels are measured, it is possible to determine the squamous cell carcinoma of the cancer tissue type. A kit for detecting flat epithelial cancer, specifically. It is selected from at least one of the following (a) to (f). (a) A part of the nucleotide sequence of the nucleotide sequence described in the sequence number 1, 3, 5, 7, or 13 of the sequence list to a length of 15 to 30 bases of the entire specific width can be continuous Oligonucleotide primers; (b) Detects the polynucleoside by hybridizing it with the nucleotide sequence described in sequence number 1, 3, 5, 7, or 13 of the sequence listing under severe conditions Consecutive polynucleotide probes of 15 nucleotides or more in acid; (c) Immobilized reagents in which the substance described in any one of (a) or (b) has been immobilized. (d) an antibody specifically binding to the polypeptide of the amino acid sequence described in sequence number 2, 4, 6, 8, or 14 of the sequence listing; (e) two of which binds to the antibody described in (d) Secondary antibody; (f) Immobilization reagent 200413526 1 1 .gp44 cancer-promoting effect of any of the substances described in (d) or (e), g P 44 has platelet aggregation promoting function. Gp 4 4 expressing cells are administered to the blood vessels of non-human mammals. Due to the agglutination function of g p 4 4 platelets, the administered cells will form clumps. The clot is blocked in the capillaries, and a nodule (tumor) is formed there. The nodules thus formed are called metastatic nodules. That is, when gp44-expressing cells are administered, nodules are produced in non-human mammals. For example, CHO cells expressing the gp44 human homolog gp36 gene are administered intravenously to the tail vein of mice, compared with the case where gP36 cells are not expressed, which can increase the number of nodules. Metastatic nodules can be observed and administered to human homologous cells expressing gp36 to various tissues such as the lung, liver, and lymph nodes of non-human mammals. For example, when CHO cells expressing gp36 are administered intravenously, the lungs can be confirmed by visual observation generate. 12. Using the number of metastatic nodules to determine the method of screening for platelet aggregation promoting activity or platelet aggregation inhibiting active substances. When a large number of cells expressing gp44 are administered to non-human mammals, nodules can be caused by platelet aggregation and cell aggregation. Therefore, based on the results of measuring nodules, it can be determined whether the test substance has platelet aggregation promotion activity or blood platelet aggregation inhibition activity. Therefore, by measuring the number of nodules, it can be determined whether the test substance is a cancer metastasis inhibiting substance or a substance that promotes cancer metastasis. Substances with these properties can be specifically obtained by the following screening methods. A. Screening method including the following projects (1) to (3): -84-200413526 (1) administering gp44-expressing cells to non-human mammals in the presence of the test substance; (2) to (1) Determine the number of nodules from non-human mammal tissue; (3) Based on the measurement result of (2), determine whether the test substance has platelet aggregation promotion activity or platelet aggregation inhibition activity. B · Screening method including the following projects (1) to (3): (1) administering gp44-expressing cells to non-human mammals in the presence of the test substance; (2) lactating from non-humans in (1) Determine the number of nodules in animal tissues. Lu (3) Based on the measurement results in (2), determine whether the test substance has cancer metastasis inhibiting activity or cancer metastasis promoting activity. The screening method used the test substance and gp44-expressing cells as described in "1. Screening method using cultured cells" above. When gp44-expressing cells are administered to a non-human mammal in the presence of the test substance, the test substance can be judged to have platelet inhibition activity when the number of nodules is reduced compared to the case where the test substance is not administered. When the number of metastatic nodules decreases, the test substance can be determined to be a cancer metastasis inhibiting substance. © Furthermore, when a cell expressing gp44 is administered to a non-human mammal in the presence of the test substance, the test substance can be judged to have platelet aggregation promotion activity when the number of nodules is increased compared to the case where the test substance is not administered. When the number of metastatic nodules increases, the test substance can be determined to be a cancer metastasis-promoting substance. 1 3 · Diagnostic methods for diseases related to platelet agglutination or platelet dysfunction. Extract mRNA or polypeptides and reagents from specimens obtained from patients and normal persons, according to the methods described in "3." or "4." above. By measuring the expression level of the mRNA related to the present invention-85-200413526 or the polypeptide of the present invention, the expression level from a patient specimen and a normal human specimen can be compared to diagnose platelet aggregation or platelet dysfunction-related diseases. That is, when the expression level of the mRNA related to the present invention or the polypeptide related to the present invention in the specimen obtained from the patient is higher than the expression level of the mRNA or the polypeptide in the specimen obtained from the normal person, it can be diagnosed. Platelet agglutination promotes the disease or is more likely to develop the disease. Conversely, when the expression level of the mRNA related to the present invention or the polypeptide related to the present invention in the specimen obtained from the patient is lower than the expression level of the mRNA or the polypeptide in the specimen obtained from the normal person, it can be diagnosed Platelet aggregation inhibits the disease or the likelihood of developing the disease. Furthermore, the expression level of m R N A of the present invention or a polypeptide related to the present invention is measured by long-term observation. The diagnostic method of the present invention is also applicable to mammals according to the specimens obtained from the tested animals and normal animals; in the above, "normal person" or "normal animal" means a person or animal without abnormal platelet aggregation activity. 1 4 · For the measurement of a platelet aggregation-promoting or platelet-agglutination-inhibiting active substance A platelet aggregation-promoting or platelet aggregation-inhibiting substance can be detected using a kit containing at least one of the following substances (1) to (5). (1) A part of the nucleotide sequence of the nucleotide sequence described in the sequence number 1, 3, 5, 7, or 13 of the sequence list to a length of 15 to 30 bases of the specificity can be used. Consecutive oligonucleotide primers; (2) a polynucleotide that hybridizes with a nucleoside-86-200413526 acid sequence described in sequence number 1, 3, 5, 7 or 13 of the sequence listing, Can detect continuous polynucleotide probes of 5 or more nucleotides of the polynucleotide; (3) Amino acid sequences described in sequence number 2, 4, 6, 8 or 14 of the sequence listing Polypeptide-specific antibodies; (4) Secondary antibodies obtained by binding to the antibodies described in (3); (5) Immobilization of the substances described in any of (1) to (4) Reagent. The primers described in (1) above are designed in accordance with the information using commercially available primer design software (for example, Wisconsin GCG package Version 10 · 2) and the like according to the polynucleotide base sequence of the present invention. The probe described in the above (2) is not particularly limited except for a polynucleotide which can specifically hybridize with the polynucleotide of the present invention. The length of these probes is preferably 100 to 1500 bases, more preferably 300 to 600 bases. Such primers and probes can also be appropriately labeled (for example, enzyme-labeled, radio-labeled or fluorescent-labeled, etc.), and linkers can also be added. Among the immobilization reagents described in (5), the primers described in (1) or the probes described in (2), the primers or probes can be fixed by a glass plate, nylon, or other methods. Fixedly made. The reagent immobilization method can be carried out in the same manner as described in (Immobilization reagent preparation) in the above (1-2-5). The antibodies described in the above (3) and (4) can be performed in the same manner as described in the above 12 (4-3). The antibody can also be appropriately labeled (for example, enzyme label, radioactive label, fluorescent label) by the same method described in (4_4) above. Furthermore, among the immobilization reagents of (5), the antibody immobilization reagents can be performed by the method described in 1. (4-2) above as a guideline -87-200413526. In this set, the above methods can be used as a guideline. As the detection of the polynucleotides of the present invention or the peptides of the present invention, the screening of platelet aggregation promoting or inhibiting active substances is performed. Or platelet aggregation promotes or inhibits the diagnostic use of the disease. 15. Pharmaceutical composition The polynucleotide or peptide of the present invention as an active ingredient of the pharmaceutical composition of the present invention can be prepared according to a conventional method, for example, 'culturing a cell that produces the polynucleotide or peptide, since Cells are extracted and purified, or the polynucleotide is amplified in a test tube, or the polypeptide is expressed. When the obtained antibody is used as a medicine for humans, it is desirable to prepare a human-type anti-human gp44 antibody due to the problem of antigenicity. The human type anti-human gp44 antibody can be prepared by the following method. ① Human lymphocytes were collected from human peripheral blood or spleen, and infected with gp44 as an antigen in vitro and in the presence of IL-4. Infected human lymphocytes and hybrid fusion tumors (Heterohybrid oma) K6H6 / B5 (ATCC CRL 1 823) to perform cell fusion to produce a fusion tumor producing a human type anti-gp44 monoclonal antibody. An antibody neutralizing human gp44 activity was selected from the antibodies produced by the fusion tumor to obtain a human type anti-human gP44 antibody. The human-type anti-human gp44 monoclonal antibody obtained from the above can be highly affinityd by the following method. The human type anti-human gP44 monoclonal antibody obtained according to the above method introduced mutations in its CDR domain (specifically CDR-3) at the opportunity. The mutated antibody was expressed as a phage, and a phage display method was performed using a human gp44 immobilized plate to select a phage -88-200413526 that closely bound to the human gP44 antigen. This bacteriophage is bred in E. coli and has a high affinity CDR amino acid sequence determined from its base sequence. The genes encoding the obtained human-type anti-gp44 monoclonal antibodies were combined with expression vectors for mammalian cells generally used to express human-type anti-human gp44 monoclonal antibodies. From this antibody, human gP44 biological activity was neutralized, and a high-affinity human type anti-gp44 monoclonal antibody was selected to obtain a desired high-affinity antibody. ② Using Balb / c mice, a mouse-type anti-human gp44 monoclonal antibody was prepared according to the usual method (Kohler et al .: Nature 256, p. 49 5-497, 1 97 5).

For human gp44 biological activity, select a high affinity human type anti-gp44 monoclonal antibody. The CDR-domain (CDR-1, 2 and 3) of this high-affinity mouse-type anti-human gp44 monoclonal antibody was transplanted into human IgG by CDR-grafting (Winter and Milstein: Nature 349, p293-299, 1991). In the CDR field, high affinity antibodies are produced. ③ Transplant human terminal hemolytic lymphocytes into severe combined immune defiiency (SCID) mice. To produce human type C antibodies in SCID mice (Mosier DE et al. · Nature 335, p 2 5 6-259, 1988; Duchosal MA et al ·: Nature 355, P25 8-262, 1 992), to Human gp44 was used as an antigen to infect the mouse, and screening was performed to select a specific human type monoclonal antibody against human gp44. The obtained lymphocytes were subjected to cell fusion with the hybrid fused tumor K6H6 / B5 (ATCC CRL 1 823) of mouse and human in the same manner as in the above-mentioned ①, and the obtained fused tumor was screened to obtain a fused tumor producing a human monoclonal antibody of interest. From the above-mentioned cultured fusion tumors, a large amount of human type monoclonal antibodies of interest are produced. -89-200413526 Furthermore, the gene (cDNA) encoded by the human type monoclonal antibody for the purpose of selection is genetically engineered in combination with an appropriate vector and expressed in various animal cells, insect cells, or E. coli hosts. Manufacture of recombinant human monoclonal antibodies. Moreover, the anti-gp44 monoclonal antibody obtained by this method can obtain an antibody that neutralizes the biological activity of gP44. The neutralized gp44 bioactive antibody thus obtained, and gP44 bioactivity in vivo, i.e., due to its cell platelet aggregation inhibitory activity, can be used as medicine, particularly for the treatment and / or prevention of diseases related to platelet aggregation. The neutralizing activity of the gp44 biological activity of the in vitro anti-gp44 antibody is determined by measuring the platelet aggregation inhibitory activity of gp44 overexpressing cells according to the method described in 7. above. In vivo experimental animals are aimed at the treatment and / or prevention of platelet aggregation-related diseases using anti-gP44 antibodies. For example, the same gp 4 4 antibody is administered to transgenic animals with g P 4 4 overexpression to determine platelet aggregation-related diseases. The changes are determined. Furthermore, according to the above method, an antibody that promotes gP44 activity can also be obtained. This antibody, because of its platelet aggregation promoting activity, can be used as medicine, especially as a therapeutic and / or preventive agent for diseases related to platelet dysfunction. The g p 4 4 promoting activity of the anti-g P 4 4 antibody in vitro can be determined by measuring the platelet aggregation promoting activity of the overexpressing cells according to the method described in 7. above. In vivo experimental animals use platelet aggregation with anti-gP44 antibodies to treat diseases and / or preventive effects. For example, the same gP44 antibody is administered to a transgenic animal with insufficient expression of gp44 to determine the changes in disease related to platelet aggregation. When the polynucleotide is used as an active ingredient, a gene therapy agent -90-200413526 is usually introduced into cells in a conventional manner. For example, these methods may use a viral vector method or a non-viral introduction method (Nikkei Science, April 1994, pp. 20-45, Journal of Experimental Medicine, 12 (1 5) (1 994), The experimental sidebook "Basic Technology of Gene Therapy" Yangtusha (I 9 9 6)) and so on. Methods for introducing genes by viral vectors, for example, DNA viruses such as retrovirus, adenovirus, adenovirus, herpes virus, vaccinia virus, pox virus, polio virus, or RNA virus, encoded with TR4 or TR4 Method of DNA combination introduction. It should be retrovirus, adenovirus, adenovirus or vaccinia virus. The non-viral gene introduction method is: a method for directly expressing plastids into the tissue (DNA vaccine method), microliposome method, Lipofection method, microinjection method, calcium phosphate method or electroporation, etc., preferably , DNA vaccine method or microliposome method. In addition, the gene therapy agent is actually used as medicine to in vivo DNA (invivo), which can directly introduce DNA into the body, or to take out cells from human organs or tissues and introduce the DNA into the cell, and then implant the cell into the body. In vitro method (e X viv 〇) (Nikkei Science, April, 1994, pp. 20-45, Intermonthly _ Pharmaceutical Affairs, 36 (1), 8723-48 (1994), Experimental Medicine Journal, 1 2 (1 5) (1994)). For example, when the gene therapy agent is administered in vivo, it can be administered by an appropriate administration route from a vein, artery, subcutaneous, intradermal, or intramuscular (including muscle) according to a disease or symptom. Furthermore, when administered in vivo, the gene therapy agent can be administered in accordance with a general injection or the like, and a conventional carrier can be added if necessary. In addition, when it is in the form of microlipids or membrane-fused microlipids (Sendai virus-microlipids, etc.) -91-200413526 ′, it may be a microlipid formulation such as a suspending agent, a refrigerant, or a centrifugal concentrated refrigerant. A nucleotide sequence complementary to the nucleotide sequence of sequence number 1, 3, 5, 7, or 13 or a partial sequence thereof is used as antisense therapy. Antisense molecules usually use 15 to 30 mer DNA, or stable DNA derivatives such as phosphorothioate, methyl phosphonate or morpholinyl derivatives, and stable RNA derivatives such as 2-0-alkyl RNA. . Such antisense molecules can be introduced into cells by microinjection, using microlipid-containing capsules or vectors containing antisense sequences, etc., in a conventional manner within the technical scope of the present invention. These antisense therapies can reduce the activity of the protein encoded by the nucleotide sequence shown in the sequence number 1, 3, 5, 7, or 13 of the sequence listing as a platelet aggregation to promote the treatment of related diseases. A pharmaceutical composition containing the above-mentioned antisense oligonucleotide is prepared by a conventional method, such as mixing with a pharmaceutically acceptable carrier (for example, thunderton p h a r m a c e u t i c a 1 S c i e n c e s). The seal δ tolerance is a platelet aggregation-promoting activity-related disease that contains the nucleotide sequence of the nucleotide sequence shown in sequence number i, 3, 5, 7, or i 3 and whose gene product is abnormal in activity. Oligonucleotide pharmaceutical composition. The effective amount varies depending on the patient's state, weight, gender, and age, and different administration methods, such as subcutaneous, local, oral, and intramuscular (including muscle). For example, when administered intravenously, from 0.2 to 0.2 mg / kg / hour for 2 hours, and when administered subcutaneously, from 1 to 200 mg / m2 / day. When the polypeptide is used as the active ingredient, the administration form is, for example, lozenge, capsule, granule, powder, syrup, or the like, or parenteral such as injection, suppository, or the like. These formulations can be mixed with appropriate excipients (for example, lactose, sugar 'glucose', mannose, sorbose and other sugar derivatives; corn starch, mayonnaise-92-200413526 potato starch 'α starch, dextrin and other starch derivatives; crystals Cellulose derivatives such as cellulose; acacia gum; polydextrose; polytriglucose and other organic excipients; and light silicic anhydride, synthetic derivatives such as aluminum silicate, calcium silicate, magnesium aluminum silicate, and other derivatives ; Phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; inorganic excipients such as sulfates such as calcium sulfate; lubricants (for example, stearic acid such as stearic acid, calcium stearate, and magnesium stearate) Metal salts; Talc; colloidal silica; propolis, cetyl wax, etc; boric acid; fatty acids; sulfates such as sodium sulfate; ethylene glycol; fumaric acid; sodium benzoate; DL leucine; fatty acid sodium salt; dodecyl Sodium sulfate; Dodecyl sulfate such as sodium dodecyl sulfate, magnesium dodecyl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; and the above starch derivatives) 'Binders (for example, hydroxypropyl fiber Cellulose, hydroxypropyl methylcellulose, polyethylene Pyridone, polyethylene glycol, and the aforementioned excipients, etc.), disintegrating agents (eg, low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, internally cross-linked carboxymethyl fiber Cellulose derivatives such as Sodium Sulfate; Carboxymethyl Starch, Sodium Phosphate, Chemically Modified Starch Cellulose such as Cross-linked Polyvinylpyrididone), Stabilizers (Methyl Paraben, Parabens such as propyl parabens; alcohols such as chlorobutanol 'T alcohol, phenethyl alcohol; benzane ammonium chloride: phenols such as phenol, cresol; thimerosal; dehydroacetic acid; and sorbic acid ), Flavoring agents (for example, customary sweeteners, sour ingredients, flavors, etc.), additives such as diluents, are manufactured according to conventional methods. The amount used varies according to symptoms and administration methods, such as the lower limit of 0.1 mg / kg body weight (preferably 0.1 ing / kg body weight) and the upper limit of 100 mg / kg body weight (preferably 10 mg / kg) Weight), the lower limit of each administration is 0.00 1mg / kg body weight (preferably 0.0 1mg / kg body weight), the upper limit] 0mg / kg body weight (-93-200413526 should lmg / kg body weight), daily points] ~ several times Depending on the symptoms. When used as an active ingredient of the antibody, it can be formulated for oral or parenteral administration. The pharmaceutical composition may be in the form of an injectable solution containing a drop, a nasal preparation, a nasal preparation, a sublingual preparation, a percutaneous absorption preparation, and the like. Monoclonal antibodies are unstable due to their high-molecular-weight proteins adsorbing on glass containers such as vials and syringes. They are liable to lose their activity due to various physical and chemical factors, such as heat, pΗ, and humidity. Stabilizers can be added. Stabilized forms such as pH adjusters, buffers, solubilizers, and surfactants are formulated. Stabilizing agents include amino acids such as glycine 'alanine, sugars such as polydextrose 40 and mannose, sugar alcohols such as sorbitol' and mannitol, xylitol, and the like. You can also use the above two types. Such stabilizers are added in an amount of 0.01 to 100 times the weight of the antibody, preferably 0.1 · 10 times the amount of the antibody. The buffering agent is, for example, a phosphoric acid phosphate buffer solution, a citric acid buffer solution, or the like. The amount of the buffering agent to be added, for example, in a liquid preparation or a freeze-dried preparation, is preferably 110 mM with respect to water. The surfactant is preferably polysorbate 20 'PluronicF-68, polyethylene glycol, etc., and particularly suitable is polysorbate 80, or two or more kinds can be used. The surfactant can be added in an amount of 0.0010% to the weight of water after the liquid preparation or lyophilized preparation is redissolved. When used as a medical or animal injection, the osmotic pressure ratio is preferably 1 to 2. The osmotic pressure ratio can be adjusted by reducing sodium chloride during formulation. The content of the antibody in the preparation may vary depending on the applicable disease, the applicable administration route, etc. The amount of human-type antibody administered to humans depends on the affinity of the antibody for human gP44, that is, the dissociation number of human gp44 (Kd 値) When the affinity is high (Kd 値 is low), when the dosage of human is low, the drug effect can be exerted. The human type anti-gP44 antibody is administered at about 0.1 to 100 mg / kg in humans, and it is administered at a dose of 1 to 94-200413526 within 1 to 30 days. [Embodiments] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the examples. Furthermore, the operations related to genetic manipulations in the following examples are not limited to those shown, and may be "Molecular Cloning" (Sambrook, J., Fritsch, EF, and Maniatis, T. Cold Spring Harbor Laboratory Press issued in 1989), or when using commercially available reagents and kits according to the instructions on the market. Lu (Example 1) Detection of gp3 8P expression in cancer cells caused by platelet aggregation (1) Cell culture of mouse brain capillary endothelial cells MBEC-4 (Tatsuta T, Naito M, Oh-hara T, Sugawara I, Tsuruo T J Biol Chem. 1992; 267: 203 8 3-9 1), mouse lymph node stromal cells CA-12 (Fujita N,

Kataoka S, N a i t ο M, H e i k e Y, B o k u N, N ak aj i n a M,

TsiimoT. Cancer Res. 1 993; 5 3: 5022-7), mouse colon cancer cell book colon26 and its aromatic strains NL-14, NL-17, NL-44 (Watanabe M,

