JP2008301776A - New peptide and method for using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new peptide which specifically binds to a blood coagulation factor: von Willebrand factor to inhibit its degradation, to provide a method for using the same, to provide a von Willebrand factor-cleaving enzyme activity inhibitor, and to provide a primary hemostatic thrombus formation promoter. <P>SOLUTION: This peptide has a specific amino acid sequence and an activity for binding to the von Willebrand factor. This peptide also comprises a specific amino acid sequence which is a part of the amino acid sequence of human ADAMTS13 protein and has 1 to 22 amino acids added to the N-terminal side or 1 to 3 amino acids added to the C-terminal side, and has activity for binding to the von Willebrand factor. A medicine containing the peptide can be used as a von Willebrand factor-cleaving enzyme activity inhibitor, or a primary hemostatic thrombus formation promoter. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel peptide that specifically binds to von Willebrand factor (vWF) and inhibits degradation, a method of using the same, an inhibitor of von Willebrand factor cleaving enzyme activity, and primary hemostatic thrombus It relates to a formation accelerator.

  When bleeding occurs, a blood component is solidified, and a defense mechanism (hemostatic mechanism) that operates to minimize bleeding by forming a hemostatic thrombus is activated.

  Various factors are involved in such a hemostatic mechanism, one of which is the von Willebrand factor (vWF). vWF is a protein produced by vascular endothelial cells and bone marrow megakaryocytes, which forms platelet thrombus (primary hemostatic thrombus formation) and promotes the growth of coagulated thrombus at that site (secondary hemostatic thrombus formation) It plays an important role (see Non-Patent Document 1, for example). Specifically, vWF acts as a glue necessary for the binding of platelet membrane receptors and collagen, and stabilizes and transports factor VIII when platelets adhere to subendothelial tissues (eg, collagen). To do.

On the other hand, in the body, ADAMTS13 (a disintegrin-like and metalloprotease with the enzyme that works to degrade vWF and reduce the polymer (multimer) size in order to prevent the formation of platelet thrombus in blood vessels. thrombospondin type 1 motifs 13) exists (see, for example, Non-Patent Document 2).
Fujimura Y, Titani K: Structure and function of von Willebrand factor.In Haemostasis and Thrombosis.Bloom Al, et al, ed.Churchill Livingstone., 379-395 (1994) Soejima K, et al, Anovel human metalloprotease synthesized in the liver and secreted into the blood: possibly, the von Willebrand factor-cleaving protease ?, J Biochem, 130, 475-480 (2001)

  Therefore, it was thought that if the degradation of von Willebrand factor was suppressed, it would be useful for hemostasis and coagulation.

  The present invention provides a novel peptide that specifically binds to von Willebrand factor and inhibits degradation, a method for using the same, a von Willebrand factor cleavage enzyme activity inhibitor, and a primary hemostatic thrombus formation promoter. With the goal.

  As shown in the following examples, the present inventors have identified a site that strongly binds to von Willebrand factor in ADAMTS13 protein, and found that the peptide corresponding to that site inhibits cleavage of vWF by ADAMTS13, The present invention has been completed.

That is, the peptide according to the present invention is characterized by the following peptide (a), (b) or (c).
(A) A peptide having the amino acid sequence of SEQ ID NO: 1.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and having a binding activity to von Willebrand factor (vWF).
(C) a part of the amino acid sequence of human ADAMTS13 protein, which consists of an amino acid sequence in which 1 to 22 amino acids are added to the N-terminal side in the amino acid sequence of SEQ ID NO: 1; A peptide consisting of an amino acid sequence to which three amino acids are added and having a binding activity to von Willebrand factor.

Moreover, the peptide concerning this invention is the peptide of the following (a), (b) or (c), It is characterized by the above-mentioned.
(A) A peptide having the amino acid sequence of SEQ ID NO: 2.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2, and having a binding activity to von Willebrand factor.
(C) a part of the amino acid sequence of human ADAMTS13 protein, consisting of an amino acid having 1 to 2 amino acids added to the N-terminal side in the amino acid sequence of SEQ ID NO: 2, or 1 to 12 C-terminal side A peptide having an amino acid sequence to which is added an amino acid and having binding activity to von Willebrand factor.