Okochi E, Sugimoto Y, Tsuruo T. Cancer Res. 1988; 48: 64 1 1-6), mouse B 1 6 melanoma cell strains b 16 / F1 and B16 / F10 (Watanabe M, Sugimoto Y, Tsuruo T. Cancer Res. 1 990; 50: 665162) 'and Chinese golden rat ovary fibroblasts (CHO) are contained in 2mM L-Melanine, 100 # g / mi anti-namycin (1 ^ 1 ^ 11 ^ (: 丨 11) and 10% heat-deactivated? 83 feet? 1 ^ 11640 medium, cultured at 37 -95-200413526 ° C, 95% humidity, 5% C02. Mouse bones Bud cell MC3T3-E1 (Sakamoto, S. and Sakamoto, M. Biochem. Int., 9, 5 bu 58, 1984), human fetal kidney cells 293T containing amine acid,] 00 // g / m] anti-mold And 10% heat-deactivated FBS in DMEM medium, cultured at 37 ° C, 95% humidity, 5% C02. Phage in the normal abdominal cavity of mice can be obtained according to the following method. BALB / c females at 8 weeks Mice were injected with 1 mL of a 3% ethyl mercaptan solution into the abdominal cavity. After 4 days, 5 mL of PBS was injected. The abdominal fluid was carefully absorbed from the abdomen. After centrifuging the cells, the cells were washed twice with PBS to obtain cells (see Gallily R, Feldman M., Immunology, 1 9 6 7 Feb; 1 2 (2): 197-206) ° ( 2) Western blot method (MBEC-4, CA-12, MC3T3-E1 cells) Each 1 X 107 cell of MBEC-4, CA-12 and MC3T3-E1 cultured according to the method described in (1), suspended in 500 // 1 lysate B (25mM Tris (ρΗ7.4), 50mM NaCl, 0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% SDS), and stir once within 10 minutes, Place on ice for 1 hour. After quantifying protein by BCA-protein assay (manufactured by Pieree), add SDS sample buffer (containing 2-thiohydroethanol) 20 // 1, and boil for 5 minutes, using 10% polypropylene% Amine gel was used for electrophoresis. It was transferred to PVDF membrane, and after blocking, it was a monoclonal antibody that specifically reacted with gp44, namely 8F11 antibody (10 # g / ml) (Watanabe et al, Cancer Res, 1991), / 3-muscle. The protein antibody (manufactured by Sigma) was reacted at room temperature for 1 hour, washed with PBS containing 0.05% Tween20 for 10 minutes and 3 times, and reacted with HRP-labeled secondary antibody. After washing in the same way, ECL detection was performed. The results are shown in Figure 1A. Figure 1A shows that the 8F1 1 antibody recognizes about 44 kDa protein on -96-200413526 MC3T3-E1. In addition, 8F1 1 antibody-recognizable antigens include mouse brain capillary endothelial cells MB EC-4, mouse lymph node stromal cells CA-12, and mouse normal intraperitoneal phage. (3) RT-PCR (NL-17, NL-14, NL-44, B16F1, B16F10 cells) (NL-17, NL-14, NL-44, B16F1, B16F10 cells to be cultured according to the method described in (1) For each lxlO7 cell, RNA was prepared using the RNA PCR group (AMV) Ver. 2.1 (Takara Shuzo Co.). RT-PCR was performed according to a known method (Morinaga Y, Fujita N 5 Ohishi K) Tsuruo T. Int J Cancer, 1997, 71 422-8). The amount of mRNA was corrected according to the expression of / 3-actin antibody in the control group. The β-actin synonymous primers in the primers used were 5, gatatcgctgcgctggtcgtcgac-3 '(sequence listing sequence No. 15) Polynucleotide of the nucleotide sequence shown in the figure, the antisense primer is 5, caagaaggaaggctggaaaagagc-3 '(sequence list sequence number 16) The synonymous primer of the polyglylic acid' gp 3 8 P sequence is not 5'-tcaagatgtg gaccgtgccagtg-3 '(sequence list sequence number 9) a polynucleotide of the nucleotide sequence shown in Figure 1 (Figure 1B) or 5'-ctcaagcttc aagatgtgga ccgtgccagt g-3' (sequence list sequence number 1) 1) Polynucleotide of the nucleotide sequence shown (Figure 1C), the antisense primer is 5'-cgccatgggt catcttcctc cac-3 '(sequence Table sequence number 10) Polynucleotide of the nucleotide sequence shown in Fig. 1 (Figure 1B) or, 5? -Gaggaattcg ggcgagaacc ttccagaaat c-3, (order 歹 ij list 歹 ij number -97-200413526 code 1 2) Polynucleotide of the nucleotide sequence shown (Figure 1C). Results are shown in Figures 1B and C. Figure 1B shows gp44 (mouse colorectal cancer cell ( C ο 1 ο η 2 6) and its adjacent strains) and gp 4 0 (when mouse melanoma cells) express the full-length mRNA that recognizes gp38P. Furthermore, the mRNA expression of gp3 8P in the large intestine of normal mice The amount is lower than that of cancer cells. Furthermore, Figure 1C shows that the 8F1 1 antibody Western blot method shows that compared with NL-17 cells, the platelet agglutination capacity of NL-14 cells is lower and the expression of gP44 is lower. Less, it is known that the mRNA expression level of gp3 8P is also small, which was confirmed by RT-PCR method. Among them, the 8F11 antibody is a molecule that can recognize gp38P (or gP3 8). (Example 2) The flow cell technology is represented by SEQ ID No. 13 The nucleotide sequence of the nucleotide sequence was taken from the cDNA of gp3 8P, the mRNA of NL-17 cells was used as a template, and the synonym primer was 5'- ctcaagetteaagatgtggaccgtgccagtg-3 '(sequence list sequence number Code 17) The polynucleotide of the nucleotide sequence shown in FIG. 5 was antisense primer 5′-ttacttgtcgtcatcgtcUtgtagtcgggcgagaaccttccagaaatc-3 (SEQ ID NO: 18) and the polynucleotide was subjected to PCR. The obtained PCR product was purified by Concert Rapid PCR Purification Syslem (manufactured by Gibco) and adhered with a p3T vector (manufactured by MoBiTec) cut with Xcml to obtain p3T / gp38P. Then P3T / gP38P was cut off with Hindlll and Notl, and adhered to the EcoRI-Notl site of the PcDNA3 carrier -98-200413526 (manufactured by Invitrogen) to obtain pcDNA3-gp3 8P. The resulting pcDNA3-gp38P plastids were introduced into CHO cells and selected with G4 18 (1 mg / ml) to establish a CHO / gp38P cell line that constantly expressed gp38P. After CHO cells and CHO / gp38P cells were cultured according to the method described in Example 1, they were treated with trypsin, washed with PBS, and recovered by centrifugation. To each 1 × 10 6 cells, 8F 1 1 antibody 1 // g was added and reacted on ice for 1 hour. After washing with PBS, anti-mouse IgG-FITC (manufactured by CapPel) was diluted 100-fold, 50 μl / sample was added, and the reaction was performed on ice for 30 minutes. After washing with PBS, it was measured by FACS Caliber (Becton Dickinson). The results are shown in Figure 2A. 8F1 1 antibody can react with samples derived from CHO / gp38P cells, but not with samples derived from the parent strain CC0, showing that 8F1 1 antibodies can react with gp44 and g38P. It is shown that gP38P is the corresponding substance of gp44. (Example 3) Western blot method. CHO cells with gene introduction were selected from G418 (1 mg / mL), and a transgenic strain (CHO / Mock) with constant introduction of null genes was obtained. The human homolog gp 3 6 encoding gene of the nucleotide sequence shown in SEQ ID NO: 7 was taken from the human lung c DNA library (manufactured by Cosmobio) as a template, and 5'-ctcaagcttgaacgatgtggaaggtgtcagc-3 '(sequence listing sequence number) was used as a template. 19) The nucleotide sequence shown in the nucleotide sequence, antisense primer, 5'-gaggaattcgggcgagtaccttcccgaca-3 '(sequence 歹 ij table sequence 歹 [J number 20) was subjected to PCR. The obtained p c R product uses -99-200413526

Purified by Concert Rapid PCR Purification System (manufactured by Gibco), cut ends with Hindlll and EcoRI, and pcDNA3-FLAG cleaved with existing Ecol and Hindlll (the EcoRI-Notl site of pcDNA3 vector (manufactured by Invitrogen) is inserted into the FLAG-tag sequence) The vector was bound to obtain pcDNA3-gp36. PcDNA3-gp36 was used to establish a stable transfectant (CHO / gp36). Introduced into human fetal kidney cells 293T cells to temporarily express pcDNA3-gp38P, pcDNA3-gp36, pFLAG-CMV-2-gp38P (using pcDNA3-gp38P as a template for PCR, and in the EcoRI position of Sigma's pFLAG-CMV-2 vector The PCR products were adhered to prepare each plastid with the FLAG-tag gp38P-expressing plastid) gene at the N-terminus. CHO cells, CHO / gP38P cells, CHO / gp36 cells, and CHO / Mock cells have 293T cells that temporarily express the gp38P-encoding gene of the nucleotide sequence shown in SEQ ID NO: 13 and temporarily express the nucleotides of SEQ ID NO: 7 The 29 3T cells encoding the sequence gp36 gene were cultured according to the method of Example 1, and each 1 X 107 cell was collected and suspended in 5 00 // 1 lysate B (25mM Tris (ρΗ7.4), 50mM NaCl, 0.5% Sodium deoxycholate, 2% NonidetP-40, 0.2% SDS), stir once within 10 minutes, and place on ice for 1 hour. After quantifying the protein by the B C A-protein assay (manufactured by Pierce Corporation), add SDS sample buffer (containing 2-thiohydroethanol) 20 // 1, boil for 5 minutes, and perform electrophoresis using 10% polypropylene ammonium gel. It was transferred to a PVDF membrane, and after blocking, it was reacted with 8F11 antibody (10 // g / ml) and / 3-actin antibody (manufactured by Sigma) at room temperature for 1 hour. Wash with 0.05% Tween20 in PBS for 10 minutes and 3 times, and react with HRP-labeled secondary antibodies. After washing in the same way, ECL detection was performed. -100- 200413526 The results are listed in Figure 2B. The 8F1 1 antibody recognizes about 44 kDa protein of CHO / gP38P cells, but does not react with the mother strain, the control strain, and the human homolog gp36 gene expression strain. Furthermore, it was confirmed that in response to the introduction of the approximately 44 kD a protein temporarily expressing the gp38P gene in human 29 3T cells. In other aspects, it was confirmed that the transgenic strain gp: 38P could recognize the antigen of the 8F1 1 antibody by reacting with cells that did not introduce the gp36 gene of a homologue, suggesting that it was not a secondary protein expression accompanied by gP38P expression. (Example 4) Immunoprecipitation-Western blotting method After CHO cells and CHO / gp38P cells were cultured according to the method of Example 1, each cell was collected 1 × 107 and suspended in a 500 // 1 lysate (25mM Tris (ρΗ7 · 4), 50mM NaC, 0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% SDS), stir once within 10 minutes, and place on ice for 1 hour. Immunoprecipitation of CHO mother strain and CHO / gp3 8P cell lysate containing FLAG-tag using 8F11 antibody, anti-FLAG antibody, normal mouse antibody (for control group), 8 F 1 1 antibody or recognizable g P Anti-gp 4 4 monoclonal antibody to 4 4 is 20A1 1 antibody (refer to Watanabe M, Okochi E,

Sugimoto Y, Tsuruo T. Cancer Res. 1988; 48 · 6411-6), followed by Western blotting method. 8F11 antibody, anti-FLAG-M2 antibody (manufactured by Sigma), and a normal mouse IgG antibody (NR IgG; Sigma) 10 // g were used in the control group, together with protein G-agarose 4B gel (manufactured by Zymed) 1 〇 // 1 react on ice for 1 hour, wash with PBS, add 100 V 1 of lysate for each cell, and react on ice for 1 hour. After washing with PBS, add 20 1 SDS sample buffer (containing 2-thiohydroethanol), and boil.

For 5 minutes, electrophoresis was performed using a 10% polypropylene gel. Transfer to PVDF-101-200413526 membrane, and block with 8 F 1 1 antibody (1 0 // g / m 1), 20 A 1 1 antibody (culture 淸) at room temperature for 1 hour. The PVDF membrane was washed with 0.05% Tween20 in PBS for 10 minutes and 3 times to react with anti-mouse IgG-HRP (manufactured by DAKO). After washing, perform ECL-detection. The results are shown in Figure 2C. Among them, those using the 8F] 1 antibody in the Western blot method are listed on the left of Figure 2C, and those using the monoclonal antibody 20A1 1 are listed on the right of Figure 2C. The immunoprecipitated gP38P with anti-FLAG antibody can recognize 8F11 antibody and 20A1 1 antibody, which proves that the molecule recognized by gP44 is the same as gP38P. (Example 5) Platelet agglutination experiment Blood was collected from mice using heparin, centrifuged at 900 rpm for 10 minutes, and platelet-rich plasma (PRP) was collected. PRP was diluted to 3 times the volume with PBS, and PRP was used for platelet aggregation. Cells used for platelet agglutination were recovered by trypsin treatment and frozen overnight in a cell bank. In fact, the mouse PRP was dispensed into 200 // 1, 2 × 10 7 cells / ml of the CHO mother strain and CHO / gp38P cells were each added to 10 // 1, using Hema tracer (NKK HEMA TRACER I MODEL PAT-4M; Niko Bioscientific CO, Tokyo, Japan) to record the state of platelet aggregation (see Watanabe M, Okochi E, Sugimoto Y, Tsuruo T.

Cancer Res. 1 98 8; 4 8: 64 1 1-6). When examining the effect of platelet aggregation inhibition in 8 F 1 1 antibody, C Η 〇 / gp 3 8 P cells were treated with Norma 1 R at IgG ( Control group antibody; manufactured by Sigma) was used as a control group or 8F1 1 antibody (5 00 // g) for 20 minutes on an ice bath, and then the above platelet aggregation experiment was performed.

Results are shown in Figures A and B. In Figure 3A, the platelet aggregation property of CHO / gp38P-102-200413526 cells was determined, and gp38P was active on platelet aggregation. In Fig. 3B, in the agglutination reaction of CHO / gp38P cells, the cells can inhibit the agglutination reaction by reacting with the 8F11 antibody. The results show that the platelet agglutination line expresses the gp38 gene product. (Example 6) Preparation of anti-human gP36 polyclonal antibody (TT679) In Japanese white rabbits, a peptide composed of the following amino acid sequence and Keyhole Limpet Hemocyanin (KLH) co-bonded peptide as a carrier :

Cys Glu Gly Gly Val Ala Met Pro Gly Ala Glu Asp Asp Val Val (Sequence Listing No. 21) called for immunization 6 times to recover blood pupa. Blood pupae were purified on an affinity column fixed with agarose sugar by the above peptide. (Example 7) Western blot method was used to express CHO cells without genes pcDNA3 (Mock), pcDNA3-gp38P, pcDNA3-gp36, or gp38P-M41A cDNA with a point variant of gp38P ii Λ pcDNA3 ^ If (P c DNA 3-gp 3 8 P -M4 1 A). After 24 hours, lxl07 cells were recovered and suspended in 500 // 1 lysate B (25mM Tris (pH7.4), 50mM NaCl, 0.5% sodium deoxycholate®, 2% Nonidet P-40, 0.2% SDS), vortex once every 10 minutes, and placed on ice for 1 hour. After the protein was quantified by Pierce's BCA-protein analysis method, SDS sample buffer (containing 2-thiohydroethanol) 20 // 1 was added, boiled for 5 minutes, and electrophoresis was performed on a 10% polypropylene ammonium gel. It was transferred to a PVDF membrane, and after blocking, it was reacted with an anti-gp36 polyclonal antibody (TT6 79) and an 8F11 antibody (10 V g / ml) at room temperature for 1 hour. Wash with PBS containing 0.05% Tween20 for 10 minutes for 3 times, and react with HRP-labeled secondary antibodies. After washing according to the same method -103-200413526, use the ECL test. The results are shown in Figure 4A. Anti-gp 36 antibody (τ T 6 7 9) can react with samples from CHO / gp36 cells, but not with CHO / Mock, CHO / gp3 8P, CHO / gp3 8P-M41 A, 8F1 1 antibody can It reacts with samples from CHO / gp38P source, but does not react with CHO / Mock, CHO / gp38P-M41A, CHO / gp36. The anti-gP36 polyclonal antibody (TT679) can specifically react with the human homologue gP36. (Example 8) Mouse-human platelet agglutination experiment Blood was collected from mice or healthy people using heparin, centrifuged at 900 rpm for 10 minutes, and platelet-rich plasma (PRP) was collected. PRP was diluted to 3-fold volume with PBS, and PRP was used for platelet aggregation. Cells used for agglutination of blood platelets are used after trypsin recovery. Add 2x1007ce] ls / ml CHO parent strain, CHO / gp38P, CHO / gp36 (10 // 1 each) to mouse or human PRP 200 # 1, using Hema tracer (NKK HEMA TRACER I MODEL PAT-4M; Niko Bioscientifie CO, Tokyo, Japan) to record the state of platelet aggregation (see Watanabe M 'Okochi E, Sugimoto Y, Tsuruo T. Cancer Res. 1988; 48: 6411-6). The results are shown in Figure 4, B, C, and D. Figure 4B shows that CHO / gp38P cells expressing the mouse gp3 8P gene, and CHO / gp36 cells expressing the human homolog (gp36 gene) of gp38P have an induction activity on mouse platelet aggregation. Figure 4C shows that CHO / gP38P cells expressing mouse gp38P gene have agglutinating activity on human platelets. Furthermore, Figure 4D shows that CHO / gp36 cells expressing the human gp36 gene have agglutination of human platelets > 104-200413526. From this result, it is known that g p 3 8 P and human homolog g g 3 6 have platelet aggregation-inducing activity in mice and humans. (Example 9) Inhibition of platelet aggregation by 8F11 antibody Blood was collected from mice using heparin, centrifuged at 90 pm for 10 minutes, and platelet-rich blood aggregate fractions were collected (P) ate 1 et-richp 1 asma: PRP). Pr p was diluted to 3 times the volume with PBS and used as PRP for platelet aggregation. CHO / gp38P cells used for platelet aggregation were recovered after trypsin treatment and used. Actually, the mouse PRP was dispensed into 200 // 1 and each 8F1 1 antibody or control group was added with 1 mg of Normal Rat IgG (control antibody; manufactured by Sigma) and reacted at 37 ° C for 30 minutes. Add 2 X 107cells / ml of CHO / gp38P 10 // 1 and use Hema tracer (NKK HEMA TRACER I MODEL PAT-4M; Niko Bioscientific CO, Tokyo, Japan) to record the state of blood / J and plate agglutination (see Watanabe M, Okochi E, Sugimoto Y, T suru ο T. Cancer Res. 1988; 48: 6411-6). The results are shown in Figure 5A. It can be seen that the 8F1 1 antibody can inhibit the gp38P-dependent platelet agglutination activity of CHO / gP38P cells that are constantly expressed by gP38P. The normal rat IgG (control antibody) in the control group does not inhibit platelet agglutination activity. It can be seen that the 8F1 1 antibody binds to recognize gp38P. To neutralize platelet agglutination activity. (Example 10) PCR was performed using gp 3 8P fragment to identify 8F 11 antibody binding site. A plastid containing the full-length gp38P gene was used as a template for PCR. The PCR product was treated with EcoRI and Nhel 'and pET-21a vector (manufactured by Novagen). The plastids with FLAG-tag encoding genes are adhered. E. coli with this plastid transformation was cultured and IPTG was added to induce expression. Collect the bacterial cells of Enterobacter large -105-200413526 by centrifugation, directly add 50 # 1 SD sample buffer (containing 2-thiohydroethanol), boil for 5 minutes, and perform electrophoresis on a 0% polypropylene amidamine gel. It was transferred to a PVDF membrane, and after blocking, it was reacted with an anti-flag_m2 antibody (manufactured by Sigma) and an 8F1 1 antibody (10 μg / ml) at room temperature for 1 hour. With 0.05% Ding ~ 661120? Wash 63 times and 10 minutes for 3 times, and react with ^ 111? Labeled secondary antibody. After washing in the same way, the test was performed with e C L. The results are shown in Figures B and c. Figure 5B shows that the 8FU antibody has a strong response to the polypeptide generated by the gp3 8P gene fragment from the N-terminus of gp3 8P since the number 46 (sequence number 14 amino acid number 46), but it has a strong response to the sequence number 1 4 Polypeptides of the amino acid sequence shown by amino acid numbers 1 to 43 are weakly reactive. Furthermore, the amino acid of sequence number 14 in the sequence listing, that is, the expression peptide of the amino acid sequence shown in numbers 1 to 43 can be recognized with the anti-FL A G antibody. The 8F 11 antibody does not react with the amino acid sequence polypeptide shown in amino acid numbers 1 to 40 of sequence number 2 in the sequence listing. It is shown in FIG. 5C that it does not react with the amino acid sequence polypeptide shown in the sequence table sequence number 14 amino acid number 41 to 172 at the end deletion, but it does not react with the sequence table sequence number 1 4 amino acid number 3 9 to The polypeptide of the amino acid sequence shown in 172 has a strong reaction. The reactivity of the polypeptide generated from the gp3 8P gene fragment to the 8F1 1 antibody is shown in Figure 5D. It is speculated that the recognition site on gP3 8P for the 8F11 antibody is the sequence listing sequence number 14 amino acid number 3 9 to 44 peptide of the amino acid sequence shown in 9 to 44. (Example 1 1) gp38P point variant was used to confirm 8F1 1 The gp38PcDNA (WT) was used as the antibody binding site, and the glycine-encoding gene of sequence number 14 amino acid number -106-200413526 code 38 of the sequence listing was replaced by the gene encoding alanine G3 8 A (ie, sequence number 22 of the sequence listing Nucleotide sequence, amino acid sequence of sequence number 23), glycine-encoding gene of sequence number 1 4 amino acid number 40 and gene G40 A encoded by alanine (ie, sequence number 24 of sequence listing Nucleotide sequence, amino acid sequence of 25), the methionine-encoding gene of sequence number 1 4 amino acid number 4 1 is replaced by the gene M41A of alanine-encoding gene (that is, the nucleotide number of sequence number 26 in the sequence listing Sequence, amino acid sequence of 27) 'sequence number 1 4 amino acid number 42 amino acid coding gene is replaced by alanine gene V42 A (that is, the nucleotide sequence of sequence number 28 in the sequence listing' 29 of Amino acid sequence), sequence number 1 4 amino acid number 4 3 proline The coding gene is replaced by the alanine coding gene P4 3 A (that is, the nucleotide sequence of SEQ ID NO: 30 and the amino acid sequence of 31), and the glycine coding gene of sequence number 1 4 and amino acid number 45 The alanine-encoding gene G45 A (that is, the nucleotide sequence of the sequence listing sequence number 3 2 of the amino acid sequence of 3 3) 'sequence number 1 4 the amino acid-encoding gene of 49 is replaced by alanine Acid coding gene K4 9A (the nucleotide sequence of sequence number 34 in the sequence listing, amino acid sequence of 35), the glutamic acid coding gene of sequence number 14 amino acid number 34 is replaced by the alanine coding gene T34A ( That is, the nucleotide sequence of sequence number 36 in the sequence listing, and the amino acid sequence of 3 7) 'SEQ ID No. 1 4 The amino acid encoding gene of 3 7 is replaced by the alanine encoding gene T3 7 A (sequence listing SEQ ID NO: 3 8 nucleotide sequence, 3 9 amino acid sequence) 'Sequence number 1 4 amino acid number 51 1 glutamic acid encoding gene replaced by alanine encoding gene T 5 1 A (sequence listing sequence Nucleotide sequence of number 40, amino acid sequence of 41), sequence number 1 of amino acid number 4 5 2 The glutamic acid encoding gene was replaced by C-107-200413526 The amino acid encoding gene T5 2 A (the nucleotide sequence of SEQ ID No. 42 in Sequence 15 and the amino acid sequence of 43) uses Quick Change Mutagenesis Kit (manufactured by Stratagene). After the mutation introduced in this variant was confirmed by DNA sequencing, a PcDNA3 vector was implanted to guide the gene to CHO cells. 24 hours after gene introduction, CHO cells were recovered for 1 X 1 07 ce 11 s and suspended in 500 μl of Lysate B (2.5 μM Tris (pH 7.4), 50 mM NaCl, 0.5% sodium deoxycholate). , 2% Nonidet P-40, 0.2% SDS), vortex once every 10 minutes, and placed on ice for 1 hour. After quantifying the protein by Pierce's BCA-protein analysis method, add SDS sample buffer (containing 2-thiohydrin ethanol) 20 // 1, boil for 5 minutes, and perform electrophoresis on a 10% polypropylene gel. It was transferred to a PVDF membrane, and after blocking, it reacted with an anti-FLAG-M2 antibody (manufactured by Sigma) and an 8F11 antibody (10 // g / ml) at room temperature for 1 hour. Wash with 0.05% Tween20 in PBS for 3 times for 10 times, and react with HRP-labeled secondary antibodies. After washing in the same way, use ECL to test. The results are shown in Figure 6A. 8F1 1 antibody does not react with G40A, M41A, V42A, and P43A variants, and has weak reactivity with G38A variants. Other variants respond strongly to the 8F11 antibody. Combining the results in Figure 5 above, the 8F11 antibody can recognize the peptide sequence of gp 3 8P amino acid numbers 39 to 44 (sequence list sequence number 14 amino acid numbers 39 to 44). (Example 12) ELISA confirmed that the 8F11 antibody reaction site was due to amino acid numbers 39 to 47 of the gp 3 8P protein (sequence list sequence number 14 amino acid numbers 39 to 47). Amino acid sequence of amino acid (C): -108-200413526

AspGlyMelVal ProProGlylleGluCys (SEQ ID NO. 44) is a peptide (WT), and the amino acid sequence of alanine with methionine of amino acid number 41 is:

Asp G] y Ala Val Pro Pro Gly lie Glu Cys sequence:

Asp Gly Met Ala Pro Pro Gly lie Glu Cys (Sequence 歹 ij List 歹 IJ No. 46) is a peptide (VUApt). The proline of amino acid number 43 is replaced by the amino acid sequence of alanine:

Asp Gly Met Val Ala Pro Gly lie Glu Cys (sequence number 47) is a peptide (P43 Apt), the amino acid number 44 of the proline is replaced with the amino acid sequence of alanine:

The peptide (P44Apt) made from Asp Gly Met Val Pro Ala Gly lie Glu Cys (sequence number 48) was synthesized using a peptide synthesizer and fixed on a 96-well plate. According to the usual method (References: Kato, Y., Fujita, N., Y an ο 9 Η, and Tsuruo, T. Cancer Res. 5 57, 3040-3045, 1 997), 8F11 antibody and control were measured by ELISA Reactivity of mouse antibodies (manufactured by Sigma) to peptides. The results are shown in Figure 6B. Figure 6B shows that 8F11 antibody is reactive to WT peptides, but not reactive to other mutant peptides. The results of Figure 6A are reproduced in another experiment, confirming that 8F1 1 antibody can recognize the amino acid number of gP38P 39 To 44 (sequence listing sequence number 14 amino acid numbers 39 to 44). (Example 13) Mechanism of platelet aggregation inhibition of gp38P due to 8FH. Antibody -109-200413526 Discussion of the mechanism of the peptide sequence recognized by 8FH antibody for platelet aggregation activity of gp38P, the amino acid sequence at this site was introduced The mutant gp3 8P is required for platelet aggregation activity. The lymphatic blood of mice was collected, centrifuged at 90 r pm for 10 minutes, and the platelet-rich blood fraction (? 1 & 616 ^ rich plasma: PRP) was collected. PRP was diluted to 3 times the volume with PBS, and the platelets were agglutinated with PRP. Cells used for platelet aggregation were recovered after trypsin treatment. In fact, the mouse PRP was dispensed at 200 // 1, and the 2x107 cel] s / ml CHO mother strain, CHO / gp38P and gp38P number 41 were replaced with alanine M41A-gp38P (sequence list sequence number 27) CHO / M41A with constant expression of 10 μ1 each, using Hematracer (NKK HEMA TRACER I MODEL PAT-4M i Niko B ioscientific CO, Tokyo, Japan) to record the state of platelet aggregation (refer to Watanabe M, Okochi Ε, Sugimoto Y, Tsuruo T. Cancer Res. 1 988; 48: 6411-6) The results are shown in Figure 6C and D. As shown in FIG. 6C, not only CHO / gp38P cells expressing the mouse gp38P gene, but also replaced the methionine in gp38P number 41 (amino acid number 41 in the sequence listing 1 to 4) with M of alanine. C 1 0 / M 4 1 A cells stably introduced with 4 1 A-gp 3 8 P have inducing activity on mouse platelet aggregation. As shown in Fig. 6D, the 8 F 11 antibody may have a steric disorder of gp 3 8P sugar chains at the recognition site of 8 F 11 and may inhibit sugar chain-dependent platelet aggregation. (Example 14) Identification of the site required for platelet aggregation on the gp38P protein will contain the amino acid sequence of sequence number 14 in the sequence listing, and wild type-110-200413526 gP3 8P amino acid number 4 of the methionine The coding gene is replaced by the alanine-encoding gene mutant protein M4 1 A, the amino acid number 34 of the glutamic acid encoding gene is replaced by the alanine-encoding gene protein T4 A (SEQ ID NO: 37), amine The glutamic acid encoding gene of amino acid number 37 was replaced by the alanine encoding gene variant protein T37 A (sequence list sequence number 39), and the glutamic acid encoding gene of amino acid number 51 was replaced by the alanine encoding gene The mutant protein T5 1 A (sequence list sequence number 41), and the amino acid code 5 2 of the glutamic acid encoding gene was replaced by the alanine encoding gene variant protein T5 2 A (sequence list sequence number 43). The pcDNA3 vector of the cDNA encoded by each variant protein was introduced into CHO cells, and each gene was expressed on the membrane surface. After 24 hours, it was recovered by trypsin treatment. Mice collected blood with heparin, centrifuged at 900 rpm for 10 minutes, and collected plasma-rich plasma (PRP). PRP was diluted to 3 times the volume with PBS and used as PRP for platelet aggregation. Temporary expression mutant protein M4 1A CHO / M41 A, temporary expression mutant protein T34A CHO / T34A, and temporary expression mutant protein T37A CHO / T3 7A were added to mouse PRP 200μ 1 at a concentration of 2 × 107 cells / ml each. CHO / T51A, which temporarily expresses the mutant protein T51A, and CHO / T5 2A25 (10 // 1), which temporarily expresses the mutant protein T52A, using Hema tracer (NKK HEMA TRACER I MODEL PAT-4M; Niko Bioscientific CO, Tokyo, Japan ) To record the state of platelet aggregation (see Watanabe M, Okochi E, Sugimoto Y, Tsuruo T.

Cancer Res. 1988; 48: 6411-6). The results are shown in Figure 7A. As shown in Figure 7A, the CHO / T34A cells that temporarily expressed the mutant egg-111-200413526 white matter T34A did not induce mouse platelet aggregation, and it was found that the number 34 (sequence number 14 amino acid number 34) was short. Amino acid is an essential site for platelet aggregation. This site is shown in Figure 7B. The site where glutamic acid is connected to alanine is the site where sugar chains can be added (References: Yoshida, A., Suzuki, M ·, Ikenaga, Η ·, and Takeuchi, MJ Biol. Chem. 272: 16884-16888, 1997). This 咅 [position 5 (* in Figure 7B) is not only mouse gp38P, but also in other humans (sequence number 8 amino acid number 52) · mouse (sequence number 4 amino acid number 34) · Dogs (sequence number 6 amino acid number 41 or amino acid number 50) are conserved among homologues. The amino acid sequence comparison is shown in Figure 7B, which shows the important site of platelet aggregation activity. (Example 15) Identification of the site required for platelet aggregation on human homolog gp36 protein The wild-type gp36 cDNA (WT) and its number 52 (sequence list sequence number 8 amino acid number 52) were replaced by the glutamic acid encoding gene Alanine coding gene gp 3 6-T52 A (nucleotide sequence of SEQ ID NO: 49, amino acid sequence of 50), sequence listing sequence sequence number 8 amino acid number 5 5 glutamic acid coding gene replaced by Alanine-encoding gene gp36-T55A (nucleotide sequence of SEQ ID NO: 51, amino acid sequence of 52) Each variant encodes the pcDNA3 vector of cDN, each introduces a gene in CHO cells, and each expresses a protein on the membrane surface After 24 hours, it was recovered by trypsin treatment. Mice collected blood with heparin, centrifuged at 900 rpm for 10 minutes, and collected platelet-rich plasma (PRP). PRP was diluted to 3 times the volume with PBS and used as PRP for platelet aggregation. In fact, the mouse PRP was subdivided into -112-200413526 200 / il, 2xl07 cells / ml was temporarily expressed as CHO / gp36 of wild type gP36, and gp36 was temporarily expressed as number 52 (Sequence 歹 U Table 歹 Sho No. 8 amino acid Number 52) is replaced by glutamic acid of alanine, CHO / gp36-T52A of gP36-T52A, and glutamic acid of gp36 number 55 (sequence 歹 IJ table sequence 歹 [J number 8 amino acid number 55). Replace it with CHO / gp36-T55 A of Alanine P36-T55A and enter 10/1/1 with Hema tracer (NKK HEMA TRACER I MODEL PAT-4M; Niko B ioscientific CO, Tokyo, Japan) The state of platelet aggregation was recorded (see Watanabe M, Okochi E, Sugimoto Y, Tsuru T. Cancer Res. 1988; 48: 6411-6). The results are shown in Figure 7C. As shown in FIG. 7C, it can be seen that CHO / gp3 6-T5 2A cells temporarily expressing gp3 6-T52A have no induction activity on platelet aggregation in mice. It is found that the glutamic acid of amino acid number 52 in sequence number 8 is An essential site for platelet aggregation. As shown in Figure 7B, the site is highly homologous to gP3 8P, and is the same as gP38P, and it is highly likely that the glycine chain is attached to the site following glutamic acid (see Reference: Yoshida, A. , Suzuki, M., Ikenaga, R., and Takeuchi, MJ Biol. Chem. 272: 16884-16888, 1997). Regarding dog homologues, the possibility that this glycosyl acid binds to sugar chains cannot be read by Edman-decomposed sequences (References: Zimmer, G., Lottspeich, F., Maisner, A., Klenk, HD, and Herrlei ·, G .. Biochem. J. 3 26: 99-108, 1997). Therefore, it is implied that this part of the superspecies (the marked part in Fig. 7B) is extremely important for platelet aggregation-inducing activity. (Example 16) Expression of human homolog gp36 gene in colorectal cancer increased -113-200413526

Cancer Profiling Array II (manufactured by BD Biosciences) extracts 1TlRNA from cancerous sites (T) and non-cancerous sites (N) of various cancer patients (160 cases), converts them into cDNA, and converts 4 types of homekeeping genes. After the cDNA amount was corrected, it was immobilized on a nylon membrane. This Cancer Profiling Array II membrane was hybridized with a human homolog gP36 gene probe calibrated with P32. The radioactivity at each point was developed and quantified with a BAS 1 000 phosphoimage analyzer (manufactured by Fujifilm Corporation) to express the gp36 gene in cancer cells. Discussion of quantity. The basic operation method can follow the operation template of BD Bioseiences (Reference: Wiechen. K ·, Lu

Diatchenko, L., Agoulnik, A., Scharff, K. M. 5 Schober, H., Arlt, K., Zhumbarayeva, B., Sieb e r t, P. D.,

Dietel j M. »Schafer j R. 5 and Sers» C. Am. J. Pathol. 159, 1 63 5- 1 643, 2001). The difference in expression at the cancerous site (T) and non-cancerous site (N) can cause the Cancer Profiling Array II membrane to hybridize to the control probe of the human ubiquitin gene labeled with 32P, and develop and quantify it with a BAS 1000 phosphoimage analyzer. The radioactivity of gp36 gene was corrected, and the expression ratio of cancer site (T) and non-cancer site (N) in each patient was calculated. The cDNAs of colorectal cancer patient No. 5 and small bowel cancer patient No. 2 immobilized in Cancer Profiling Array II were purchased from I BD Bioseiences as templates. The expanded primers of gp36 gene were: 5, -TGTGGAAGGTGTCAGCTCTG-3, (synonym: sequence歹 [J SEQ ID NO: 5 3) 5, -TTAGGGCGAGTACCTTCCCG-3, (Antisense: SEQ ID NO: 54) -114-200413526 The riboprotein S9 gene was used for the control group, and the primer 5'-GATGAGAAGGACCCACGGCGTCTGTTCG_3 was expanded with (Synonym sequence list sequence number 5 5) 5, -GAGACAATCCAGCAGCCCAGGAGGGACA-3 '(antisense sequence list sequence number 56) into fj PCR. PCR conditions can be according to the BD Biosciences operating template '94 ° C for 30 seconds, 94 ° C for 30 seconds, 68 ° C for 2 minutes, 35 cycles of PCR are performed, and finally 5 minutes at 68 ° C , Perform agar electrophoresis. The results are shown in Figures A, B, C and D. As shown in Figure 8A, when the gp36 gene probe is used, it is shown that the gp36 gene is in cancer (T) and non-cancer (N) areas of Colon, Rectum, and S ma 11 intestine (small intestine). By comparison, its expression increased by more than 90%. In Lung, Breast, Ovary cancer (T) compared with non-cancer (N), there was no change in the expression of gp36 gene, indicating that the expression of gp 36 gene is specific to colon cancer rise. Furthermore, the ubiquitin gene was used as a control probe to correct the amount of cDNA on the membrane. The calculation results of the expression levels of the gp36 gene in the cancer site (T) and non-cancer site (N) of each patient are shown in Figure 5B. The calculation results of gP3 6 gene expression at the cancer site of the organ are shown in Fig. 5C. This figure shows the increase of gP36 gene expression in Col on, Recect, S ma 11 intestine (small intestine), T estis (睪 九) cancer site (T), so the expression of gP36 can be diagnosed Whether there is bowel and testis cancer. The results of Cancer Profiling Array II show that when using Cancer Profiling Array II immobilized colorectal cancer patient No. 5 small bowel cancer patient No. 2 cDN A (sample of circled numbers in Figure 8A), gp36 gene primers were used to perform PCR results. It was confirmed that the cDNA from cancer site -115-200413526 (T) had higher expression of gp36 gene than non-cancer site (N) (Figure 8 D). In addition, as a result of performing PCr with the amplified primer of riboprotein S9 in the control group, the amounts of cDNA of the cancerous site (τ) and non-cancerous site (N) used in the PCR were the same (Fig. 8D, lower panel). (Example 17) Expression of human homolog gp36 protein in colon cancer In order to investigate the expression of gP36 protein in human colon cancer, sections of human colon cancer tissue and normal colon tissue were stained by immunohistochemistry. Specifically, the Land Mark Tissue Microarrays Low Density Colorecta purchased from Ambion was used for immunostaining using the above-mentioned TT6 7 9 antibody against multiple human gP36 antibodies. This Micro Array is a needle-rolled colon cancer and normal tissue with a diameter of 600 // m. Sample blocks of tissue sections fixed with formalin are embedded in paraffin. This tissue section was reacted with the primary antibody of TT679 antibody, the secondary antibody was labeled with biotin-labeled anti-rabbit antibody, and the peroxidase-labeled streptavidin (DAKO company, LSAB 2 group) was reacted, and then the DAB matrix was added. In color, the reaction site with the antibody is brown. The hematoxylin-eosin staining (H & E) staining chart of each tissue is the result of staining each sample by using H & E staining of Ambion Corporation. The results are shown in Figure 9. In Figures A and C, human colon cancer tissue sections have strong reactivity to TT679 antibody. The reactivity to normal colon tissue sections is shown in Figure 9E, and the amount of gP36 gene is shown in Figure 8. The protein quality is determined in The expression of gp36 is increased in cancer, and in the diagnosis of intestinal cancer, the expression of gp36, a homologue of gp44, can be used as a marker. (Example 18) Correlation between expression level of human homolog gP36 and colon cancer metastasis -116-200413526

Cancer Profiling Array (manufactured by BD Biosciences) is extracted from cancerous sites (T) and non-cancer sites (N) of various cancer patients (241 cases) and metastatic sites (μ) in several cases in accordance with the above Cancer Profiling Array II. The mRNA was transformed into cDNA, and the cDNAs of the four family genes were corrected for immobilization on a nylon membrane. This Cancer Profiling Array membrane was hybridized with the human gp36 gene probe calibrated by P32. The radioactivity of each point was developed and quantified with a BAS 1000 phosphoimage analyzer (manufactured by Fujifilm Corporation), and the primary and metastatic lesions of the gp36 gene were investigated. The amount of expression. The basic operation method can follow the operation template of BD Bioseiences (Reference: Wiechen. K., Diatchenko, L., Agoulnik, A ·, Scharff, K · M ·, Schober, H ·, Arlt, K., Zhumabayeva, B ·, Si eb ert, P · D ·, Dietel, M., Schafer, R., and Sei.s, C. A m · J. P ath ο 1 · 1 5 9, 1 6 3 5-1 6 4 3, 2 0 0 1). Differences in expression at cancerous sites (T), non-cancerous sites (N), and metastatic sites (M) can cause Cancer Profiling Array membranes to be hybridized with control probes of the human ubiquitin gene labeled with 32P and developed with a BAS1000 phosphoimage analyzer. Quantitatively, to correct the specific radioactive activity of the gp36 gene, the expression level of non-cancerous site (N) in each patient was set to 1, and the expression ratio of cancer site (T) and metastatic site (M) was calculated. The results are shown in Figures 10 and A and B. In Fig. 10A, when the human homolog gp36 gene is used as a probe, the expression of gp36 gene is more in the cancerous part (T) of colorectal cancer than in the non-cancer part (N). Furthermore, the expression of the gpS6 gene in the metastatic site (M) was also increased. Using the ubiquitin gene as a control probe to correct the amount of cDNA on the membrane, the gp36 gene expression level in each patient's non-cancerous site (N) was determined to be 1 -117-200413526 'Calculate the cancer site (T) and metastatic site (Μ), the results are listed in Figure 10 ° B ° The results show that 'metastasis site (gp36 expression is higher than cancer site (τ), and then the non-cancerous site (N) is increased, this result shows g ρ The expression of 36 genes is positively correlated with colorectal cancer metastasis. (Example 19) The expression of human homolog gp36 is accompanied by the addition of lung metastasis. According to the method described in Example 3, the nucleotide sequence shown in sequence number 7 in the sequence listing is included. The pcDNA3-gP36 plastid of the human homolog gp36 gene and the pcDNA3 plastid of the control group not containing the gp: 36 gene were introduced into CH0 cells, and a resistant strain was selected with G418 (1 mg / mL). The cell line expressing the human homolog gp36 was named CHO / gp36 cells, and the pP3-containing pcDNA3 cell line without the gP36 gene was named CHO / mock cells. CHO / gp36 cells and CHO / mock cells were described according to Example 1. After incubation, treat with trypsin, wash with PBS, and recover by centrifugation. 〇 / g ρ 3 6 cells and C Η 〇 / m 〇ck cells each with Hanks, Balanced Salt Solution (without magnesium · Ming: 0.40g / L KC1, 0.06g / L KH2P04, 8.0g / L NaCl, 0.35 g / L NaHC03, 0.048 g / L Na2HP04, 1.0 g / L D-glucose, 0.00 g / L phenol red) to make 1 mL of a 2.5 × 10 06 cell concentration solution. Each cell solution will be prepared Nude mice implanted with BALB / c (purchased from Charles River, Japan, 5 weeks 齢 female tail vein 2 0 0 // L (5 X 105 cells / mouse) (Reference: Sugim 〇t 〇, Y ., Watanabe, M., oh-h ar a, Ding ·, sat ο, S ·, I soe, T., and Tsuruo, T. Cancer Res. 51: 92 1-925, 1991). Cell transplantation After 19 days, nude mice were killed under ether mochi and the lungs were removed. The lungs from 200413526 were fixed in saturated picric acid to visualize the lung nodules. The results are shown in Figure 11 compared to CHO / There were almost no metastatic nodules in the lungs of mock cells transplanted into nude mice. The lungs of nude mice transplanted with CHO / gp36 cells not only had larger lungs but also had more nodules on the surface of the lungs. When transplanting CHO / mock cells, it is 2-4, and when transplanting CHO / gP36 cells, it is 78 ~ 106. It can be seen that the expression of gP36 is accompanied by a sharp increase in lung metastasis. related. (Example 20) Correlation between lung cancer tissue type and human homolog gp3 6 gene expression According to the method described in Example 16, Cancer Profiling Array II (manufactured by BD Biosciences) and Cancer Profiling Array (manufactured by BD Biosciences) were calibrated with P32. The homologue gp36 gene and the human ubiquitin gene probe labeled with P32 were hybridized. The radioactivity of each point was developed and quantified with a BAS1000 phosphoimage analyzer (manufactured by Fujifilm Corporation) to investigate the expression level of gp36 gene in lung cancer. Refer to Cancer Profiling Array II and Cancer Profiling Array for more information □ Each cancer tissue type to explore the relationship between gp36 gene expression and lung cancer tissue. The results are shown in Figure 12. In addition to the Cancer Profiling Array II and Cancer Profiling Array replicates, the expression of the human homolog gP36 gene in 13 cases of adenocarcinoma and 15 cases of squamous cell carcinoma was plotted. The results showed that there were more cases of gp 3 6 expression in flat epithelial cancer than in adenocarcinoma. With a cut-off of 1.5, the expression of cancer sites increased in 200413526 0 in adenocarcinoma cases, and gp36 gene expression increased in 10 of 15 cases in squamous cell carcinoma. (Example 21) Correlation between lung cancer tissue type and human homolog gp36 expression To investigate the expression of the gp36 protein in human lung cancer, human lung cancer tissue and normal lung tissue sections were immunostained. Specifically, immunostaining was performed using the Landmark Low Density Lung Tissue Microarmy s (Cat. 3172, Lot-122P 14 A) described in Example 17 and the anti-gP36 polyclonal antibody (TT 679 antibody) described in Example 6. This macro array is a sample block of needle breast cancer and normal lung tissue with diameter 600 / m, as in Example 17, and a formalin fixed tissue section sample block, embedded with paraffin. This tissue section was reacted with the primary antibody of TT 679 antibody, and the secondary antibody was reacted with biotin-labeled anti-rabbit antibody, biotin-labeled peroxidase-streptavidin complex (DAKO, CSASystem), and then reacted with biotin After reacting with T iramide and peroxidase-labeled streptavidin, D is added to the DAB matrix to develop a color, and it can be stained with the antibody reaction site. The results are shown in Figure 13. In the first three figures, most of the flattened epithelial cancers are stained with cancer cells, and they are all stained in adenocarcinoma and normal lungs. It can be seen that flattened flatepithelial cancer expresses gp36 specifically. Among the 8 cases of Landmark Low Density Lung Tissue Microarrays, 8 cases of flat epithelial cancer had positive expression of P36, and 13 cases of adenocarcinoma had positive expression of gp36 and 2 cases (15.4%). As a result, it was found that the expression of gp36 protein in the gp44 human homologue was specifically increased in flat epithelial cancer. (Preparation Example 1) A soft capsule is prepared by degrading an oily substance, such as soybean oil, cottonseed oil, or olive oil. * · 120-200413526 is added to any of the nucleotide sequences described in any of (a) to (ii) below. Polynucleotide mixtures of sense sequences can be pumped into gelatin to obtain soft capsules containing 10 mg of active ingredient. After washing and drying: (a) Sequence Listing Sequence Number] Nucleotide Numbers 1 to 5 〖9 Nucleotide sequence

(b) the nucleotide sequence 1 to 501 of nucleotide number 1 to 501 in sequence number 3 of the sequence listing

(d) Nucleotides 1 to 48 of 9 in Sequence Listing Sequence Number 7

(e) the nucleotide sequence 1 to 540 of nucleotide number 1 to 540 in sequence number 13 of the Sequence Listing [J (0 a nucleoside complementary to the nucleotide sequence described in any of (a) to (e) above Polynucleotides formed from acid sequences hybridize under severe conditions, and have platelet aggregation-enhancing active peptide-encoded nucleotide sequences; (g) amino acid numbers 1 to 172 in sequence number 2 of the sequence listing Nucleotide sequence encoded by amino acid sequence poly-peptide; (h) Nucleotide sequence encoded by amino acid sequence poly-peptide shown by amino acid numbers 1 to 166 in Sequence Listing Sequence Number 4; (i) SEQUENCE LISTING SEQUENCE NUMBER 6 Amino Acid Numbers 1 to 169 Nucleotide Sequences Encoded by Polypeptide; (j) Sequence Listing Sequence Number 8 Amino Acid Numbers 1 to 1 62 Polypeptide-encoded nucleotide sequence; -121-200413526 (k) Sequence table sequence number 1 4 Amino acid numbers 1 to 7 2 Nucleotide sequence encoded by peptide sequence; (1 ) The amino acid sequence described in any one of (g) to (k) above, which is missing, substituted or added] or has an amino acid sequence formed by several amino acids and has blood Nucleotide sequence encoded by a polypeptide that promotes platelet aggregation-promoting activity. (Preparation Example 2) Tablets According to the usual method, oligonucleotide 2 is described in 10 mg of Formulation Example 1, and 0.2 mg of gelatinous dioxide is used. 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 1 mg of starch, and 98.8 mg of lactose are produced. Also, the agent skin may be coated as needed. (Formulation Example 3) The suspension agent is prepared in 5 ml and contains 10%. The preparation of mg in Example 1, 100 mg of sodium hydroxymethylcellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution (Nippon Pharmacopoeia), and 0.25 ml of vanilla extract are described. 4) Injection The 1.5% by weight of the oligonucleotide described in Formulation Example 1 was stirred in 10% by volume of propylene glycol. Then, it was added to a certain volume with water for injection. It can be obtained by sterilization. The industrial peptide of the present invention has platelet agglutination activity. Therefore, the polypeptide of the present invention and the gene encoded by the peptide can be used as a platelet aggregation promoter and as a treatment for diseases related to platelet dysfunction. The polypeptides and genes of the present invention can be used to obtain platelets Methods for the use of substances used as therapeutic or preventive agents for diseases related to agglutination or platelet dysfunction, methods for measuring platelet aggregation -122-200413526 to promote and block active substances, and methods for diagnosing platelet aggregation or platelet dysfunction related diseases. The invention is that platelet aggregation activity or inhibitory active substance can be used as a platelet aggregation promoter or inhibitor. [Simplified illustration of the figure] In Figure 1, A is the mouse brain capillary endothelial cells MB EC-4, mouse lymphoid Ganglia stromal cells CA-12, mouse osteoblasts MC3T3-EI, normal mouse phages and lysates of LPS-activated mouse phages, Western blotting results with 8F11 antibody. From the left, MBEC-4, CA-12, MC3T3-E1 normal mouse phages, and LPS activated mouse phages. B is a colon cancer cell line NL-17, NL-44,

Col on2 6, mouse melanoma B16 cell lines B16 / F1, B16 / F10 and normal mouse large intestine-modulated mRNA. Results of RT-PCR using gp38P primer. Marked from the left (603bp), NL-17, NL-44, Colon26, B16 / F1, B16 / F10, normal mouse large intestine. C is the result of RT-PCR of mRNA prepared from NL-17 and NL-14 cells using ® gP3 8P and Lu-actin primers. In Fig. 2, A is the result of the flow cell technique using CHO / gp38P cells and the parent strain CHO. The vertical axis is the number of cells, and the horizontal axis is the fluorescence intensity. The mother strain did not respond to the 8F11 antibody, so the cells with strong fluorescence intensity did not exist (8F11 antibody discriminator) (the peak is on the left). CHO / gp38P cells responded to the 8F1 1 antibody, so there were many cells with strong fluorescence intensity. (Spikes move to the right). B is the result of using western ink stains. From the left, CHO / Mock -123-200413526 cells, CHO / gp38P cells, CHO / gp36 cells, 29 3T cells temporarily express pcDNA3-FLAG-gp38P, the same pcDNA3- FLAG-gp36 cells, and pFLAG-CMV-2 -Results for gp38P cells (right column 2). C is the result of western blotting after immunoprecipitation of CHO / gp3 8P cells and parental CHO cells. The left is the result of western blotting using 8F1 1 antibody, and the right is the western blotting result using 20A1 1 antibody. Immunoprecipitation samples of 8F1 1 antibody, anti-FLAG antibody, and normal mouse IgG antibody are shown in every 2 columns from the left. Among the immunoprecipitation samples, the left is the CHO / gp38P sample, and the right is the sample of the parent strain CHO. Figure 3 is the platelet agglutination force of gP3 8P. The mother plate CHO and CHO / g38PP cells were used to investigate normal platelets prepared with mouse blood. Agglutination activity. A is the parent strain CHO and CHO / gp3 8P cells introduced with the gp38P gene described in SEQ ID NO: 13, and the platelet agglutination inhibitory activity was measured. B is the result of measuring the platelet aggregation inhibitory activity of the 8F 11 antibody when the CHO / gp3 8P cells reacted with the 8F11 antibody. Figure 4 shows the platelet agglutination ability of mouse gp38P and human gp36. Using parent strain CHO and CHO / gP3 8P and CHO / gp36 introduced into CHO cells, we explored the normal platelets prepared by blood in mice and humans. Agglutination activity. A: CHO cells were temporarily introduced into the empty pcDNA3 vector (Mock), pcDNA3-gp38P, pcDNA3-gp38P-M41A, pcDNA3-gp36 gene-124-200413526, and each cell lysate was uniquely identified as g P 3 6 of τ Τ 6 7 More than 9 strains of antibodies and 8F1 1 antibodies were the result of western blotting. TT679 antibodies can react with cells into which the pcDNA3-gp36 gene is introduced, and 8 F1] antibodies can react with cells into which the pcDNA3-gp38P gene is introduced. B is a parent strain CHO and a CHO / gp38P cell with the gp38P gene introduced in SEQ ID NO: 13 and a CHO / gP36 cell with the gp36 gene introduced in SEQ ID NO: 7; C is the parent strain CHO and the CHO / gp3 8P cell introduced with the gp38P gene described in SEQ ID NO: 13, and the results of activity measurement with human platelet agglutination were measured using a platelet agglutination meter. D is the parent strain CHO and the C H O / g p 36 cell with the gp36 gene introduced in SEQ ID NO: 7 and the results of measuring platelet aggregation with human platelet aggregation. Fig. 5 shows the results of the investigation of gp 3 8P recognition sites by the 8F11 antibody, which can inhibit gp38P platelet aggregation induction activity. A is a graph showing the results of measuring the platelet aggregation inhibitory activity of the 8F1 1 antibody with platelet agglutination when the CHO / g38P cell reacts with 1 mg of the 8F1 1 antibody or the control antibody. B is E. coli, and a pET-21a vector (manufactured by Novagen) containing a gene encoding the gp 3 8P gene fragment of SEQ ID NO: 13 was introduced, and the protein encoded by the gene was expressed in E. coli. Results of Western blotting of E. coli lysate using 8F1 1 antibody or anti-FLAG antibody. From the left, the E. coli expressing the sequence number 14 and the g p 3 8 P protein amino acid -125-200413526 number 1 4 0,] 4 3, 1 4 6, 1 4 9, 1 5 4, 16 0. C is the result of Western blotting of the E. coli lysate using 8F 11 antibody or anti-FLAG antibody as described above. From left to right, E. coli expressing the amino acid numbers 37-1, 72-39, 172, and 41-172 of the gp38P protein are shown in sequence number 14. D is a graph showing the reactivity of the gP38P protein fragment in E. coli to the 8F11 antibody. Those who are not reactive are white columns, those who are weakly reactive are gray columns, those who are strongly reactive are ocher columns. From this result, it is presumed that the recognition site of the 8F11 antibody is the amino acid number 39 to 44 described in SEQ ID NO: 14. Figure 6 shows the correlation between gp38P recognition site and platelet aggregation activity of the 8F11 antibody. A is the introduction of empty pcDNA3 (Mock) into CHO cells, containing pcDNA3 (WT) containing gp3 8P gene with sequence number 13, or gp3 8P gene containing sequence number 13 with various variants of alanine Western blotting results of gp3 8P pcDNA3, its cell lysate and 8F1 1 antibody or anti-FLAG antibody. B is the sequence number of the sequence list 1 4 amino acid number 3 9 to 47 amino acid sequence ® C-terminal peptide with cysteine amino acid sequence (WTpt: sequence number 44), amino acid number 4 The methionine of 1 is replaced by the peptide of the amino acid sequence of alanine (M41 Apt sequence list sequence number 45), and the amino acid of the amino acid number 42 is replaced by the amino acid sequence of alanine. Peptide (V42Apt: Sequence Listing No. 46), Proline from Amino Acid No. 43 was replaced with the amino acid sequence of Alanine (P4 3 Apt: Sequence Listing No. 47), Amino Acid No. 44 Peptide-126-200413526 (P44Apt: SEQ ID NO: 48) of proline was replaced with amino acid sequence of alanine, and was fixed on a 96-well plate, and the 8F11 antibody and the control group antibody were examined by ELIS A method. Reactive results of peptides. C is the parent strain CHO and CHO / gP3 8P cells introduced with the sequence number 13 described in gP38P gene, and the methionine number 41 in gp38P was replaced by alanine M4 1 A-gp3 8P, and CHO / M41A was expressed in a constant manner. The results of platelet aggregation measurement and platelet aggregation inhibitory activity are shown. D is the recognition site of gP3 8P and the 8F11 antibody that can inhibit platelet aggregation, and 8F 11 antibody and the pattern diagram of the aggregation inhibition mechanism. Figure 7 shows the identification of the necessary sites of platelet aggregation induction activity in gP3 8P and gp36. A is the mutant protein M41A containing the amino acid sequence shown in sequence number 14 in the sequence listing, and the methionine encoding gene of wild-type gp 3 8P amino acid number 41 substituted with alanine. The glutamate-encoding gene of 34 is a mutant protein T3 4 A with an alanine-encoding gene, and the glutamic acid-encoding gene of alanine number 37 is a mutant protein T37A with an amino acid number. The glutamate coding gene of 51 is substituted with the alanine coding gene to obtain the mutant protein T5 1 A, and the glutamic acid coding gene of the amino acid number 5 2 is substituted with the alanine coding gene to obtain the mutant protein T52A. The pc DNA 3 vector encoding the cDNA of each variant protein was introduced into CΗ0 cells to temporarily express each protein on the membrane surface, and the platelet aggregation-inducing activity of each cell was measured with a platelet agglutination meter. B is a diagram of alignment of -127-200413526 in the necessary site of platelet aggregation on the mouse g p 3 8 P identified in A, with homologous proteins of other species. The site required for platelet agglutination on mouse gp 3 8P is not only highly preserved in other congeners, but especially the glutamic acid (sequence number 14 amino acid number 34 in the sequence listing of the mouse gp 3 8P; Figure 7B) *), Others in humans (sequence number 8 amino acid number 52) · mice (sequence number 4 amino acid number 34) · dogs (sequence number 6 amino acid number 4 1 or Homologs of amino acid number 50) are also preserved. C is the gp36-T52A obtained by replacing the glutamic acid encoding gene in the wild type gP36 cDNA (WT) and its number 52 (amino acid number 52 in sequence number 8 of the sequence listing) with the alanine acid encoding gene. Number 8 amino acid number 5 5 Replace the alanine-encoding gene with alanine-encoding gene to obtain gp 3 6-T 5 5 A Each variant encodes a cDNA pcDNA3 vector, and introduces the gene into CHO cells to temporarily attach it to the membrane Each protein is expressed on the surface, and the platelet aggregation-inducing activity of each cell is measured by a platelet agglutination meter. Figure 8 is a graph examining the increase in gp 36 gene expression in colorectal cancer. A is hybridized with P32-labeled human gp36cDN A (sequence number 7) or human ubiquitin cDNA probe in Cancer Profiling Array II membrane (manufactured by BD Biosciences), and each cancer site (τ) and The radioactivity in non-cancer areas (N) was visualized and quantified by a BAS1000 phosphoimage analyzer (manufactured by Fujifilm Corporation), and the expression level of gp 36 gene in cancer cells was examined. B is the difference in expression between cancerous site (T) and non-cancerous site (n) in Jian 疋 A. The radioactivity of human ubiquitin c DNA is used to correct the cancer site (τ) and non-cancerous site (N) of each patient. The expression ratio is quantified and the results are expressed in dots. The level of cancer in all organs -128-200413526 is shown in a bar chart. C is the quantitative result of gp36 gene expression in various organ cancers, such as B. The average 値 of each organ cancer is represented by a bar graph. D is the Cancer Profiling Array II immobilized colorectal cancer patient No. 5 and small bowel cancer patient No. 2 cDN A (sample of circled numbers in Figure 8A) was purchased from BD Biosciences, using it as a template, and the gp36 gene was used for amplification Primers (sequence list sequence numbers 5 3 and 5 4) or the control group R ib 0s 0ma 1 protein S9 gene was amplified primers (sequence list sequence numbers 55 and 56), and the results of PCR were performed. _ Figure 9 shows gp36 protein expression in g-inch colon cancer. Landmark Tissue Microarrays Low Density Colorectal was purchased from Ambion, and the samples were immunostained with TT679 antibody against multiple gp36 antibodies (A, C, E). Hematoxylin-eosin staining (H & E) was used to stain each sample (B, D, F) using Ambion's H & E staining. Figure 10 shows the increase of gp 3 6 gene expression in the metastatic site of colon cancer. Figure A shows the human gp 3 6 c DNA (sequence list sequence number 7) or human labeled with p3 2 in Cancer Profiling Array (manufactured by BD Biosciences). The ubiquitin cDNA probe was hybridized, and the radioactivity in each cancer site (τ), non-cancer site (N), and metastasis site (M) was visualized and quantified using a BAS 1000 phosphoimage analyzer (manufactured by Fujifilm Corporation). 36 gene expression in cancer cells. B is to identify the difference between the expression of -129-200413526 in the cancer site (T) and non-cancer site (N) in A and the metastasis site (M). The specific radioactivity of human ubiquitin cDNA was used to correct the cancer site of each patient ( T) and non-cancer site (N), and metastatic site (M) expression ratios were quantified. In metastatic lesions, and then in cancer sites, there are many cases of increased expression of g p 3 6 gene. Figure 11 is a graph of the nodule of delta towel in mice transplanted with CHO / mock cells and mice transplanted with CHO / g36 cells. Figure 12 is a graph showing the difference in gp36 mRNA expression in cancerous sites and non-cancer g positions in human lung epithelial carcinoma and adenocarcinoma. Fig. 13 shows gP36 expression in human lung flat epithelial carcinoma 'adenocarcinoma and normal lung. There are gp36 expression parts can be stained.

-130-200413526 Preface List

SEQUENCE LISTING &lt; n〇 &gt; Sankyo Co., Ltd. 1 Peptide for promoting platelet aggregation &lt; 130> FP0335 &lt; 150 &gt; IP 2002-298400 &lt; 151> 2002-10-11 &lt; 150 &gt; JP 2003-129954 &lt; 151> 2003-05-08 &lt; 160 &gt; '56 &lt; 170 &gt; Patentln version 3.1 &lt; 210> 1 &lt; 211> 519 &lt; 212 &gt; DNA <川 &gt; Mouse &lt; 220 &gt; ^ <223 &gt; Inventors: Tsurumi Takashi, Fujita Naoya, Osawa Suzuki, Kato Kosei &lt; 220〉 ◊ 21 &gt; CDS &lt; 222 &gt; (1) · (519) &lt; 223 &gt;-&lt; 400〉 1 atg tgg acc gtg cca gtg ttg ttc tgg gtt ttg ggg age gtt tgg ttc Met Trp Thr Yal Pro Val Leu Phe Trp Val Leu Gly Ser Val Trp Phe 1/66 200413526 15 10 15 tgg gac tct gcg cag gga ggg act ata ggc gtg aat aaa aat aat att 96

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val Asn Lys Asn Asn lie 20 25 30 gtg acc cca ggt aca gaa aac ggc atg gig ccc cca ggl ata gaa gac 144

Val Thr Pro Gly Thr Glu Asn Gly Met Val Pro Pro Gly lie Glu Asp 35 40 45 aaa ata aca acc aca ggt get act gga ggg ett aat gaa tct act ggc 192

Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly

50 55 60 aag gca cct ctg gta cca aeg cag aga gag cgt ggg aeg act cct ccc 240

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80 tta gag gaa ctg lec acc tea gca acc tea gac cat gat cac aga gaa 288

Leu Glu Glu Leu Ser. Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95 cac gag agt aca acc act gtc aaa gtg gtg act age cac tel gtg gac 336

His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser His Ser Val Asp

100 105 110 aag aaa aca agt cac ccc aat aga gat aat gca ggg gat gaa aeg cag 384

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115 120 125 aca aca gat aag aaa gat ggc ttg cca gta gtc acc ctg gtt gga ate 432

Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 140 ata gtt ggc gtc ttg tta gcc att ggc ttc gtc gga ggg ate ttc alt 480 lie Yal Gly Yal Leu Leu Ala lie Gly Phe Val Gly Phe lie 2/66 200413526 145 150 155 160 gtt gtt atg aag aag att tct gga agg ttc teg ccc taa Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 &lt; 210〉 2 &lt; 211 &gt; 172 &lt; 212 &gt; PRT &lt; 213〉 mouse <400> 2

Met Trp Thr Val Pro Yal Leu Phe Trp Val Leu Gly Ser Yal Trp Phe 15 10 15

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val Asn Lys Asn Asn lie 20 25 30

Val Thr Pro Gly Thr Glu Asn Gly Met Val Pro Pro Gly lie Glu Asp 35 40 45

Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95 3/66 200413526

His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser His Ser Val Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115 120 125

Thr Thr Asp Lys Lys Asp Gly Leu Pro Yal Yal Thr Leu Val Gly lie 130 135 140 lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly Gly lie Phe lie 145 150 155 160

Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 &lt; 210 &gt; 3 &lt; 211> 501 &lt; 212) DNA &lt; 2Π &gt; Mice &lt; 220 &gt; 〈221〉 CDS 〈222〉 (1) ·· (501) <223> <400> 3-atg tgg acc gcg cca gtg ttg etc tgg gtt ttg gga age gtt tgg ttc Met Trp Thr Ala Pro Yal Leu Leu Trp Yal Leu Gly Ser Val Trp Phe 15 10 15 4/66 200413526 tgg gac tea geg cag gga ggg gee ate ggt geg eta gaa gat gat ct t 96 Trp Asp Ser Ala Gin Gly Gly Ala lie Gly Ala Leu Glu Asp Asp Leu 20 25 30 gtg acc ccg ggt cca gga gat gac atg gtg aac cca ggt eta gaa gac 144 Val Thr Pro Gly Pro Gly Asp Asp Met Yal Asn Pro Gly Leu Glu Asp 35 40 45 aga ata gaa acc aeg • gac act aca gga gaa ctg gat aag tea act gee 192 Arg lie Glu Thr Thr Asp Thr Thr Gly Glu Leu Asp Lys Ser Thr Ala 50 55 60 aag geg cct ctg gta cca aca cag cct ccc at t gag gaa ctg ccg acc 240 Lys Ala Pro Leu Val Pro Thr Gin Pro Pro lie Glu Glu Leu Pro Thr 65 70 75 80 tea gga acc tee gac cac gai cac aaa gaa cat gag agt aeg acc act 288 Ser Gly Thr Ser Asp His Asp His Lys Glu His Glu Ser Thr Thr Thr 85 90 95 gtc aaa geg gtg acc age cac tee aeg gac aag aaa aca act cac ccc 336 Val Lys Ala Val Thr Ser His Ser Thr Asp Lys Lys Thr Thr His Pro 100 105 110 aat aga gat aac gca ggc ggt gaa aeg cag acc aca gat aag aaa gat 384 Asn Arg Asp Asn Ala Gly Gly Glu Thr Gin Thr Thr Asp Lys Lys Asp 115 120 125 ggc ttg get gta gtg acc ctg gtc gga ate ata att ggc gtc ttg tta 432 Gly Leu Ala Val Val Thr Leu Val Gly He lie lie Gly Val Leu Leu 130 • 135 140 gee att ggc ttc att gga ggg ate ate att gtg gtt atg agg aag att 480 Ala lie Gly Phe lie Gly Gly lie lie lie Val Yal Met Arg Lys lie 145 150 155 160

5/66 200413526 tct gga agg ttc teg ccc taa Ser Gly Arg Phe Ser Pro 165 &lt; 210> 4 &lt; 211> 166 &lt; 212 &gt; PRT 〈213> Rat &lt; 400 &gt; 4

Met Trp Thr Ala Pro Val Leu Leu Trp Val Leu Gly Ser Val Trp Phe 15 10 15

Trp Asp Ser Ala Gin Gly Gly Ala lie Gly Ala Leu GIu Asp Asp Leu 20 25 30

Val Thr Pro Gly Pro Gly Asp Asp Met Yal Asn Pro Gly Leu Glu Asp 35 40 45

Arg lie Glu Thr Thr Asp Thr Thr Gly Glu Leu Asp Lys Ser Thr Ala 50 55 60

Lys Ala Pro Leu Val Pro Thr Gin Pro Pro lie Glu Glu Leu Pro Thr 65 70 75 80

Ser Gly Thr Ser Asp His Asp His Lys Glu His Glu Ser Thr Thr Thr 85 90 95 6/66 200413526

Val Lys Ala Val Thr Ser His Ser Thr Asp Lys Lys Thr Thr His Pro 100 105 110

Asn Arg Asp Asn Ala Gly Gly Glu Thr Gin Thr Thr Asp Lys Lys Asp 115 120 125

Gly Leu Ala Val Val Thr Leu Val Gly lie lie lie Gly Val Leu Leu 130 135 140

Ala lie Gly Phe lie Gly Gly lie lie lie Val Val Met Arg Lys lie 145 150 155 160

Ser Gly Arg Phe Ser Pro 165 〈210〉 5 &lt; 211 &gt; 510 &lt; 212 &gt; DNA &lt; 213> dog &lt; 220 &gt; &lt; 221 &gt; CDS 〈222> (1): (510) &lt; 223 &gt; &lt; 400 &gt; 5-atg tgg agg gtg cca gtc ctg etc ttg gtt ttg ggc ggc geg ggg etc Met Trp Arg Val Pro Val Leu Leu Leu Val Leu Gly Gly Ala Gly Leu 15 10 15 7/66 200413526 egg gtc ccg gca gca gga gee age aca gtc egg cca gat gac ata ata 96

Arg Val Pro Ala Ala Gly Ala Ser Thr Yal Arg Pro Asp Asp lie lie 20 25 30 cca ggt gta gaa gac age gtg gtg acc cca ggc aca gaa gac age gtg 144

Pro Gly Val Glu Asp Ser Val Val Thr Pro Gly Thr Glu Asp Ser Val 35 40 45 gtg aeg cca ggc gca gaa gat aac gtg gtg act gac ggt gee act gaa 192

Val Thr Pro Gly Ala Glu Asp Asn Val Val Thr Asp Gly Ala Thr Glu 50 55 60 gag cct tat gag tet ggc tta acc cct ctg gtg aca aaa aat aca gag 240

Glu Pro Tyr Glu Ser Gly Leu Thr Pro Leu Val Thr Lys Asn Thr Glu 65 70 75 80 agt gta aca gac tta cac tta gag gat ggg ccg act caa gaa age aca 288

Ser Val Thr Asp Leu His Leu Glu Asp Gly Pro Thr Gin Glu Ser Thr 85 90 95 gtc cat gee aaa gaa gag age cag age acc aca acc ttg aat gta gtg 336

Val His Ala Lys Glu Glu Ser Gin Ser Thr Thr Thr Leu Asn Val Val 100 105 110 act agt cac tee aga gag aaa gta ggt gaa gac acc gag aca aeg gtt 384

Thr Ser His Ser Arg Glu Lys Val Gly Glu Asp Thr Glu Thr Thr Val 115 120 125 gag aaa gat ggc ctg gca aca gtg acc ctg gtt ggg ate ata git gga 432

Glu Lys Asp Gly Leu Ala Thr Yal Thr Leu Val Gly lie lie Val Gly 130 135 140 gtc tta eta gee ati gga ttc att ggt ggg ate ate att gtg gtt get 480

Val Leu Leu Ala lie Gly Phe lie Gly Gly ly lie lie Yal Val Ala 145 150 155 160 8/66 200413526 cga aaa atg tea gga agg tac teg ccc taa 510

Arg Lys Met Ser Gly Arg Tyr Ser Pro 165 <210> 6 &lt; 211> 169 &lt; 212 &gt; PRT &lt; 213> dog &lt; 400> 6

Met Trp Arg Val Pro Val Leu Leu Leu Val Leu Gly Gly Ala Gly Leu 15 10 15

Arg Val Pro Ala Ala Gly Ala Ser Thr Val Arg Pro Asp Asp lie lie 20 25 30

Pro Gly Yal Glu Asp Ser Val Val Thr Pro Gly Thr Glu Asp Ser Val 35 40 45

Val Thr Pro Gly Ala Glu Asp Asn Val Val Thr Asp Gly Ala Thr Glu 50 55 60

Glu Pro Tyr Glu Ser Gly Leu Thr Pro Leu Val Thr Lys Asn Thr Glu 65 70 75 80

Ser Yal Thr Asp Leu His Leu Glu Asp Gly Pro Thr Gin Glu Ser Thr 85 90 95 9/66 200413526

Val His Ala Lys Glu Glu Ser Gin Ser Thr Thr Thr Leu Asn Val Val 100 105 110

Thr Ser His Ser Arg Glu Lys Val Gly Glu Asp Thr Glu Thr Thr Val 115 120 125

Glu Lys Asp Gly Leu Ala Thr Val Thr Leu Val Gly lie lie Val Gly 130 135 140

Val Leu Leu Ala lie Gly Phe lie Gly Gly lie lie lie Val Val Ala 145 150 155 160

Arg Lys Met Ser Gly Arg Tyr Ser Pro '165 &lt; 210> 7 &lt; 211 &gt; 489 &lt; 212> DNA &lt; 213> person <220> &lt; 221 &gt; CDS &lt; 222 &gt; (1) .. (489) ) &lt; 223〉 &lt; 400 &gt; 7 atg tgg aag gtg tea get ctg etc ttc gtt ttg gga age geg teg etc 48

Met Trp Lys Val Ser Ala Leu Leu Phe Val Leu Gly Ser Ala Ser Leu 15 10 15 tgg gtc ctg gca gaa gga gcc age aca ggc cag cca gaa gat gac act 96 10/66 200413526

Trp Val Leu Ala Glu Gly Ala Ser Thr Gly Gin Pro Glu Asp Asp Thr 20 25 30 gag act aca ggt ttg gaa ggc ggc gtt gcc atg cca ggt gcc gaa gat 144 Glu Thr Thr Gly Leu Glu Gly Gly Val Ala Met Pro Gly Ala Glu Asp 35 40 45 gat gtg gtg act cca gga acc age gaa gac ege tat aag tet ggc ttg 192 Asp Val Val Thr Pro Gly Thr Ser Glu Asp Arg Tyr Lys Ser Gly Leu 50 55 60 aca act ctg gtg gca aca agt gtc aac agt gta aca ggc att ege ate 240 Thr Thr Leu Val Ala Thr Ser Val Asn Ser Val Thr Gly lie Arg lie 65 70 75 80 gag gat ctg cca act tea gaa age aca gtc cac geg caa gaa caa agt 288 Glu Asp Leu Pro Thr Ser Glu Ser Thr Val His AlaGln Glu Gin Ser 85 90 95 cca age gcc aca gcc tea aac gtg gcc acc agt cac tee aeg gag aaa 336 Pro Ser Ala Thr Ala Ser Asn Val Ala Thr Ser His Ser Thr Glu Lys 100 105 110 gtg gat gga gac aca cag aca aca gtt gag aaa gat ggt ttg tea aca 384 Yal Asp Gly Asp Thr Gin Thr Thr Val Glu Ly s Asp Gly Leu Ser Thr 115 120 125 gtg acc ctg gtt gga ate ata gtt ggg gtc tta eta gcc ate ggt t tc 432 Val Thr Leu Val Gly lie lie Yal Gly Val Leu Leu Ala lie Gly Phe 130 135 140 att ggt gga ate ate git gtg gtt atg ega aaa atg teg gga agg tac 480 lie Gly Gly lie lie Yal Val Val Met Arg Lys Met Ser Gly Arg Tyr 145 150 155 160 teg ccc taa 489

11/66 200413526

Ser Pro &lt; 210〉 8 &lt; 211〉 162 &lt; 212〉 PRT &lt; 213〉 person &lt; 400〉 8

Met Trp Lys Val Ser Ala Leu Leu Phe Yal Leu Gly Ser Ala Ser Leu 1.5 10 15

Trp Yal Leu Ala Glu Gly Ala Ser Thr Gly Gin Pro Gtu Asp Asp Thr 20 25 30

Glu Thr Thr Gly Leu Glu Gly Gly Val Ala Met Pro Gly Ala Glu Asp 35 40 45

Asp Val Val Thr Pro Gly Thr Ser Glu Asp Arg Tyr Lys Ser Gly Leu

Thr Thr Leu Val Ala Thr Ser Yal Asn Ser Val Thr Gly lie Arg lie 65 70 75 80

Glu Asp Leu Pro Thr Ser Glu Ser Thr Yal His Ala Gin Glu Gin Ser 85 90 95

Pro Ser Ala Thr Ala Ser Asn Yal Ala Thr Ser His Ser Thr Glu Lys 12/66 200413526 1.00 1 05 1 10

Yal Asp Gly Asp Thr Gin Thr Thr Val Glu Lys Asp Gly Leu Ser Thr 115 120 125

Val Thr Leu Val Gly lie lie Val Gly Val Leu Leu Ala lie Gly Phe 130 135 140 lie Gly Gly lie lie Val Val Val Met Arg Lys Met Ser Gly Arg Tyr 145 150 155 160

Ser Pro &lt; 210 &gt; 9 <211> 23 &lt; 212 &gt; DNA &lt; 213> Artificial &lt; 220 &gt; <223> gp38P PCR synonym primer &lt; 400 &gt; 9 tcaagatgtg gaccgtgcca gtg &lt; 210> 10- &lt; 211> 23 &lt; 212 &gt; DNA &lt; 213〉 Artificial 13/66 200413526 &lt; 220 &gt; &lt; 223〉 gp38P PCR antisense primer &lt; 400 &gt; 10 cgccatgggt catcttcctc cac 〈210〉 11 &lt; 211 &gt; 31 &lt; 212> DNA &lt; 213> artificial &lt; 220> <223> gpMP synonymous primer &lt; 400 &gt; .11 ctcaagct tc aagatgtgga ccgtgccagt &lt; 210> 12 &lt; 211> 31. &lt; 212 &gt; DNA 〈213> artificial &lt; 220 &gt; 〈 223> Antisense primer &lt; 400 &gt; 12 gaggaattcg ggcgagaacc ttccagaaat 〈210〉 13-〈211〉 540 〈212〉 DNA 〈213〉 Mus musculus 200413526 &lt; 220> &lt; 221 &gt; CDS &lt; 222 &gt; (1): (534) &lt; 223 &gt; &lt; 400> 13 tcc aac gag acc'aag atg tgg acc gig cca gtg ttg ttc tgg gtt ttg 48

Ser Asn Glu Thr Lys Met Trp Thr Yal Pro Yal Leu Phe Trp Val Leu 15 10 15 ggg age gtt tgg ttc tgg gac tet geg cag gga ggg act ata ggc gtg 96

Gly Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Yal 20 25 30 aat gaa gat gat att gtg acc cca ggt aca gga gac ggc atg gtg ccc 144

Asn Glu Asp Asp lie Val Thr Pro Gly Thr Gly Asp Gly Met Val Pro 35 40 .45 cca ggt ata gaa gac aaa ata aca acc aca ggt get act gga ggg ett 192

Pro Gly lie Glu Asp Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu 50 55 60 aat gaa tet act ggc aag gca cct ctg gta cca aeg cag aga gag cgt 240

Asn Glu Ser Thr Gly Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg 65 70 75 80 ggg aeg aag cct ccc tta gag gaa ctg tcc acc tea gca acc tea gac 288

Gly Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp 85 90 95 cat gat cac aga gaa cac gag agt aca acc act gtc aaa gtg gtg act 336

His Asp Hi-s Arg Glu His Glu Ser Thr Thr Thr Val Lys Val Val Thr 100 105 110 age cac tet gtg gac aag aaa aca agt cac ccc aai aga gat aat gca 384

Ser His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala 15/66 200413526 115 120 125 ggg gat gaa aeg cag aca aca gat aag aaa gat ggc ttg cca gta gtc 432 Gly Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val 130 135 140 acc ctg gtt gga ate ata gtt ggc gtc ttg t ta gcc att ggc t tc gtc 480 Thr Leu Val Gly lie lie Val Gly Val Leu Leu Ala lie Gly Phe Val 145 150 155 160 gga ggg ate t tc att gtt gtt atg aag aag at t tet gga agg ttc teg 528 Gly Gly He Phe lie Val Val Met Lys Lys lie Ser Gly Arg Phe Ser 165 170 175 ccc taa agaget 540 Pro

&lt; 210 &gt; 14 &lt; 211 &gt; 177 &lt; 212〉 PRT &lt; 213〉 mouse

&lt; 400 &gt; 14

Ser Asn Glu Thr Lys Met Trp Thr Val Pro Val Leu Phe Trp Val Leu 15 10 15

Gly Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val -20 25 30

Asn Glu Asp Asp lie Val Thr Pro Gly Thr Gly Asp Gly iMet Val Pro 35 40 45 16/66 200413526

Pro Gly lie Glu Asp Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu 50 55 60

Asn Glu Ser Thr Gly Lys Ala Pro Leu Yal Pro Thr Gin Arg Glu Arg 65 70 75 80

Gly Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp 85 90 95

His Asp His Arg Glu His Glu Ser Thr Thr Thr Yal Lys Val Val Thr 100 105 110

Ser His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala 115 120 125

Gly Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Yal 130 135 140

Thr Leu Val Gly lie lie Val Gly Val Leu Leu Ala lie Gly Phe Val 145 150 155 160

Gly Gly lie Phe lie Val Yal Met Lys Lys lie Ser Gly Arg Phe Ser • 165 170 175

Pro 17/66 200413526 &lt; 210 &gt; 15 &lt; 211> 24 &lt; 212) DNA &lt; 213> Artificial &lt; 220 &gt; &lt; 223 &gt; / 3-Actin PCR antisense primer &lt; 400 &gt; 15 gatatcgctg cgctggtcgt cgac &lt; 210 &gt; 16 &lt; 211〉 24 &lt; 212) DNA &lt; 213〉 artificial &lt; 220〉 &lt; 223〉 0-actin PCR antisense primer &lt; 400 &gt; 16 caagaaggaa ggctggaaaa gage 24

twenty four

&lt; 210 &gt; 17 &lt; 211 &gt; 31 &lt; 212) DNA &lt; 213〉 artificial &lt; 220〉-&lt; 223〉 gP38P PCR synonym primer &lt; 400> 17 ctcaagcttc aagatgtgga ccgtgccagt g 18/66 31 200413526 &lt; 210> 18 &lt; 211 &gt; 49 &lt; 212> DNA 〈213 &gt; artificial 〈220〉 〈223〉 gp38P PCR antisense primer 〈400〉 18 ttacttgtcg tcatcgtctt tgtagtcggg cgagaacctt ccagaaatc 49

&lt; 210 &gt; 19 <211> 31 &lt; 212> DNA &lt; 213 &gt; Artificial &lt; 220 &gt; &lt; 223> gp36 PCR synonym primer &lt; 400 &gt; 19 ctcaagcttg aacgatgtgg aaggtgtcag &lt; 210 &gt; 20 &lt; 211> 29 &lt; 212> DNA &lt; 213 &gt; Artificial &lt; 220 &gt; &lt; 223> PCR antisense primer for gp36 <400> 20 gaggaattcg ggcgagtacc Itcccgaca 31

29 19/66 200413526 &lt; 210) 21 &lt; 211 &gt; 15 &lt; 212〉 PRT &lt; 213〉 Artificial &lt; 220 &gt; &lt; 223〉 Preparation of partial peptide of antisense gP36 polyclonal antibody &lt; 400> 21

Cys Glu Gly Giy Val Ala Met Pro Gly Ala Glu Asp Asp Val Val • 1 5 10 15 <210> 22 &lt; 211 &gt; 540 &lt; 212 &gt; DNA &lt; 213> Artificial &lt; 220>

&lt; 223〉 G38A &lt; 220〉 &lt; 221〉 CDS &lt; 222〉 (16) ·. (534) &lt; 223〉 &lt; 400〉 22 tccaacgaga ccaag atg tgg acc gtg cca gtg ttg ttc tgg gtt ttg ggg Met Trp Thr Yal Pro Val Leu Phe Trp Val Leu Gly 1 5 10 age gtt tgg ttc tgg gac tet geg cag gga ggg act ata ggc gtg aat Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Yal Asn 20/66 200413526 15 20 25 gaa gat gat att gtg acc cca ggt aca gca gac ggc atg gtg ccc cca 147 Glu Asp Asp lie Val Thr Pro Gly Thr Ala Asp Gly Met Val Pro Pro 30 35 40 ggt ata gaa gac aaa ata aca acc aca ggt get act gga ggg ct t aat 195 Gly lie Glu Asp Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn 45 50 55 60 gaa tct act ggc aag gca cct ctg gta cca acg cag aga gag cgt ggg 243 Glu Ser Thr Gly Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly 65 70 75 acg aag cct ccc t ta gag gaa ctg tcc acc tea gca acc tea gac cat 291 Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His 80 85 9 0 gat cac aga gaa cac gag agt aca acc act gtc aaa gtg gtg act age 339 Asp His Arg Glu His Glu Ser Thr Thr Thr Val Lys Val Yal Thr Ser 95 100 105 cac tct gtg gac aag aaa aaa aca agt cac ccc aat aga gat aat gca ggg 387 His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 110 115 120 gat gaa acg cag aca aca gat aag aaa gat ggc itg cca gta gtc acc 435 Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr 125 130 135 140 ctg gtt gga ate ala gtt ggc gtc ttg tta gcc att ggc ttc gtc gga 483 Leu Val Gly lie lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly 145 150 155 ggg ate ttc att gtt gtt atg aag aag att tct gga agg ttc teg ccc 531 Gly lie Phe lie Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro

21/66 200413526 160 165 170 taa agagct &lt; 210) 23 &lt; 211〉 172 &lt; 212) PRT &lt; 213〉 manual &lt; 220 &gt; &lt; 223〉 G38A &lt; 220> 〈221〉 MUTAGEN &lt; 222〉 (38) &lt; 400> 23

Met Trp Thr Yal Pro Val Leu Phe Trp Val Leu Gly Ser Val Trp Phe 15 10 15

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val Asn Glu Asp Asp lie 20 25 30

Val Thr Pro Gly Thr Ala Asp Gly Met Val Pro Pro Gly lie Glu Asp 35 40 45

Lys lie Th-r Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 22/66 200413526 65 70 75 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95

His Glu Ser Thr Thr Thr Val Lys Val Yal Thr Ser His Ser Val Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115 120 125

Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 140,

He Yal Gly Yal Leu Leu Ala lie Gly Phe Val Gly Gly lie Phe lie 145 150 155 160

Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 &lt; 210 &gt; 24 &lt; 211〉 540 &lt; 212〉 DNA &lt; 213〉 artificial &lt; 220〉

&lt; 223〉 G40A &lt; 220 &gt; 23/66 200413526 &lt; 221 &gt; CDS &lt; 222〉 (16): (534) &lt; 223〉 &lt; 400〉 24 tccaacgaga ccaag atg tgg acc gig cca gtg ttg ttc tgg gtt ttg ggg 51

Met Trp Thr Yal Pro Val Leu Phe Trp Yal Leu Gly 1 5 10 age gtt tgg ttc tgg gac tet geg cag gga ggg act ata ggc gig aat 99

Ser Yal Trp Phe Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val Asn

15 20 25 gaa gat gat att gtg acc cca ggt aca gga gac gcc atg gtg ccc cca 147

Glu Asp Asp lie Val Thr Pro Gly Thr Gly Asp Ala Met Yal Pro Pro 30 35 40 ggt ata gaa gac aaa ata aca acc aca ggt get act gga ggg ett aat 195

Gly lie Glu Asp Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn 45 50 55 60 gaa tet act ggc aag gca cct ctg gta cca aeg cag aga gag cgt ggg 243

Glu Ser Thr Gly Lys Ala Pro Leu Yal Pro Thr Gin Arg Glu Arg Gly 65 70 75

aeg aag cct ccc tta gag gaa ctg tee acc tea gca acc tea gac cat 291

Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His 80 85 90 gat cac aga gaa cac gag agt aca acc act gtc aaa gtg gtg act age 339

Asp His Arg Glu His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser 95 · 100 105 cac tet gtg gac aag aaa aca agt cac ccc aat aga gat aat gca ggg 387

His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 110 115 120 24/66 200413526 gat gaa acg cag aca aca gat aag aaa gat ggc ttg cca gta gtc acc Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Yal Val Thr 125 130 135 140 ctg gtt gga ate ata gti ggc gtc ttg tta gee att ggc ttc gtc gga Leu Val Gly lie lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly 145 150 155 ggg ate ttc att gtt atg aag aag att tet gga agg ttc teg ccc Gly lie Phe lie Yal Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 160 165 170 taa agaget 〈210〉 25 &lt; 211 &gt; 172 &lt; 212 &gt; PRT ◊ 13 &gt; artificial &lt; 220 &gt; &lt; 223 &gt; G40A <220> &lt; 221> MUTAGEN &lt; 222> (40) &lt; 400> 25

Met Trp Thr Yal Pro Val Leu Phe Trp Val Leu Gly Ser Val Trp Phe 1-5 10 15

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val Asn Glu Asp Asp lie 20 25 30 25/66 200413526

Yal Thr Pro Gly Thr Gly Asp Ala Met Val Pro Pro Gly lie Glu Asp 35 40 45

Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95

His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser His Ser Val Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115-120 125

Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 140 lie Yal Gly Yal Leu Leu Ala lie Gly Phe Val Gly Gly lie Phe lie 145-150 155 160

Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 26/66 200413526 &lt; 210 &gt; 26 &lt; 211〉 540 &lt; 212) DNA &lt; 213〉 artificial &lt; 220〉

&lt; 223 &gt; M41A &lt; 220 &gt; <221> CDS <222> (16) .: (534) &lt; 223> <400> 26 tccaacgaga ccaag atg tgg acc gtg cca gtg ttg ttc tgg gtt itg ggg Met Trp Thr Yal Pro Yal Leu Phe Trp Val Leu Gly 1 5 10 age gtt tgg ttc tgg gac tet geg cag gga ggg act ata ggc gtg aat

Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Tly lie Gly Val Asn 15 20 25 gaa gat gat att gtg acc cca ggt aca gga gac ggc geg gtg ccc cca

Glu Asp Asp lie Val Thr Pro Gly Thr Gly Asp Gly Ala Val Pro Pro 30 35 40 ggt ata gaa gac aaa ata aca acc aca ggt get act gga ggg ett aat

Gly lie Glu Asp Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn 45 50 55 60 gaa tet act ggc aag gca cct ctg gta cca aeg cag aga gag cgt ggg

Glu Ser Thr Gly Lys Ala Pro Leu Yal Pro Thr Gin Arg Glu Arg Gly 65 70 75 27/66 291200413526 acg aag cct ccc tta gag gaa ctg tcc acc tea gca acc tea gac cat Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His 80 85 90 gat cac aga gaa cac gag agl aca acc act gtc aaa gtg gtg act age Asp His Arg Glu His Glu Ser Thr Thr Thr Val Lys Yal Val Thr Ser 95 100 105 cac tet gtg gac aag aaa aca agt cac ccc aat aga gat aat gca ggg His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 11.0 115 120 gat gaa acg cag aca aca gat aag aaa gat ggc ttg cca gta gtc acc Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr 125 130 135 140 ctg gtt gga ate ata gtt ggc gtc ttg tta gcc attgge tie gtc gga Leu Val Gly lie lie Yal Gly Val Leu Leu Ala lie Gly Phe Val Gly 145 150 155 ggg ate ttc att gtt gtt atg aag aag att tet gga agg ttc teg ccc Gly lie Phe lie Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 160 165 170 339 387 387 435 483 531 taa agaget 540 &lt; 210 &gt; 27 〈211〉 172 &lt; 212 &gt; PRT &lt; 213〉 Manual &lt; 220 &gt; &lt; 223〉 M41A &lt; 220 &gt; 28/66 200413526 &lt; 221 &gt; MUTAGEN &lt; 222 &gt; (41) &lt; 400 &gt; 27

Met Trp Thr Val Pro Val Leu Phe Trp Yal Leu Gly Ser Val Trp Phe 1 5 10 15

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Yal Asn Glu Asp Asp lie 20 25 30

Val Thr Pro Gly Thr Gly Asp Gly Ala Val Pro Pro Gly lie Glu Asp 35 40 45

Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95

His Glu Ser Thr Thr Thr Yal Lys Yal Val Thr Ser His Ser Val Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115 120 125 29/66 200413526

Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 140 lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly Gly lie Phe lie 145 150 155 160

Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 &lt; 210〉 28 &lt; 211 &gt; 540 &lt; 212 &gt; DNA &lt; 213〉 artificial &lt; 220〉 &lt; 223〉 V42A, &lt; 220〉 &lt; 221 &gt; CDS. &Lt; 222〉 (16) · (534) &lt; 223〉 &lt; 400〉 28. Tccaacgaga ccaag atg tgg acc gtg cca gtg ttg ttc tgg gtt ttg ggg Met Trp Thr Val Pro Val Leu Phe Trp Val Leu Gly 1 5 10 age gtt tgg ttc tgg gac let geg cag gga ggg act ata ggc gtg aat Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val Asn 15 20 25 gaa gat gat att gtg acc cca ggt aca gga gac ggc atg geg ccc cca 30/66 200413526

Glu Asp Asp lie Yal Thr Pro Gly Thr Gly Asp Gly Mel Ala Pro Pro 30 35 40 ggt ata gaa gac aaa ata aca acc aca ggt get act gga ggg ett aat

Gly lie Glu Asp Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn 45 50 55 60 gaa tet act ggc aag gca cct ctg gta cca aeg cag aga gag cgt ggg

Glu Ser Thr Gly Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly 65 70 75 aeg aag cct ccc tta gag gaa ctg tee acc tea gca acc. Tea gac cat

Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His 80 85 90. Gat cac aga ga a cac gag agt aca acc act gtc aaa gtg gtg act age

Asp His Arg Glu His Glu Ser Thr Thr Thr Val Lys Yal Val Thr Ser 95 100 105 cac tet gtg gac aag aaa aca agt cac ccc aat aga gat aat gca ggg

His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 110 115 120 gat gaa aeg cag aca gat aag aaa gat ggc ttg cca gta gtc acc

Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Yal Val Thr 125 130 135 140 ctg gtt gga ate ata gtt ggc gtc ttg tta gee att ggc ttc gtc gga

Leu Val Gly lie He Yal Gly Val Leu Leu Ala lie Gly Phe Val Gly 145 150 155 ggg ate ttc att gtt gtt atg aag aag att tet gga agg ttc teg ccc

Gly lie Phe lie Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 160 165 170 taa agaget 31/66 200413526 &lt; 210> · 29 &lt; 211 &gt; ^ 172 &lt; 212) PRT &lt; 213〉 labor & 220 〉

&lt; 223 &gt; V42A &lt; 220〉 &lt; 221) MUTAGEN &lt; 222〉 (42) &lt; 400 &gt; 29

Met Trp Thr Val Pro Val Leu Phe Trp Val Leu Gly Ser Val Trp Phe 15 10 15

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Yal Asn Glu Asp Asp lie 20 25 30

Val Thr Pro Gly Thr Gly Asp Gly Met Ala Pro Pro Gly He Glu Asp 35 40 45

Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80 32/66 200413526

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95

His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser His Ser Val Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115 120 125

Thr Thr Asp Lys Lys Asp Gly Leu Pro Yal Val Thr Leu Val Gly lie 130 135 140 lie Val Gly Val Leu Leu Ala He Gly Phe Val Gly Gly lie Phe lie 145 150 155 160

Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170

&lt; 210> 30 &lt; 211> 540 <212> DNA &lt; 213> Artificial &lt; 220 &gt; &lt; 223 &gt; P43A <220>

<221> CDS &lt; 222> (16) ·. (534) &lt; 223> 33/66 200413526 &lt; 400> 30 tccaacgaga ccaag atg tgg acc gtg cca gtg ttg ttc tgg gtt ttg ggg 51

Met Trp Thr Val Pro Yal Leu Phe Trp Val Leu Gly 1 5 10 age gtt tgg ttc tgg gac tet geg cag gga ggg act ata ggc gtg aat 99

Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Tly lie Gly Val Asn 15 20 25 gaa gat gat att gtg acc cca ggt aca gga gac ggc atg gtg geg cca 147

Glu Asp Asp lie Val Thr Pro Gly Thr Gly Asp Gly Met Yal Ala Pro 30 35 40 ggt ata gaa gac aaa ata aca acc aca ggt get act gga ggg ett aat 195

Gly lie Glu Asp Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn 45 50 55 60 gaa tel act ggc aag gca cct ctg gta cca aeg cag aga gag cgt ggg 243

Glu Ser Thr Gly Lys Ala Pro Leu Val Pro Thr. Gin Arg Glu Arg Gly 65 70 75 aeg aag cct ccc tta gag gaa ctg tee acc tea gca acc tea gac cat 291

Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His

80 85 90 t gat cac aga gaa cac gag agt aca acc act gtc aaa gtg gtg act age 339

Asp His Arg Glu His Glu Ser Thr Thr Thr Yal Lys Val Val Thr Ser 95 100 105 cac tet gtg gac aag aaa aca agt cac ccc aat aga gat aat gca ggg 387

His Ser Va-1 Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 110 115 120 gat gaa aeg cag aca gat aag aaa gat ggc ttg cca gta gic acc 435

Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Yal Val Thr 34/66 200413526 125 130 135 140 ctg git gga a (c aia gtt ggc gic itg tia gcc ait ggc iic gic gga

Leu Yal Gly lie He Val Gly Val Leu Leu Ala lie Gly Phe Val Gly 145 150 155 ggg ate ttc att gtt gtt atg aag aag att tet gga agg ttc teg ccc Gly lie Phe lie Yal Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 160 165 170 taa agaget &lt; 210> 31 <211> 172 &lt; 212> PRT <213 &gt; Manual &lt; 220 &gt; &lt; 223 &gt; P43A <220>

&lt; 221> MUTAGEN <222> (43), &lt; 400> 31

Met Trp Thr Val Pro Val Leu Phe Trp Val Leu Gly Ser Val Trp Phe Γ 5 10 15

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val Asn Glu Asp Asp lie 20 25 30

Val Thr Pro Gly Thr Gly Asp Gly Met Yal Ala Pro Gly lie Glu Asp 35/66 200413526 35 40 45

Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu

His Glu Ser Thr Thr Thr Yal Lys Val Yal Thr Ser His Ser Yal Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115 120 125

Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 140 lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly Gly lie Phe lie 145 150 155 160

Val Val Me-t Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 &lt; 210〉 32 36/66 200413526 &lt; 211) 540 &lt; 212) DNA &lt; 213〉 artificial &lt; 220 &gt; ’

<223> G45A &lt; 220 &gt; <221> CDS &lt; 222> (16) · (534) &lt; 223 &gt; &lt; 400 &gt; 32 tccaacgaga ccaag atg tgg acc gtg cca gtg ttg ttc tgg gtt ttg ggg Met Trp Thr Val Pro Val Leu Phe Trp Yal Leu Gly 1 5 10 age gtt tgg ttc tgg gac tet geg cag gga ggg act ata ggc gtg aat

Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Yal Asn 15 20 25 gaa gat gal att gtg acc cca ggt aca gga gac ggc atg gtg ccc cca

Glu Asp Asp lie Val Thr Pro Gly Thr Gly Asp Gly Met Val Pro Pro 30 35 40 get ata gaa gac aaa ata aca acc aca ggt get act gga ggg ett aat

Ala lie Glu Asp Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn 45 50 55 60 gaa tet act ggc aag gca cct ctg gta cca aeg cag aga gag cgt ggg

Glu Ser Thr Gly Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly-65 70 75 aeg aag cct ccc tta gag gaa ctg tee acc tea gca acc tea gac cat

Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His '80 85 90 37/66 200413526 gat cac aga gaa cac gag agt aca acc act gtc aaa gig gig act age

Asp His Arg Glu His Glu Ser Thr Th: Thr Val Lys Val Val Thr Ser 95 100 105 cac tet gtg gac aag aaa aca agt cac cat aat aga gat aat gca ggg

His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 110 115 120 gat gaa aeg cag aca gat aag aaa gat ggc ttg cca gta gtc acc

Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Yal Val Thr 125 130 135 140 ctg gtt gga ate ata gtt ggc gtc ttg tta gee att ggc ttc gtc gga

Leu Val Gly lie lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly 145 150 155 ggg ate ttc att gtt gtt atg aag aag att tet gga agg ttc teg ccc

Gly lie Phe lie Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 160 165 170 taa agaget &lt; 210 &gt; 33 <211> 172 &lt; 212 &gt; PRT 〈213 &gt; manual &lt; 220>

&lt; 223〉 G45A &lt; 220 &gt; &lt; 221 &gt; MUTAGEN &lt; 222〉 (45) 38/66 200413526 &lt; 400〉 33

Met Trp Thr Yal Pro Val Leu Phe Trp Val Leu Gly Ser Val Trp Phe 15 10 15

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Yal Asn Glu Asp Asp lie 20 25 30

Yal Thr Pro Gly Thr Gly Asp Gly Met Val Pro Pro Ala lie Glu Asp

Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95

His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser His Ser Val Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 11-5 120 125

Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 140 39/66 200413526 lie Va! Gly Val Leu Leu Ala lie Gly Phe Val Gly Gly lie Phe lie 145 150 155 160

Val Yal Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 &lt; 210) 34 &lt; 211 &gt; 540 〈212〉 DNA 〈213〉 Artificial 〈220〉 〈223〉 K49A lt 220 and 221 cds &lt; 222〉 (16): (534) &lt; 223〉 &lt; 400〉 34 tccaacgaga ccaag atg tgg acc gtg cca gtg ttg ttc tgg gtt ttg ggg Met Trp Thr Val Pro Val Leu Phe Trp Yal Leu Gly 1 5 10 age gtt tgg ttc tgg gac tet geg cag gga ggg act ata ggc gtg aat

Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Tly lie Gly Val Asn 15 20 25 gaa gat gat att gtg acc cca ggt aca gga gac ggc atg gtg ccc cca

Glu Asp Asp lie Val Thr Pro Gly Thr Gly Asp Gly Met Val Pro Pro 30 35 40 40/66 200413526 ggt ata gaa gac gca ata aca acc aca ggt get act gga ggg c 11 aat 195 Gly He Glu Asp Ala lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn 45 50 55 60 gaa tci act ggc aag gca cct ctg gta cca acg cag aga gag cgt ggg 243 Glu Ser Thr Gly Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly 65 70 75 acg aag cct ccc t ta gag gaa ctg tcc acc tea gca acc tea gac cat 291 Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His 80 85 90 gat cac aga gaa cac gag agt aca acc act gtc aaa gtg gtg act age 339 Asp His Arg Glu His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser 95 100 105 cac tct gtg gac aag aaa aca agt cac ccc aat aga gat aat gca ggg 387 His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 110 115 120 gat gaa acg cag aca aca gat aag aaa gat ggc ttg cca gta gtc acc 435 Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr 125 130 135 140 ctg gtt gga ate ata gtt ggc gtc ttg tta gec att ggc ttc gtc gga 483 Leu Val Gly lie lie Yal Gly Yal Leu Leu Ala lie Gly Phe Val Gly 145 150 155 ggg ate t t att gtt gtt atg aag aag at t tct gga agg t tc teg ccc 531 Gly lie Phe lie Val Yal Met Lys Lys lie Ser Gly Arg Phe Ser Pro 160 165 170 taa agaget 540

&lt; 210〉 35 41/66 200413526

&lt; 211〉 172 &lt; 212) PRT &lt; 213〉 manual &lt; 220 &gt; &lt; 223 &gt; K49A &lt; 220〉 &lt; 221〉 MUTAGEN &lt; 222 &gt; (49)

&lt; 400 &gt; 35

Met Trp Thr Val Pro Val Leu Phe Trp Val Leu Gly Ser Yal Trp Phe 15 10 15

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Yal Asn Glu Asp Asp lie 20 25 30

Yal Thr Pro Gly Thr Gly Asp Gly Met Val Pro Pro Gly lie Glu Asp 35 40 45

Ala lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75. 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95 42/66 200413526

His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser His Ser Val Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp .Glu Thr Gin 115 120 125

Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 140 lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly Gly lie Phe lie 145 150 155 160

Val Yal Met Lys Lys lie Ser Gly Arg Phe Ser Pro .165 170 〈210〉 36 &lt; 211〉 540 &lt; 212 &gt; DNA &lt; 213> Artificial &lt; 220 &gt; &lt; 223〉 T34A 〈220〉 &lt; 221 &gt; CDS &lt; 222〉 (1 &): (534) &lt; 223〉 &lt; 400〉 36 tccaacgaga ccaag atg tgg acc gtg cca gtg ttg ttc tgg gtt ttg ggg 43/66 200413526

Met Trp Thr Val Pro Yal Leu Phe Trp Val Leu Gly 15 10 age gtt tgg ttc tgg gac tet geg cag gga ggg act ata ggc gtg aat

Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Tly lie Gly Yal Asn 15 20 25 gaa gat gat att gtg gcc cca ggt aca gga gac ggc atg gtg ccc cca

Glu Asp Asp lie Val Ala Pro Gly Thr Gly Asp Gly Met Val Pro Pro 30 35 '40 ggt ata gaa gac aaa ata aca acc aca ggt get act gga ggg ett aat

Gly lie Glu Asp Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn 45 50 55 60 gaa tet act ggc aag gca cct ctg gta cca aeg cag aga gag cgt ggg

Glu Ser Thr Gly Lys Ala Pro Leu Val Pro Thr Gin1 Arg Glu Arg Gly 65 70 75 aeg aag cct ccc tta gag gaa ctg tee acc tea gca acc tea gac cat

Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His 80 85 90 gat cac aga gaa cac gag agt aca acc act gtc aaa gtg gtg act age

Asp His Arg Glu His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser 95 100 105 cac tet gtg gac aag aaa aca agt cac cat aat aga gat aat gca ggg

His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 110 115 120 gat gaa aeg cag aca gat aag aaa gat ggc ttg cca gta gtc acc

Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Yal Thr 125 130 135 140 ctg gtt gga ate ata gtt ggc gtc tig tta gcc att ggc ttc gtc gga 44/66 200413526

Leu Val Gly lie lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly 145 150 155 ggg ate ttc att gtt gtt atg aag aag att tet gga agg ttc teg ccc 531 Gly lie Phe lie Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 160 165 170 taa agaget 540

&lt; 210 &gt; 37 &lt; 211 &gt; 172 &lt; 212 &gt; PRT &lt; 213〉 manual &lt; 220> &lt; 223 &gt; T34A &lt; 220> &lt; 221 &gt; MUTAGEN 〈222〉 (34) 〈400〉 37

Met Trp Thr Val Pro Val Leu Phe Trp Val Leu Gly Ser Val Trp Phe 15 10 15

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val Asn Glu Asp Asp lie 20 25 30

Val Ala Pro Gly Thr Gly Asp Gly Met Val Pro Pro Gly lie Glu Asp 35 40 45 45/66 200413526

Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60

Lys Ala Pro Leu Yal Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95

His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser His Ser Val Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115 120 125

Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 140 lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly Gly lie Phe lie 145 150 155 160

Val Yal Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 &lt; 210> 38 &lt; 211 &gt; 540

&lt; 212) DNA 〈213〉 Artificial 46/66 200413526 &lt; 220 &gt; &lt; 223 &gt; T37A 〈220〉 〈221〉 CDS, 〈222〉 (16) · (534) 〈223〉 〈400〉 38 tccaacgaga ccaag atg tgg acc gig cca gtg ttg ttc tgg gtt ttg ggg 51

Met Trp Thr Val Pro Val Leu Phe Trp Val Leu Gly 1 5 10 age gtt tgg ttc tgg gac tet geg cag gga ggg act ata ggc gtg aat 99

Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Tly lie Gly Yal Asn 1 5 20 '.25 gaa gat gat att gtg acc cca ggt gca gga gac ggc atg gtg ccc cca 147

Glu Asp Asp lie Val Thr Pro Gly Ala Gly Asp Gly Met Val Pro Pro 30 3540 ggt ata gaa gac aaa ata aca acc aca ggt get act gga ggg ett aat 195

Gly lie Glu Asp Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn

45 50 55 60 gaa tet act ggc aag gca cct ctg gta cca aeg cag aga gag cgt ggg 243

Glu Ser Thr Gly Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly 65 70 75 aeg aag cct ccc tta gag gaa ctg tee acc tea gca acc tea gac cat 291

Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His 80 85 90 gat cac aga gaa cac gag agt aca acc act gtc aaa gtg gtg act age 339

Asp His Arg Glu His Glu Ser Thr Thr Thr Val Lys Val Yal Thr Ser 47/66 200413526 95 100 105 cac tct gig gac aag aaa aca agt cac ccc aat aga gat aat gca ggg 387 His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 110 115 120 gat gaa aeg cag aca aca gat aag aaa gat ggc ttg cca gta gtc acc 435 Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr 125 130 135 140 ctg gtt gga ate ata gtt ggc gtc ttg t ta gcc ait ggc t tc gtc gga 483 Leu Val Gly lie lie lie Gly Val Leu Leu Ala lie Gly Phe Val Gly 145 150 155 ggg ate t tc at t gtt gtt atg aag aag at t tct gga agg ttc teg ccc 531 Gly lie Phe lie Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 160 165 170 taa agaget 540

&lt; 210> 39 &lt; 211 &gt; 172

&lt; 212 &gt; PRT <213 &gt; manual &lt; 220>

<223> T37A &lt; 220 &gt; &lt; 221 &gt; MUTAGEN &lt; 222> (37)-&lt; 400> 39

Met Trp Thr Val Pro Val Leu Phe Trp Val Leu Gly Ser Val Trp Phe 48/66 200413526 15 ίο

Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val AsnGlu Asp Asp lie 20 25 30

Val Thr Pro Gly Ala Gly Asp Gly Met Val Pro Pro Gly lie Glu Asp 35 40 45

Lys lie Thr Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly

Lys Ala Pro Leu Val Pro Thr GlnArg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95

His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser His Ser Val Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115 120 125

Thr Thr As-p Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 · 140 lie Val Gly Val Leu Leu Ala He Gly Phe Val Gly Gly lie Phe lie 49/66 200413526 145 150 155 160

Yal Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 &lt; 210 &gt; 40 &lt; 211 &gt; 540 &lt; 212 &gt; DNA &lt; 213> Artificial <220> &lt; 223> T51A <220> &lt; 221 &gt; CDS <222> (16) .. (534) &lt; 223> &lt; 400 &gt; 40 tccaacgaga ccaag atg tgg acc gtg cca gtg ttg ttc tgg gtt ttg ggg Met Trp Thr Yal Pro Val Leu Phe Trp Val Leu Gly 1 5 10 age gtt tgg ttc tgg gac tet geg cag gga ggg act ata ggc gtg aat Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Yal Asn 15 20 25 gaa gat gat att gtg acc cca ggt aca gga gac ggc atc gtg ccc

Glu Asp Asp lie Val Thr Pro Gly Thr Gly Asp Gly Met Val Pro Pro 30-35 40 ggt ata gaa gac aaa ata gca acc aca ggt get act gga ggg cti aat

Gly lie Glu Asp Lys lie Ala Thr Thr Gly Ala Thr Gly Gly Leu Asn 45 50 55 60 50/66 200413526 gaa tct act ggc aag gca cct ctg gta cca acg cag aga gag cgt ggg

Glu Ser Thr Gly Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly 65 70 75 acg aag cct ccc tta gag gaa ctg tcc acc tea gca acc tea gac cat

Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His 80 85 90 gat cac aga gaa cac gag agt aca acc act gtc aaa gtg gtg act age

Asp His Arg Glu His Glu Ser Thr Thr Thr Val Lys Val Yal Thr Ser 95 100 105 cac tct gtg gac aag aaa aca agt cac ccc aat aga gat aat gca ggg His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 110 115 120 gat gaa acg cag aca aca gat aag aaa gat ggc ttg cca gta gtc acc

Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr 125 130 135 H〇 ctg gtt gga ate ata gtt ggc gtc ttg tta gcc att ggc ttc gtc gga

Leu Val Gly lie lie Yal Gly Val Leu Leu Ala lie Gly Phe Yal Gly 145 150 155 ggg ate ttc att gtt gtt atg aag aag att tct gga agg ttc teg ccc

Gly lie Phe lie Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 160 165 170 taa agaget &lt; 210 &gt; 41 &lt; 211〉 172 &lt; 212〉 PRT &lt; 213〉 labor 51/66 200413526 &lt; 220 &gt; '

&lt; 223> T51A &lt; 220> &lt; 221 &gt; MUTAGEN <222> (51) &lt; 400> 41

Met Trp Thr Val Pro Val Leu Phe Trp Val Leu Gly Ser Yal Trp Phe 1 5 10 15

Trp Asp Ser Ala Gin Gly Gly Thr. Lie Gly Val Asn Glu Asp Asp lie 20 25 30

Val Thr Pro Gly Thr Gly Asp Gly Met Val Pro Pro Gly lie Glu Asp 35 40 45

Lys lie Ala Thr Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95

His Glu Ser Thr Thr Thr Yal Lys Val Yal Thr Ser His Ser Val Asp 100 105 110 52/66 200413526

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115 120 125

Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 140 lie Val Giy Val Leu Leu Ala lie Gly Phe Val Gly Gly lie Phe lie 145 150 155 160

Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 〈210〉 42 &lt; 211 &gt; 540 〈212〉 DNA 〈213〉 artificial 〈220〉

&lt; 223〉 T52A &lt; 220〉 &lt; 221〉 CDS &lt; 222 &gt; (16). (534) 〈223〉 &lt; 400 &gt; 42-tccaacgaga ccaag atg tgg acc gtg cca gtg ttg ttc tgg gtt ttg ggg 51

Met Trp Thr Yal Pro Val Leu Phe Trp Yal Leu Gly 1 5 10 53/66 200413526 age git tgg tic tgg gac tet geg cag gga ggg act ata ggc gtg aat 99 Ser Val Trp Phe Trp Asp Ser Ala Gin Gly Gly Thr lie Gly Val Asn 15 20 25 gaa gal gat alt gtg acc cca ggt aca gga gac ggc atg gtg ccc cca 147 Glu Asp Asp lie Val Thr Pro Gly Thr Gly Asp Gly Met Val Pro Pro 30 35 40 ggt ata gaa gac aaa ata aca gee aca ggt get act gga ggg ct t aat 195 Gly lie Glu Asp Lys lie Thr Ala Thr Gly Ala Thr Gly Gly Leu Asn 45 50 55 60 gaa tet act ggc aag gca cct ctg gta cca aeg cag aga gag cgt ggg 243 Glu Ser Thr Gly Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly 65 70 75 aeg aag cct ccc tta gag gaa ctg tee acc tea gca acc lea gac cat 291 Thr Lys Pro Pro Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His 80 85 90 gat cac aga gaa cac gag agt aca acc act gtc aaa gtg gtg act age 339 Asp His Arg Glu His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser 95 100 105 cac tet gtg gac aag aaa aca agt cac ccc aat aga gat aat gca ggg 387 His Ser Val Asp Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly 110 115 120 gat gaa aeg cag aca aca gat aag aaa gat ggc ttg cca gta gtc acc 435 Asp Glu Thr Gin Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr 125, 130 135 140 ctg gtt gga ate ata gtt ggc gtc ttg tta gee att ggc t tc gtc gga 483 Leu Val Gly lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly 145 150 155

54/66 200413526 ggg ate ttc alt gtt git atg aag aag att tel gga agg tie teg ccc Gly lie Phe He Yal Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 160 165 170 taa agaget &lt; 210〉 43 &lt; 211〉 172 &lt; 212 &gt; PRT &lt; 213〉 Manual &lt; 220〉 &lt; 223〉 Ding52A &lt; 220〉 &lt; 221 &gt; MUTAGEN &lt; 222〉 (52) &lt; 400 &gt; 43

Met Trp Thr Val Pro Val Leu Phe Trp Val Leu Gly Ser Val Trp Phe 15 10 15

Trp Asp Ser Ala Gin Gly Gly Thr He Gly Val Asn Glu Asp Asp lie 20 25 30

Val Thr Pro Gly Thr Gly Asp Gly Met Val Pro Pro Gly lie Glu Asp 35 40 45

Lys lie Thr Ala Thr Gly Ala Thr Gly Gly Leu Asn Glu Ser Thr Gly 50 55 60 55/66 200413526

Lys Ala Pro Leu Val Pro Thr Gin Arg Glu Arg Gly Thr Lys Pro Pro 65 70 75 80

Leu Glu Glu Leu Ser Thr Ser Ala Thr Ser Asp His Asp His Arg Glu 85 90 95

His Glu Ser Thr Thr Thr Val Lys Val Val Thr Ser His Ser Val Asp 100 105 110

Lys Lys Thr Ser His Pro Asn Arg Asp Asn Ala Gly Asp Glu Thr Gin 115 120 125

Thr Thr Asp Lys Lys Asp Gly Leu Pro Val Val Thr Leu Val Gly lie 130 135 140 lie Val Gly Val Leu Leu Ala lie Gly Phe Val Gly Gly lie Phe lie 145 150 155 160

Val Val Met Lys Lys lie Ser Gly Arg Phe Ser Pro 165 170 &lt; 210 &gt; 44 &lt; 211 &gt; 10 &lt; 212 &gt; PRT- &lt; 213> artificial &lt; 220 &gt; <223 &gt; Gp38P partial peptides of amino acids 56/66 200413526 &lt; 400> 44

Asp Gly Met Yal Pro Pro Gly lie Glu Cys 1 5 10 &lt; 210> 45 <211> 10 &lt; 212 &gt; PRT &lt; 213> artificial &lt; 220> &lt; 223> mutant peptide of gP38P; M41Apt &lt; 400〉 45

Asp Gly Ala Val Pro Pro Gly lie Glu Cys 1 5 10 &lt; 210〉 46 &lt; 211〉 10 &lt; 212 &gt; PRT &lt; 213〉 Artificial &lt; 220〉 &lt; 223> Mutant peptide of gp38P; V42Apt &lt; 400 &gt; 46

Asp Gly Met Ala Pro Pro Gly lie Glu Cys 1 5 10 &lt; 210 &gt; 47 57/66 200413526 &lt; 211 &gt; 10 &lt; 212) PRT <213 &gt; Artificial &lt; 220 &gt; &lt; m &gt; gP38P mutant peptide ; V43Apt &lt; 400 &gt; 47

Asp Gly Met Val Ala Pro Gly lie Glu Cys 1 5 10 &lt; 210 &gt; 48 <211> 10 &lt; 212) PRT 〈213〉 Artificial 〈220〉 〈223〉 mutant peptide of gP38P; V44Apt &lt; 400 &gt; 48

Asp Gly Met Val Pro Ala Gly lie Glu Cys 1 5 10 &lt; 210) 49 <211> 489 &lt; 212) DNA &lt; 213> Artificial &lt; 220 &gt;

&lt; 223〉 gp36-T52A &lt; 220) 58/66 200413526 &lt; 221 &gt; CDS &lt; 222> (1).. (489) 〈223〉 〈400〉 49 atg tgg aag gtg tea get ctg etc ttc gtt ttg gga age geg teg etc 48

Met Trp Lys Val Ser Ala Leu Leu Phe Val Leu Gly Ser Ala Ser Leu 1 5 10 15 tgg gtc ctg gca gaa gga gee age aca ggc cag cca gaa gat gac act 96

Trp Val Leu Ala Glu Gly Ala Ser Thr Gly Gin Pro Glu Asp Asp Thr 20 25 30

gag act aca ggt ttg gaa ggc ggc glt gee atg cca ggl gee gaa gat 144

Glu Thr Thr Gly Leu Glu Gly Gly Val Ala Met Pro Gly Ala Glu Asp 35 40 45 gat gtg gtg geg cca gga acc age gaa gac ege tat aag tet ggc ttg 192

Asp Val Val Ala Pro Gly Thr Ser Glu Asp Arg Tyr Lys Ser Gly Leu 50 55 60 aca act ctg gtg gca aca agt gtc aac agt gta aca ggc att ege ate 240

Thr Thr Leu Val Ala Thr Ser Val Asn Ser Val Thr Gly lie Arg lie 65 70 75 80 gag gat ctg cca act tea gaa age aca gtc cac geg caa gaa caa agt 288

Glu Asp Leu Pro Thr Ser Glu Ser Thr Val His Ala Gin Glu Gin Ser 85 90 95 cca age gee aca gee tea aac gtg gee acc agt cac tee aeg gag aaa 336

Pro Ser Ala Thr Ala Ser Asn Yal Ala Thr Ser His Ser Thr Glu Lys -100 105 110 gtg gat gga gac aca cag aca aca gtt gag aaa gat ggt ttg tea aca 384

Val Asp Gly Asp Thr Gin Thr Thr Val Glu Lys Asp Gly Leu Ser Thr115 120 125 59/66 200413526 gtg acc ctg gtt gga ate ata gtt ggg gtc tta eta gec ate ggt t tc 432 Val Thr Leu Yal Gly lie lie Val Gly Yal Leu Leu Ala lie Gly Phe 130 135 140 at t ggt gga ate ate gtt gtg gtt atg ega aaa atg teg gga agg tac 480 lie Gly Gly lie lie Val Val Yal Met Arg Lys Met Ser Gly Arg Tyr 145 150 155 160 teg ccc taa 489 Ser Pro &lt; 210 &gt; 50 &lt; 211> 162 &lt; 212 &gt; PRT 〈213 &gt; manual &lt; 220>

<223> gp36-T52A &lt; 220 &gt;

&lt; 221 &gt; MUTAGEN <222> (52) &lt; 400> 50

Met Trp Lys Val Ser Ala Leu Leu Phe Val Leu Gly Ser Ala Ser Leu 15 10 15

Trp Yal Leu Ala Glu Gly Ala Ser Thr Gly Gin Pro Glu Asp Asp Thr 20 25 30 60/66

200413526

Glu Thr Thr Gly Leu Glu Gly Gly Yal Ala Met Pro Gly Ala Glu Asp 35 40 45

Asp Yal Val Ala Pro Gly Thr Ser Glu Asp Arg Tyr Lys Ser Gly Leu 50 55 60

Thr Thr Leu Val Ala Thr Ser Val Asn Ser Val Thr Gly lie Arg lie 65 70 75 80

Glu Asp Leu Pro Thr Ser Glu Ser Thr Val His Ala Gin Glu Gin Ser 85 90 95

Pro Ser Ala Thr Ala Ser Asn Val Ala Thr Ser His Ser Thr Glu Lys 100 105 110

Val Asp Gly Asp Thr Gin Thr Thr Yal Glu Lys Asp Gly Leu Ser Thr 115 120 125

Val Thr Leu Val Gly lie lie Yal Gly Val Leu Leu Ala lie Gly Phe 130 135 140 lie Gly Gly lie lie Val Val Val Met Arg Lys Met Ser Gly Arg Tyr 145 150 155 160

Ser Pro 61/66 200413526 &lt; 210 &gt; 51 &lt; 211 &gt; 489 &lt; 212> DNA &lt; 213> Artificial &lt; 220 &gt; 〈223〉 gp36-T55A &lt; 220〉 &lt; 221〉 CDS &lt; 222> ( 1): (489) <223 &gt; '<400> 51 atg tgg aag gtg tea get ctg etc ttc gtt ttg gga age geg teg etc

Met Trp Lys Yal Ser Ala Leu Leu Phe Val Leu Gly Ser Ala Ser Leu 1 5 10 15 tgg gtc ctg gca gaa gga gee age aca ggc cag cca gaa gat gac act

Trp Val Leu Ala Glu Gly Ala Ser Thr Gly Gin Pro Glu Asp Asp Thr 20 25 30 gag act aca ggt ttg gaa ggc ggc gtt gee atg cca ggt gee gaa gat

Glu Thr Thr Gly Leu Glu Gly Gly Val Ala Met Pro Gly Ala Glu Asp 35 40 45 gat gtg gtg act cca gga geg age gaa gac ege tat aag tet ggc ttg

Asp Val Val Thr Pro Gly Ala Ser Glu Asp Arg Tyr Lys Ser Gly Leu 50 55 60 aca act ctg gtg gca aca agt gtc aac agt gta aca ggc att ege ate

Thr Thr Leu Val Ala Thr Ser Val Asn Ser Val Thr Gly lie Arg lie 65 70 75 80 gag gat ctg cca act tea gaa age aca gtc cac geg caa gaa caa agt

Glu Asp Leu Pro Thr Ser Glu Ser Thr Val His Ala Gin Glu Gin Ser 62/66 200413526 85 90 95 cca age gee aca gee tea aac gtg gee acc agt cac tee aeg gag aaa 336 Pro Ser Ala Thr Ala Ser Asn Val Ala Thr Ser His Ser Thr Glu Lys 100 105 110 gtg gat gga gac aca cag aca aca git gag aaa gat ggt ttg tea aca 384 Yal Asp Gly Asp Thr Gin Thr Thr Yal Glu Lys Asp Gly Leu Ser Thr 115 120 125 gtg acc ctg gtt gga ate ata gtt ggg gtc tta eta gee ate ggt t tc 432 Yal Thr Leu Yal Gly lie lie Val Gly Val Leu Leu Ala lie Gly Phe 130 135 140 att ggt gga ate ate gtt gtg gtt atg ega aaa atg teg gga agg tac 480 lie Gly Gly lie lie Yal Yal Yal Met Arg Lys Met Ser Gly Arg Tyr 145 150 155 160 teg ccc taa 489 Ser Pro &lt; 210 &gt; 52 &lt; 211 &gt; 162 &lt; 212 &gt; PRT 〈213〉 Labor &lt; 220 &gt;

<223> gp36-T55A &lt; 220> <221> MUTAGEN <222> (55) <400> 52 63/66 200413526

Met Trp Lys Val Ser Ala Leu Leu Phe Val Leu Gly Ser Ala Ser Leu 15 10 15

Trp Val Leu Ala Glu Gly Ala Ser Thr Gly Gin Pro Glu Asp Asp Thr 20 25 30

Glu Thr Thr Gly Leu Glu Gly Gly Yal Ala Met Pro Gly Ala Glu Asp 35 40 45

Asp Val Val Thr Pro Gly Ala Ser Glu Asp Arg Tyr Lys Ser Gly Leu 50 55 60

Thr Thr Leu Val Ala Thr Ser Val Asn Ser Val Thr Gly lie Arg lie 65 70 75 80

Glu Asp Leu Pro Thr Ser Glu Ser Thr Val His Ala Gin Glu Gin Ser 85 90 95

Pro Ser Ala Thr Ala Ser Asn Yal Ala Thr Ser His Ser Thr Glu Lys 100 105 110

Val Asp Gly Asp Thr Gin Thr Thr Val Glu Lys Asp Gly Leu Ser Thr 115 120 125

Val Thr Leu Val Gly lie lie Val Gly Val Leu Leu Ala lie Gly Phe 130 135 140 64/66 200413526 lie Gly Gly lie lie Yal Val Val Met Arg Lys Met Ser Gly Arg Tyr 145 150 155 160

Ser Pro &lt; 210> 53 &lt; 211> 20 〈212〉 DNA 〈213〉 artificial 〈220〉 〈223〉 PCR synonym primer of gp36 &lt; 400 &gt; 53 tgtggaaggt gtcagctctg 〈210〉 54 〈211〉 20 &lt; 212 &gt; DNA <213X artificial, &lt; 220 &gt; &lt; 223 &gt; PCR antisense primer for gp36 &lt; 400 &gt; 54 ttagggcgag taccttcccg

<210> 55 &lt; 211 &gt; 28 <212> DNA 65/66 200413526 &lt; 213> Artificial &lt; 220> &lt; 223>, PCR synonym primer of ribosomal protein s9 &lt; 400 &gt; 55 gatgagaagg acccacggcg tctgttcg &lt; 210 &gt; 56 &lt; 211> 28 &lt; 212 &gt; DNA &lt; 213> artificial &lt; 220> &lt; 223> PCR antisense primer of ribosomal protein S9 &lt; 400 &gt; 56 gagacaatcc agcagcccag gagggaca 66/66

Claims (1)

200413526 Patent application scope 1 · A method for screening active substances with platelet aggregation promotion or inhibition, including the following projects: (]) culturing gP44 expressing cells in the presence or absence of test substances (2) the above ( 1) The obtained cultured cells are tested for mRNA expression using the nucleotide sequence described in any one of (a) to (1) below: U) Nucleotide numbers 1 to 5 1 9 of sequence number 1 in the sequence listing Nucleotide sequence; (b) Nucleotide sequence number 1 to 501 shown in sequence number 3 in sequence listing [J; (c) Nucleotide number 1 to 510 shown in sequence number 5 in sequence listing Nucleotide sequence; (d) Nucleotide sequence number 1 to 489 shown in sequence list sequence number 7! J; (O nucleotide sequence 1 to 540 in SEQ ID NO: 13 in Sequence Listing 13; Lu (f) a core complementary to the nucleotide sequence described in any one of (a) to (e) above Polynucleotides formed by nucleotide sequences hybridize under severe conditions, and have platelet aggregation-enhancing active peptide-encoded nucleotide sequences; (g) amino acid numbers 1 to 172 in sequence number 2 in the sequence listing The nucleotide sequence encoded by the amino acid sequence poly-peptide; (h) the nucleotide sequence encoded by the amino acid sequence poly-peptide shown by amino acid numbers 1 to 166 in Sequence Listing Sequence Number 4; -131- 200413526 (i) Nucleotide sequence encoded by the amino acid sequence polypeptide shown in sequence number 6 amino acid number 1 to 169 in the sequence listing; (j) shown in sequence number 8 amino acid number 1 to 1 62 Nucleotide sequence encoded by amino acid poly-peptide; (k) Nucleotide sequence encoded by amino acid poly-peptide shown in sequence number 14 of amino acid sequence number 1 to 172; (l) above ( g) In the amino acid sequence described in any one of (k), the amino acid sequence formed by missing, replacing or adding one or more amino acids is A nucleotide sequence encoded by a polypeptide with platelet aggregation promoting activity; (3) Comparing the expression level of the mRNA detected from the above (2) to the absence of the test cell in the presence of the test substance Compared with the cells cultured in the absence of the test substance (4), the cells with lower mRNA expression were selected to have platelet aggregation inhibitory activity, or the cells with higher mRNA expression were selected to have platelet aggregation. Substances that promote activity 02. For example, the screening method in the scope of patent application No.1, the methods for testing mRNA expression include Northern blotting method (Northern B 1 0t), dot blotting method, slit blotting method, RT-PCR, ribose Nucleic acid protection assay or derivative assay. 3. A method for screening active substances with platelet aggregation promotion or inhibition, including the following processes: (1) culturing gp44 expressing cells in the presence of test substance; (2) detecting the cultured cells obtained in (1) above, detecting The expression of the polypeptide described in any one of the following (i) to (viii) -132-200413526: 之 The nucleotide sequence coded in any of the following (i-〗) to (i-5) An amino acid sequence, or a polypeptide formed from a part thereof and having platelet aggregation promoting activity; (i-1) the nucleotide sequence shown by nucleotide numbers 1 to 519 in sequence number 1 of the sequence listing; (i- 2) the nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in the sequence listing; (i-3) the nucleotide sequence shown in nucleotide number 1 to 5 10 of sequence number 5 in the sequence listing; U -4) the nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7 in sequence listing; (i-5) the nucleotide sequence shown in nucleotide number 1 to 540 of sequence number 13 in sequence listing; ( ii) A polynucleotide that is complementary to a nucleotide sequence complementary to the nucleotide sequence described in any one of (i-1) to (i-5) above under severe conditions Polynucleotide with amino acid sequence encoded by the polynucleotide encoding and having platelet aggregation promotion activity: (iii) Polyamino acid sequence polyamino acid sequence shown in SEQ ID NO: 2 in Sequence Listing 2 Peptides; (iv) amino acid sequence polypeptides shown in amino acid numbers 1 to 166 in sequence number 4 of the Sequence Listing; (v) amino acid sequences shown in amino acid numbers 1 to 169 of sequence number 6 in sequence listing Polypeptide; -133-200413526 (V i) Amino acid sequence poly-peptide shown in amino acid numbers 1 to 16 of Sequence Listing 8; (vii) Amino acid number of Sequence Listing 14 ] To 172 of the amino acid sequence polypeptide; (vii i) 'Missing, replacing, or adding 1 or more amines to the amino acid sequence of the polypeptide described in any of (iii) to (vii) above A polypeptide with an amino acid sequence formed from a basic acid and having platelet aggregation promotion activity; (3) The expression level of the peptide detected in (b) above is compared with the cells cultured in the presence of the test substance and the donor Cells cultured in the absence of the test substance; (4) Compared with cells cultured in the absence of the test substance, choose a peptide with a lower expression level The substance is a substance with platelet aggregation inhibitory activity, or a substance with a higher peptide expression is selected as a substance with platelet aggregation promoting activity. 04. If the screening method of the third item of the patent application is applied, the expression of the polypeptide is determined. Methods include Northern blotting, dot blotting, slot blotting, or fixed enzyme immunoassay (ELISA). 5 · A method for measuring the platelet aggregation promoting or inhibiting activity of a substance, comprising the following processes: (1) culturing gp44 expressing cells in the presence or absence of a test substance j (2) culturing the cultured cells obtained in (1) above To detect the expression of the nucleotide sequence mRNA containing any of the following nucleotide sequences described in (a) to (1): U) the nucleotide sequence shown in nucleotide number 1 to 5 1 9 of the sequence listing sequence number 1 ; -134-200413526 (b) the nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in the sequence listing; (c) the nucleotide sequence shown in nucleotide number 1 to 510 of sequence number 5 in sequence listing (D) the nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7 in the sequence listing; (e) the nucleotide sequence shown in nucleotide number 1 to 540 of sequence number 13 in sequence listing; (A) to (e) A polynucleotide composed of a nucleotide sequence complementary to the nucleotide sequence described in any one of the nucleotide sequences described in (a) to (e) hybridizes under severe conditions, and has a platelet aggregation-promoting active peptide-encoded core. Glycine sequence; (g) Amino acid sequence shown in SEQ ID NO: 2 in Sequence Listing 2 to 172 Nucleotide sequence; (h) Nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 166 of sequence number 4 in sequence listing; 胺 amino acid number 1 of sequence number 6 in sequence listing The nucleotide sequence encoded by the amino acid sequence poly-peptide shown to 169; the nucleotide sequence encoded by the amino acid sequence poly-peptide shown by the amino acid number 1 to 162 of the sequence table sequence number 8; k) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of sequence number 14 in the sequence listing; (l) the amino group described in any one of (g) to (k) above In the acid sequence, a nucleotide sequence encoded by a polypeptide having platelet aggregation promotion activity formed by missing, replacing, or adding 1 or more amino acid sequences formed by amino acids; -135-200413526 (3) The expression level of mRNA detected in the above (2) is compared with the cells cultured in the presence of the test substance and the cells j (4) cultured in the absence of the test substance and the cells cultured in the absence of the type 5 substance. When the expression of m RNA is low, platelet aggregation inhibition activity or mr NA expression is high Fixed with platelet aggregation promoting activity. 6 · As for the screening method in the scope of patent application No. 5, the mRNA expression detection means include Northern blotting method, dot blotting method, slit blotting method, PT-PCR, RNA protection assay or derivative assay. 0 7.  —A method for measuring the platelet aggregation promotion or inhibitory activity of a substance, comprising the following processes: (1) culturing gp44 expressing cells in the presence of a test substance; (2) detecting the following (i) The expression of the polypeptide according to any one of) to (viii): (i) the amino acid sequence encoded by the nucleotide sequence according to any one of (i-1) to (i-5), or a part thereof A polypeptide formed with platelet aggregation promoting activity: (i-1) the nucleotide sequence shown in nucleotide number 1 to 519 of sequence number 1 in the sequence listing; (i-2) sequence number 3 in the sequence listing Nucleotide sequences shown in nucleotide numbers 1 to 501; (b. 3) Nucleotide sequences shown in nucleotide number 1 to 510 in sequence number 5 in the sequence listing; (i-4) Nuclei in sequence number 7 in the sequence listing Nucleotide-136-200413526 acid sequences shown in the nucleotide numbers 1 to 489; (b 5) Nucleotide numbers of sequence number 13 in the Sequence Listing] to 540; (ii) and (il) to (I-5) Polynucleotide obtained by hybridizing a polynucleotide obtained from a nucleotide sequence complementary to the nucleotide sequence described in any one of the above under severe conditions Encoding polyamino acid peptides with platelet aggregation promoting activity; (iii) Polyamino peptides of amino acid sequences shown in amino acid numbers 1 to 172 of Sequence Listing 2; 0 (iv) sequence listing sequence The amino acid sequence polypeptide shown in amino acid numbers 1 to 166 of the number 4; (v) the amino acid sequence polypeptide shown in amino acid numbers 1 to 169 of the sequence listing sequence number 6; (vi) the sequence List of amino acid sequence polypeptides shown in amino acid numbers 1 to 162 of sequence number 8; (vii) Sequence of amino acid polypeptides shown in amino acid numbers 1 to 1 of sequence list 4 ; (Vi ii) the amino acid sequence of the polypeptide as described in any one of (iii) to (Vii) above, which is missing, substituted or added with one or more amino acid sequences Polypeptide with platelet aggregation promoting activity. (3) The above (2) detection of peptide expression, compared with cells cultured in the presence of the test substance and cells cultured in the absence of the test substance; (4) compared with cells cultured in the absence of the test substance; Comparing cells, it was judged to have platelet aggregation inhibitory activity when peptide expression was low, or platelet aggregation promotion activity was determined when peptide expression was high -137-200413526. 8 . For example, the screening method for item 7 of the scope of patent application, the method for measuring the expression level of polypeptide is Western blot method, dot blot method, slit blot method or fixed enzyme immunoassay method. 9. The method as described in any one of claims 1 to 8 of the scope of the patent application, wherein the platelet aggregation inhibitory activity is a platelet aggregation treatment effect on cancer, cancer metastasis, pulmonary thrombosis, cerebral infarction, myocardial infarction or arteriosclerosis or vascular surgery, or Preventive effect. 1 〇. The method according to any one of claims 1 to 8 in the scope of the patent application, wherein the platelet aggregation promotion activity has a therapeutic effect or a preventive effect on bleeding tendency, blood coagulation disorder, hemophilia, uremia, or chronic bone marrow colonic disease. 1 1 · A model animal related to the promotion of platelet aggregation-related diseases, a gene containing the nucleotide sequence described in any one of (a) to (1) below is introduced into a non-human gene transfection in a highly expressed form of the gene Animals: (a) Nucleotide sequence shown in nucleotide numbers 1 to 5 1 9 of sequence number 1 in the sequence listing; (b) Nucleotide sequence shown in nucleotide numbers 1 to 501 of sequence number 3 in sequence listing Sequence; (c) the nucleotide sequence shown in nucleotide number 1 to 5 10 of sequence number 5 in the sequence listing; (d) the nucleotide sequence shown in nucleotide number 1 to 4 89 of sequence listing 7 (E) Nucleoside 200413526 acid sequence shown in nucleotide number 1 to 54 0 of sequence number 13 in sequence listing; (0) Nucleus complementary to the nucleotide sequence described in any one of (a) to (e) above The nucleotide sequence of the nucleotide sequence hybridized under severe conditions, and the nucleotide sequence of the polynucleotide encoded by the active peptide with platelet aggregation promotion; (g) the amino acid of sequence number 2 in the sequence listing Nucleotide sequences encoded by amino acid polypeptides shown in numbers 1 to 1 72; (h) amino acid numbers 1 to 4 in the sequence listing Nucleotide sequence encoded by amino acid sequence polypeptide shown in 166; (i) Nucleotide sequence encoded by amino acid φ sequence polypeptide shown in amino acid number 1 to 169 of Sequence Listing 6; (j) Nucleotide sequences encoded by amino acid polypeptides shown in amino acid numbers 1 to 162 of sequence number 8 in the sequence listing; (k) amino acid numbers 1 to 172 of sequence number 14 in the sequence listing Nucleotide sequence encoded by amino acid sequence polypeptide; (l) In the amino acid sequence described in any one of (g) to (k) above, one or more amino acids are missing, substituted or added. Nucleotide sequence encoded by a polypeptide formed from the formed amino acid sequence and having platelet aggregation promoting activity. Lu 1 2 · The animal described in item 11 of the scope of patent application, wherein platelet aggregation inhibiting activity is effective against cancer and cancer. Metabolism, pulmonary thrombosis, cerebral infarction, myocardial infarction, or arteriosclerosis or vascular surgery have platelet aggregation treatment or preventive effects. 1 3 · —A test substance is used as a therapeutic or preventive agent for platelet aggregation promoting activity or platelet aggregation promoting related diseases Method of effect, including the following (2) (1) Measurement of platelet aggregation function in animals and 14; - Cheng -139,200,413,526 (1) administering a test substance to an animal, such as application 1 1 2 1 or scope of the patent is described.  A model animal related to platelet aggregation inhibition-related diseases. A gene containing the nucleotide sequence described in any one of the following U) to (1) is introduced into a non-human gene transgenic animal in a low expression form of the gene: U) The nucleotide sequence shown in nucleotide number 1 to 519 of sequence number 1 in the sequence listing; (b) the nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in sequence listing; (c) the sequence number of sequence listing Nucleotide sequence shown in nucleotide number 1 to 510 of 5; (d) Nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7 in sequence listing; (e) Nucleus of sequence number 1 to 3 in sequence listing Nucleotide sequences shown by the nucleotide numbers 1 to 540; (II) the nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e) above is severe The nucleotide sequence of the polynucleotide encoded by a polypeptide having a platelet aggregation-promoting active peptide; (g) Amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of sequence number 2 in the sequence listing The encoded nucleotide sequence; (h) the amino acid sequences shown in amino acid numbers 1 to 166 of sequence number 4 in the Sequence Listing Peptide-encoded nucleotide sequence; (i) Amino-140-200413526 sequence of amino acid numbers 1 to 169 in sequence number 6 in Sequence Listing; (j) sequence listing sequence Nucleotide sequence encoded by the amino acid sequence poly-peptide shown in amino acid numbers 1 to 162 of the number 8; (k) amino acid sequence poly-amino acid shown in amino acid numbers 1 to 172 of the sequence listing sequence number 14 Peptide-encoded nucleotide sequence; (l) the amino acid sequence described in any one of (g) to (k) above, which is formed by the absence, substitution, or addition of one or more amino acids A nucleotide sequence encoded by a polypeptide with platelet aggregation promoting activity. 1 5.  —A model animal related to platelet aggregation inhibition-related diseases, the gene containing the nucleotide sequence of any one of the following U) to (1) is a knockout non-human animal: U) a nucleoside of sequence number 1 in the sequence listing The nucleotide sequences shown in acid numbers 1 to 519; (b) The nucleotide sequences shown in nucleotide numbers 1 to 501 of sequence number 3 in the sequence listing; (c) The nucleotide numbers 1 to 5 of sequence listing 5 Nucleotide sequence shown at 510; (d) Nucleotide sequence U shown at nucleotide numbers 1 to 489 in sequence number 7 of the Sequence Listing; (e) Nucleotide number 1 to 540 at Sequence number 13 of the Sequence Listing The nucleotide sequence shown in (a) the nucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e) above hybridizes under severe conditions and has platelets Nucleotide sequence of the polynucleotide encoded by the aggregation-promoting active peptide; 200413526 (g) Nucleotide sequence of the amino acid sequence polyeptide shown in amino acid numbers 1 to 172 of the sequence listing 2 (H) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 1 of sequence number 4 in sequence listing 4; (I) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 169 of sequence number 6 in the sequence listing; (j) amino acid numbers 1 to 162 of sequence number 8 in the sequence listing The nucleotide sequence encoded by the amino acid sequence poly-peptide; (k) the nucleotide sequence encoded by the amino acid sequence poly-peptide shown by amino acid numbers 1 to 172 in sequence number 14 of the Sequence Listing; U) the above (G) to (k) A polypeptide having an amino acid sequence formed by lacking, replacing, or adding one or more amino acids and having platelet aggregation promotion activity Encoded nucleotide sequence. 16. The animal according to item 14 or 15 of the scope of the patent application, wherein the diseases associated with the inhibition of platelet aggregation are bleeding tendency, hemagglutination disorder, hemophilia, uremia or chronic myeloproliferative disease. 17 · — A test substance as a method of platelet aggregation inhibitory activity or the effect of a platelet aggregation inhibitory therapeutic or preventive agent on diseases, including the following processes (1) Administration of the animal as described in claims 14 to 16 Test substance; and (2) measuring (1) platelet aggregation function in animals. 18 · —A method for diagnosing a disease, in which a specimen is tested for mRNA expression by using the nucleotide sequence described in any one of (a) to (1) below to diagnose the specimen-142-200413526 donor or The specimen provides platelet aggregation or platelet dysfunction-related diseases in animals: (a) the nucleotide sequence shown in nucleotide numbers 1 to 5 1 of sequence number 1 in the sequence listing; (b) sequence number 3 in sequence listing Nucleotide sequences shown in nucleotide numbers 1 to 501; (c) Nucleotide sequences shown in nucleotide numbers 1 to 5 1 in sequence list 5; (d) Sequence list sequence numbers Nucleotide sequences shown in nucleotide numbers 1 to 489 of 7; (e) Nucleotide sequences shown in nucleotide numbers 1 to 540 of sequence number 13 in sequence listing; (f) As described in (a) to (e) above Polynucleotide obtained by complementing the nucleotide sequence of any one of the nucleotide sequences described in any one of the nucleotide sequences hybridizes under severe conditions, and the platelet aggregation promotes the activity of the polynucleotide encoded by the polynucleotide (G) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of sequence number 2 in the sequence listing; (h) the sequence The nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 166 of SEQ ID NO: 4; ⑴ The amino acid sequence polypeptide shown by amino acid numbers 1 to 169 of sequence list 6 Encoded nucleotide sequence; (1) Nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 162 of sequence number 8 of the sequence listing; amino acid numbers 1 to 14 of sequence number 14 in the sequence listing Nucleotide sequence encoded by amine 200413526 acid sequence polypeptide shown in 172; (1) 1 or more of the amino acid sequence described in any one of (g) to (k) above is missing, substituted or added A nucleotide sequence encoded by a polypeptide having an amino acid sequence formed from an amino acid and having platelet aggregation promoting activity. 19 · If the method in the 18th scope of the patent application, the characteristics of the method for examining the expression of mRN A include the northern ink stain method, dot ink stain method, slit ink stain method, PT-pCR, ribonucleic acid protection assay or Derivative assay. 20. A method for diagnosing a disease, in which a specimen is detected for expression of a polypeptide as described in any one of the following (i) to (viii) to diagnose platelets of the specimen provider or the stomach donor animal. Diseases related to agglutination or platelet dysfunction: The amino acid sequence encoded by the nucleotide sequence described in any one of (i-1) to (i-5) below or a part thereof has a platelet aggregation promoting activity Peptide, (i-1) Nucleotide sequence shown in nucleotide number 1 to 519 of sequence number 1 in sequence listing; (^ 2) Nucleotide sequence shown in sequence number 3 of sequence listing 3 to 501 (I-3) Nucleotide sequences shown in nucleotide numbers 1 to 510 of sequence number 5 in the Sequence Listing; (i-4) Nuclei shown in nucleotide numbers 1 to 489 of Sequence number 7 in the Sequence Listing Nucleotide sequence; U-5) Nucleotide sequence shown in nucleotide number 1 to 540 of sequence number 13 in sequence listing; • 144-200413526 (ii) as described in any one of (il) to (i-5) above Polynucleotides formed by complementary nucleotide sequences of the described nucleotide sequences hybridize under severe conditions, and have platelet aggregation-promoting active polypeptides The nucleotide sequence of the encoded polynucleotide; (iii) the amino acid sequence polypeptide shown in amino acid numbers 1 to 172 of sequence number 2 in the sequence listing; (iv) the amino acid sequence of sequence number 4 in the sequence listing Polyaminopeptides of amino acid sequences shown in numbers 1 to 166; (v) Polyaminopeptides of amino acid sequences shown in sequence numbers 6 to 6 in the sequence listing; (vi) sequence numbers 8 to 8 in the sequence listing Amino acid sequence polypeptides shown by amino acid numbers 1 to 162; (vii) Amino acid sequence polypeptides shown by amino acid numbers 1 to 172 of Sequence Listing 14; (viii) The above ( iii) the amino acid sequence of the polypeptide according to any one of (vii) [in [J] 'is missing, substituted or added with an amino acid sequence formed by one or several amino acids and has platelet aggregation promotion Active Polypeptide. Lu 21 · As for the method in the scope of patent application No. 20, the method for measuring the expression level of polypeptide uses Western blotting method, dot blotting method, slit blotting method or fixed enzyme immunoassay method as a special method. 2 2 · The method according to any one of claims 18 to 21 in the scope of patent application, wherein the platelet aggregation-related disease is cancer, cancer metastasis, pulmonary thrombosis, cerebral infarction, myocardial infarction or arteriosclerosis or platelet aggregation during vascular surgery Feature. twenty three . For example, the method of any one of claims 18 to 21, wherein the blood-145-200413526 platelet agglutination-related diseases are colon cancer, rectal cancer, small intestine cancer, seminal carcinoma, seminoma, and epithelial squamous cell carcinoma. Characteristics. 24. The method according to any one of claims 18 to 21 in the scope of patent application, wherein the blood platelet agglutination-related disease is a characteristic of bleeding tendency, blood coagulation disorder, hemophilia, uremia, or chronic myeloproliferative disease. 2 5.  -A method for isolating platelet aggregation-promoting or inhibiting active substances by contacting a polypeptide of the amino acid sequence described in any one of (i) to (viii) below with a reagent containing a test substance, and then separating Substances that bind to the polypeptide: (i) an amino acid sequence encoded by the nucleotide sequence described in any one of (1) to (i-5) below, or a part thereof and having Platelet agglutination-promoting polypeptide: (iI) the nucleotide sequences shown in nucleotide numbers 1 to 519 of sequence number 1 in the sequence listing; (i-2) nucleotide numbers 1 to 5 of sequence number 3 in the sequence listing Nucleotide sequence shown in 01; (i-3) Nucleotide sequence shown in nucleotide number 1 to 510 of sequence number 5 in the sequence listing; (B 4) Nucleotide number 1 to sequence number 7 in sequence listing Nucleotide sequence represented by 489; U-5) Nucleotide sequences represented by nucleotide numbers 1 to 540 of sequence number 13 in sequence listing; (ii) any one of (i-1) to (i-5) above Polynucleotides formed by complementary nucleotide sequences of the nucleotide sequences described in the above item are hybridized under severe conditions' and have -146-200413526 platelet aggregation promoting activity Polynucleotide sequence encoded by peptide; (iH) SEQ ID NO: 2 of amino acid sequence poly-peptide shown in amino acid numbers 1 to 172 of the sequence number 2; (iv) amino acid of sequence number 4 in the sequence listing Polyaminopeptides of amino acid sequences shown in numbers 1 to 166; (v) Polyaminopeptides of amino acid sequences shown in sequence number 6 to 6 in the sequence listing; (vi) Amines of sequence number 8 in sequence listing Amino acid sequence polypeptides shown in amino acid numbers 1 to 162; (vii) amino acid number poly-peptides shown in sequence number 14 of the sequence listing to 172; (viii) as described in (iii) above To a polypeptide according to any one of (vii), a polypeptide having a lack of, substitution, or addition of an amino acid sequence formed by one or more amino acids and having platelet aggregation promoting activity Peptide. 26 · —A method for isolating platelet aggregation-promoting or inhibiting active substances, which comprises contacting a polypeptide described in any one of the following items (Xiv) to (Xiv) with a reagent containing a test substance, and then separating the polypeptide Peptide-binding substances: (i) the amino acid sequence encoded by the nucleotide sequence described in any one of (il) to (i-5) below, or a part thereof, and having platelet aggregation promoting activity Polypeptides: (i-1) Nucleotide sequences shown in nucleotide numbers 67 to 132 of sequence number 1 in sequence listing; (i-2) Nuclei shown in nucleotide numbers 67 to 132 of sequence number 3 in sequence listing Nucleotide sequence; -147-200413526 (i-3) Nucleotide sequence shown in nucleotide number 103 to 98 of sequence number 5 in sequence listing; (i-4) Nucleotide number 88 of sequence number 7 in sequence listing (I-5) the nucleotide sequence shown in nucleotide number 82 to 186 of sequence number 13 in sequence listing; ii) in the above (i-1) to (b) The nucleotide sequence of any of the nucleotide sequences complementary to the nucleotide sequence described in any of the nucleotide sequences encoded by the polynucleotide hybridized under severe conditions, and A polypeptide with platelet aggregation promotion activity; (II, such as the sequence of the amino acid sequence of IJ table sequence number 2) [J, containing amino acid number 34 amino acid, the polymerization of more than 6 consecutive amino acid sequences Peptides; (iv) as in the amino acid sequence of SEQ ID NO: 4 in the Sequence Listing, a polypeptide containing amino acid number 34 amino acid and 6 or more consecutive amino acid sequences; (v) as the sequence listing sequence number In the amino acid sequence of 6, a polypeptide containing an amino acid number of 41, an amino acid, and more than 6 consecutive amino acid sequences; (vi) As in the amino acid sequence of sequence number 6, the amino group Acid number 50 amino acid, a polypeptide of 6 or more consecutive amino acid sequences; (vii) the amino acid sequence of sequence number 8 in the sequence listing, containing amino acid number 5 amino acid, 6 consecutive The polypeptide of the above amino acid sequence; (viii) The polypeptide containing the amino acid number 34 of the amino acid sequence of the amino acid sequence of sequence number 14 in the sequence listing; (6) IX) A polypeptide containing an amino acid sequence of 39 to 44 in the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing; -148-200413526 (X) In the amino acid sequence of SEQ ID NO: 4 in the Sequence Listing, a polypeptide containing an amino acid number of an amino acid number 39 to 44; (XI) As shown in the sequence 歹 [J Listing 歹 U number 6 of the amino group A polypeptide containing an amino acid sequence of 46 to 51 in the acid sequence 至 U; (XII) As in the amino acid sequence of sequence number 8 of the Sequence Listing, an amino group of 57 to 62 amino groups (XIII) As in the amino acid sequence of SEQ ID NO: 14 in the Sequence Listing, the polypeptide containing amino acid numbers 39 to 44; (XIV) as described in (iii) to (XIII) A polypeptide in which the amino acid sequence of the polypeptide is missing, substituted, or added with one or more amino acid sequences of the amino acid. 27 · —A method for screening active substances with platelet aggregation promotion or inhibition, including the following projects: (1) In the presence of the test substance, contact gp44 expressing cells with platelets -149-200413526 2 9 · A method for promoting or inhibiting platelet aggregation by an active substance includes the following processes: (]) gp44-expressing cells are administered to a non-human mammal in the presence of the test substance, (2) the origin of the non-human mammal as determined in (1) The number of nodules in the tissue (3) From the measurement results of (2), it is determined whether the test substance has platelet aggregation promotion activity or platelet aggregation inhibition activity. 3 〇-a method for screening for cancer metastasis-inhibiting activity or cancer metastasis-promoting active substances' Methods containing the following engineering ... (1) In the presence of the test substance, gP44 expressing cells are administered to non-human mammals, (2) Determine the number of nodules in tissues derived from non-human mammals of (1), (3) determine the results of (2) to determine whether the test substance has cancer metastasis inhibiting activity or cancer metastasis promoting activity. 3 1 · —A kit for detecting platelet aggregation-promoting or inhibiting active substances, comprising one or more of the substances described in any one of the following (1) to (3): (1) the sequence table sequence number 1, 3, A 5 to 30 base long continuous oligonucleotide primer that partially or fully amplifies one or all of the polynucleotides listed in the nucleotide sequence described in 5, 7, or 13; (2) and Polynucleotides of the nucleotide sequence described in sequence number 1, 3, 5, 7 or 13 of the sequence listing are hybridized under severe conditions, and continuous polynuclei of 15 or more nucleotides of the polynucleotide are examined. (3) The substance according to any one of (1) or (2) is a fixed immobilization reagent. -150-200413526 3 2. -A kit for detecting platelet aggregation promoting or inhibiting active substances, which contains more than one of the substances described in any one of the following (1) to (3): (1) can be specifically associated with sequence number 2, 4, Polypeptide-binding antibody with amino acid sequence according to 6, 8 or 14; (1) Secondary antibody obtained by combining with the antibody described in (1); (3) As in any one of (1) or (2) The substances described are fixed immobilization reagents. 3 3.  -A set for testing platelet aggregation-associated diseases, which contains more than one of the substances described in any one of the following (1) to (3): (1) Sequence numbers 1, 3, 5, and 7 of the sequence table can be specifically amplified Or a part of or all of the nucleotide sequence of the nucleotide sequence of 15 with a 15 to 30 base long continuous oligonucleotide primer; (2) the sequence number 1, 3, 5, 7 or 13 Polynucleotides with recorded nucleotide sequences can be hybridized under severe conditions, and the continuous polynucleotide probes with more than 15 nucleotides can be tested; (3) (1) or (2) The substance described in any one of the items is a fixed immobilizing agent. 03. A set of platelet aggregation-related diseases, including one or more items described in any one of (1) to (3) below. Substances ... (1) An antibody that specifically binds to the polypeptide of the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, or 14 of the sequence listing; (2) Combined with the antibody described in (1) to obtain two Secondary antibody; (3) The substance described in any one of (1) or (2) is a fixed immobilization reagent -151-200413526 3 5 · As described in item 3 3 or 34 of the scope of patent application The set, wherein the platelet aggregation associated disease is cancer, cancer metastasis, pulmonary embolism, cerebral infarction, myocardial infarction or arteriosclerosis special Zhi. 3 6. For example, the set described in item 33 or 34 of the scope of patent application, wherein the platelet-associated diseases are characterized by colon cancer, rectal cancer, small intestine cancer, seminal carcinoma, seminoma, and squamous cell carcinoma. 37. An antibody having platelet aggregation inhibiting activity, which can specifically bind to a polypeptide described in any one of the following (i) to (XIV): (i) any of the following (i-1) to (i-5) Record the amino acid sequence encoded by the nucleotide sequence, or a part of the polypeptide: (i -1) the nucleotide sequence shown by nucleotide number 6 7 to 1 2 in sequence number 1 of the sequence listing; U -2) Nucleotide sequence shown in nucleotide number 67 to 32 of sequence number 3 in sequence list; (i-3) Nucleotide sequence shown in sequence number 5 to nucleotide number 103 to 198 in sequence list (I_4) Nucleotide sequence shown in nucleotide number 88 to 1 86 of sequence number 7 in sequence listing; (i-5) Nucleotide sequence shown in sequence list 1 3 nucleotide number 8 2 to 1 8 6 Nucleotide sequence; (ii) A polynucleotide that is complementary to the nucleotide sequence described in any one of (i-1) to (i-5) above hybridizes under severe conditions A polypeptide made from the amino acid sequence encoded by a polynucleotide; (iii) For example, the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing contains an amino acid number of -152-200413526 with a code of 34 amino acid, continuous 6 or more amino groups (H) A polypeptide (v) containing the amino acid number 34 amino acid sequence and 6 or more consecutive amino acid sequences in the amino acid sequence of sequence number 4 in the Sequence Listing ) For example, in the amino acid sequence of sequence number 6 in the Sequence Listing, a polypeptide containing an amino acid number 41 of the amino acid sequence of more than 6 consecutive amino acids; (vi) As in Sequence Number 6 of the Sequence Listing In the amino acid sequence, a polypeptide (vii) containing an amino acid number of 50 amino acid and a sequence of 6 or more amino acids is provided. For example, the amino acid sequence of sequence number 8 in the Sequence Listing contains an amino acid. Number 52 amino acid, a polypeptide (vii i) made of more than 6 consecutive amino acid sequences, such as the amino acid sequence of sequence number 14 in the sequence listing, contains amino acid number 34 amino acid, 6 consecutive Polypeptide formed from the above amino acid sequence; (IX) For example, a polypeptide composed of the amino acid sequences of 39 to 44 in the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing; (X) For example, in the amino acid sequence of SEQ ID NO: 4 in the Sequence Listing, a polypeptide containing an amino acid number of an amino acid number 39 to 44; (XI) As shown in the Sequence Listing In the amino acid sequence of column number 6, a polypeptide containing an amino acid sequence of 46 to 51 amino acid sequence; (XII) As in the amino acid sequence of sequence number 8 of the Sequence Listing, an amino acid sequence Poly-peptide composed of amino acid sequences of numbers 57 to 62; -153-200413526 (XIII) As in the amino acid sequence of sequence number 14 in the Sequence Listing, the poly-amino acid sequence containing amino acid numbers 3 9 to 44 is included. Peptide; (XIV) the amino acid sequence of the polypeptide as described in any one of (iii) to (XΠΙ) above, which is missing, substituted or added with one or more amino acids Polypeptide. 3 8 · —Substances, such as the scope of patent application! It is screened by the method described in any one of items 4 to 27 and 30 or separated by the method described in any one of items 25 to 26 of the scope of patent application. 39. A mutant polypeptide, which is characterized by the polypeptide described in any one of the following (a) to (f): U) a variant of the amino acid sequence polypeptide shown in SEQ ID NO: 2 in the Sequence Listing, A mutant polypeptide obtained by converting an amino acid of amino acid number 34 into another amino acid; (b) a variant of the polypeptide obtained from the amino acid sequence shown in sequence number 4 in the sequence listing, which A mutant polypeptide obtained by converting an amino acid of amino acid number 34 into another amino acid; (0, a variant of the polypeptide obtained from the amino acid sequence shown in sequence number 6 in the sequence listing 'its amino group A mutant polypeptide obtained by converting an amino acid of acid number 41 to another amino acid; (d) a variant of the polypeptide obtained from the amino acid sequence shown in sequence number 6 in the sequence listing 'its amino group A mutant polypeptide obtained by converting an amino acid of acid number 50 into another amino acid; (0, a variant of the polypeptide obtained from the amino acid sequence shown in SEQ ID NO: 8 in the Sequence Listing, its amino acid number The amino acid sequence of 5 2 was converted to other amino acids and the mutant poly-peptide obtained from -154-200413526; (f) the amino acid sequence shown in sequence number 14 in the sequence listing The resulting poly-peptide variant is a mutant poly-peptide obtained by converting the amino acid of amino acid number 34 to another amino acid. 40. The mutant poly-peptide described in item 39 of the scope of patent application Peptide, in which the other amino acid is alanine. 4 1 · A DNA, which encodes a mutant polypeptide as described in item 39 or 40 of the patent application scope. 42. A pharmaceutical composition containing the following (a Polynucleotides formed by the nucleotide sequence described in any one of items) to IX are effective components: U) The nucleotide sequence shown in sequence number 1 of the sequence list 1 and the nucleotide numbers 1 to 519; (b) the sequence list Nucleotide sequence 0 shown in nucleotide numbers 1 to 501 of sequence number 3; (c) Nucleotide sequence shown in nucleotide number 1 to 5 10 of sequence list sequence number 5; (d) Sequence listing (E) the nucleotide sequence shown in nucleotide number 1 to 54 of the sequence listing sequence number 13; (f) the same as the above ( (a) to (e) The polynucleotides complementary to the nucleotide sequence described in any one of the nucleotide sequences described in the present invention hybridize under severe conditions and have platelet aggregation. Set of nucleotide sequences encoded by active peptides; (g) Amino acid-155-200413526 sequences of amino acid numbers 1 to 172 in Sequence Listing Sequence Number 2; h) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in the amino acid numbers 1 to 1 of 66 in the sequence listing sequence number 4; (i) the amine shown in the amino acid numbers 1 to 169 of the sequence listing sequence number 6 Nucleotide sequence encoded by amino acid sequence polypeptide; (j) Nucleotide sequence encoded by amino acid sequence polypeptide shown in sequence list sequence number 8 amino acid number 1 to 162; (k) sequence listing sequence The nucleotide sequence encoded by the amino acid sequence polypeptide of the amino acid sequence shown in number 1 to amino acid number 1 to 17; (l) in the amino acid sequence described in any one of (g) to (k) above, A nucleotide sequence encoded by a polypeptide having a platelet aggregation-promoting activity that is missing, substituted, or added with one or more amino acid sequences formed from amino acids. 43 · —A pharmaceutical composition containing a polynucleotide comprising the antisense sequence of the nucleotide sequence described in any one of the following (a) to (1): U) Sequence table sequence number 1 nucleoside The nucleotide sequence shown in the acid number 1 to 519; (b) the nucleotide sequence shown in the nucleotide number 1 to 501 of sequence number 3 in the sequence listing; (c) the nucleotide number of the sequence number 5 in the sequence listing Nucleotide sequences shown from 1 to 5 10; (d) Nucleotide sequences shown from nucleotide numbers 1 to 489 in Sequence Listing Sequence Number 7; (e) Nucleotide number 1 from Sequence Listing Sequence Number 1 3 The nucleotide sequence shown to 5 to 40; -156-200413526 (0 is a nucleotide sequence complementary to the nucleotide sequence described in any of (a) to (0) above. Nucleotide sequences encoded by a polypeptide that hybridizes under severe conditions and has platelet aggregation-promoting active peptides; (g) Nucleosides encoded by amino acid sequence polypeptides shown in amino acid numbers 1 to 172 of Sequence Listing sequence number 2 Acid sequence; (h) the nucleotide sequence encoded by the amino acid sequence polypeptide shown in amino acid numbers 1 to 166 in sequence number 4 in sequence listing; (i) sequence number 6 in sequence listing Nucleotide sequence encoded by amino acid sequence polypeptide shown in amino acid numbers 1 to 69; (j) Sequence listing sequence number 8 Nucleotide sequence; (k) Nucleotide sequence encoded by the amino acid sequence polypeptide shown in SEQ ID NO: 14 amino acid number 1 to 172 in the Sequence Listing; (l) any one of (g) to (k) above In the described amino acid sequence, a nucleotide sequence encoded by a polypeptide having a platelet aggregation promoting activity that is missing, substituted, or added with one or more amino acid sequences formed from an amino acid. 44 · A A pharmaceutical composition containing a polynucleotide combination carrier comprising the nucleotide sequence described in any one of the following (a) to (1): U) Sequence Listing Sequence Number 1 Nucleotide Numbers 1 to 519 Nucleosides shown. Acid sequence; (b) the nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in the sequence listing; (0 the nucleotide sequence shown in nucleotide number 1 to 5 10 of sequence listing 5; -157-200413526 (d) the nucleotide sequence shown in nucleotide number 1 to 489 of sequence number 7 in the Sequence Listing; (e) the nucleotide sequence shown in nucleotide number 1 to 540 of sequence number 13 in Sequence Listing; (f) Polynucleotides formed from nucleotide sequences complementary to the nucleotide sequences described in any one of (a) to (e) above hybridize under severe conditions, and have platelet aggregation promoting activity Nucleotide sequence of peptide-encoded polynucleotide; (g) Nucleotide sequence encoded by amino acid sequence poly-peptide shown in amino acid numbers 1 to 172 of sequence number 2 in sequence listing; φ (h) sequence The nucleotide sequence encoded by the amino acid sequence poly-peptide shown in amino acid numbers 1 to 166 of sequence number 4 in the list; (i) the sequence listing 6 Polypeptide-encoded nucleotide sequence; (j) Sequence number 8 amino acid sequence number 1 to 162 Sequence; (k) a nucleotide sequence encoded by the amino acid sequence polypeptide shown in SEQ ID NO: 14 amino acid number 1 to 172 in the sequence listing; φ (l) described in any one of (g) to (k) above 45. A nucleotide sequence encoded by a polypeptide having an amino acid sequence formed by the absence, substitution, or addition of one or more amino acids in the amino acid sequence and having platelet aggregation promoting activity. 45. A pharmaceutical composition comprising a polynucleotide combination carrier containing the antisense sequence of the nucleotide sequence described in any one of the following (a) to (1): (a) sequence table sequence number 1 nucleotide Nucleotide sequence IJ shown by numbers 1 to 5 1 9; -158-200413526 (b) Nucleotide sequence number 1 to 50 shown in sequence number 3 of the sequence listing; (C) Sequence listing Nucleotide sequence shown in nucleotide number 1 to 5] of sequence number 5; (d) Nucleotide sequence shown in nucleotide number 1 to 4 8 of sequence number 7 in sequence listing; (e) Sequence listing Nucleotide sequences shown in nucleotide numbers 1 to 54 of SEQ ID NO: 13; (ii) a polynucleotide sequence complementary to the nucleotide sequence described in any one of (a) to (e) above; Nucleotides hybridize under stringent conditions and have platelet aggregation-enhancing polypeptide-encoded nucleotide sequences; (g) the amino acid sequences shown in amino acid numbers 1 to 172 of the sequence listing sequence number 2 Polyheptide-encoded nucleotide sequence; (h) Nucleotide-encoded nucleotide sequence of amino acid sequence 1 to 166 of amino acid numbers 1 to 166 in Sequence Listing Sequence Number 4; ⑴ Sequence Listing Column No. 6 Nucleotide sequence encoded by amino acid sequence polypeptide shown by amino acid numbers 1 to 169; Lu Xun sequence listing sequence number 8 Amino acid sequence poly coded peptide shown by amino acid number 1 to 162 The nucleotide sequence of the nucleotide sequence of the amino acid number 1 to 172 of the amino acid number 1 to 172 in the sequence listing sequence number 14; U) any one of (g) to (k) above In the described amino acid sequence, a nucleotide sequence encoded by a polypeptide having platelet aggregation promotion activity formed by missing, replacing, or adding an amino acid sequence formed by one or several amino acids is added. -159-200413526 46.  -A pharmaceutical composition containing a polypeptide as described in any one of the following items (i) to (viii): (i) a nucleoside as described in any one of (i-1) to (i-5) below The amino acid sequence encoded by the acid sequence, or a part thereof, and a polypeptide with platelet aggregation promotion activity: (i -1) SEQ ID NO: 1 SEQ ID NOS: 1 to 5 1 9 Nucleotide sequence; (i-2) Nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in sequence listing; (i-3) Nucleotide number 1 to 5 in sequence listing 5 Nucleotide sequence shown in 10; (i-4) Nucleotide sequence shown in nucleotide number 1 to 4 8 in sequence number 7 in sequence listing; (i-5) Nucleotide sequence in sequence number 13 in sequence listing Nucleotide sequences represented by numbers 1 to 540; (ii) a polynucleoside composed of a nucleotide sequence complementary to the nucleotide sequence described in any one of (i-1) to (i-5) above Polyacid peptides encoded by polyacid peptides which hybridize under severe conditions and have platelet aggregation promotion activity; (iii) amines shown in amino acid numbers 1 to 172 of sequence number 2 in the sequence listing Amino acid sequence Polypeptide; (iv) Polypeptide obtained from the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing of amino acid number 1 to 166; (v) SEQ ID NO: 6 in the sequence listing of amino acid number 1 to 169 A polypeptide comprising an amino acid sequence of -160-200413526; (V i) A polypeptide comprising an amino acid sequence shown in SEQ ID NO: 8 in the sequence listing 8 amino acid numbers i to 〖6 2; (vii ) A polypeptide obtained from the amino acid sequence shown in SEQ ID NO: 14 in the sequence listing of amino acid numbers 1 to 172; (viii) the amine of the polypeptide according to any one of (iii) to (vii) above A polypeptide having an amino acid sequence formed by missing, replacing, or appending one or more amino acids in the amino acid sequence and having platelet aggregation promoting activity. 47 · A pharmaceutical composition comprising an antibody specifically binding to the polypeptide described in any one of the following (i) to (viii): (i) any one of (1-1) to (i-5) below The amino acid sequence encoded by the nucleotide sequence described in the item, or a polypeptide formed from a part thereof and having platelet aggregation promotion activity: (1 -1) Sequence Listing Sequence Number 1 Nucleotide Number 1 to 5] The nucleotide sequence shown in 9; (i_2) The nucleotide sequence shown in nucleotide number 1 to 501 of sequence number 3 in the sequence listing; (b) The nucleotide number 1 of sequence number 5 in the sequence listing 5 to 0; (i_4) Sequence number 7 to nucleotide number 1 to 489; (i-5) Sequence number 13 to nucleotide number 1 (1)) The nucleotide sequence described in any one of (i -1) to (i-5) above is complementary to the nucleotide sequence of -161-200413526 Polynucleotides hybridize under severe conditions, and have the nucleotide sequence of the polynucleotide encoded by the active peptide that promotes platelet aggregation; (iii) the amino acid numbers 1 to 2 of the sequence listing Polyaminopeptide of amino acid sequence shown at 172; (iv) Polyaminopeptide of amino acid sequence shown at sequence number 4 of Sequence Listing 4 to 166; (v) Sequence number 6 of amino acid sequence list 1 Amino acid sequence polypeptide shown to 169; 〇i) Sequence table sequence number 8 amino acid number 1 to 162 amino acid sequence polypeptide; (vii) Sequence table sequence number 14 amino acid number 1 (Vii i) the amino acid sequence of the polypeptide as described in any one of (iii) to (vii) above, which lacks, substitutes, or adds 1 or more A polypeptide formed from an amino acid sequence formed by an amino acid and having platelet aggregation promoting activity. 4 8 · — a kind of pharmaceutical composition, which contains the method selected by the method described in any one of items 1 to 4 and 27 to 30 in the scope of patent application, or as described in item 25 or 26 of the scope of patent application Any of the substances described separately. 4 9 · If the pharmaceutical composition of any one of claims 4, 2, 4 and 46 of the scope of patent application, it can promote platelet aggregation. 50. The pharmaceutical composition according to any one of claims 43, 45, 47, and 48 of the patent application scope, which can inhibit platelet aggregation. 51. Such as the application of § 靑 patent car b surrounding any one of items 43, 45, 47 and 48 of the medicine -162-413526 composition 'it can treat or prevent colon cancer, rectal cancer, small intestine cancer, seminal cancer, spermatogonia Tumor, squamous cell carcinoma, cancer metastasis, pulmonary thrombosis, cerebral infarction, myocardial infarction, arteriosclerosis, bleeding tendency, blood coagulation disorder, hemophilia, uremia or chronic bone marrow colonic disease or inhibit platelet aggregation during vascular surgery. -163-