Furthermore, the peptide concerning this invention is the peptide of the following (a) or (b), It is characterized by the above-mentioned.
(A) A peptide having the amino acid sequence of SEQ ID NO: 3.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3, and having a binding activity to von Willebrand factor.

The DNA according to the present invention is characterized by encoding the peptide.
Furthermore, the expression vector according to the present invention is characterized by expressing the peptide.

Moreover, the cell concerning this invention hold | maintains the said expression vector, It is characterized by the above-mentioned. This cell may be, for example, a prokaryotic organism such as E. coli, a unicellular eukaryotic organism such as yeast, or a cultured cell such as an insect or a mammal.
Furthermore, the von Willebrand factor cleaving enzyme activity inhibitor and the primary hemostatic thrombus formation promoting agent according to the present invention contain the peptide.

  According to the present invention, a novel peptide that specifically binds to von Willebrand factor and inhibits degradation, a method of using the same, a von Willebrand factor cleaving enzyme activity inhibitor, and a primary hemostatic thrombus formation promoter are provided. Can do.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

=== Vonville brand factor binding peptide ===
In the present invention, three types of peptides that specifically bind to von Willebrand factor and inhibit degradation are found.

The first von Willebrand factor binding peptide is the following peptide (a), (b) or (c).
(A) A peptide having the amino acid sequence of SEQ ID NO: 1.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and having a binding activity to von Willebrand factor (vWF).
(C) a part of the amino acid sequence of human ADAMTS13 protein, which consists of an amino acid sequence in which 1 to 22 amino acids are added to the N-terminal side in the amino acid sequence of SEQ ID NO: 1; A peptide consisting of an amino acid sequence to which three amino acids are added and having a binding activity to von Willebrand factor.

  The peptide may be synthesized by chemical synthesis or may be synthesized by expressing the peptide as described below using DNA encoding the peptide.

  As the DNA encoding the peptide, DNA having a sequence created from the amino acid sequence of the peptide using a codon table may be used. In order to obtain these DNAs, genomic libraries and cDNA libraries containing clones having the DNA sequence may be screened, and PCR or RACE using the genome or RNA of the species having the DNA sequence. It may be amplified by a method or the like, or these may be combined. The DNA may be prepared by chemical synthesis.

As said peptide, RPDITFTYFQPKPRQ (sequence number 1), RPDITFTYFQPKPRQAWV (PP1, sequence number 4) etc. are mentioned, for example.
The second peptide is the following peptide (a), (b) or (c).
(A) A peptide having the amino acid sequence of SEQ ID NO: 2.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2, and having a binding activity to von Willebrand factor.
(C) A part of the amino acid sequence of human ADAMTS13 protein, consisting of an amino acid having 1 to 2 amino acids added to the N-terminal side in the amino acid sequence of SEQ ID NO: 2, or 1 to 12 C-terminal side A peptide having an amino acid sequence to which is added an amino acid and having binding activity to von Willebrand factor.

  The methods for synthesizing these peptides and DNAs encoding these peptides are as described above.

  Examples of the peptide include RHMCRAIGESFIMKRGDSFLDGTR (PP2, SEQ ID NO: 2) and the like.

Furthermore, the third peptide is the following peptide (a) or (b).
(A) A peptide having the amino acid sequence of SEQ ID NO: 3.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3, and having a binding activity to von Willebrand factor.
The methods for synthesizing these peptides and DNAs encoding these peptides are as described above.
Examples of the peptide include PSLLEDGRVEYRVALTEDRLPRLE (PP3, SEQ ID NO: 3).

=== Usefulness of von Willebrand factor binding peptide ===
(1) von Willebrand factor cleaving enzyme activity inhibitor The von Willebrand factor (vWF) cleaving enzyme is also called ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs 13).

  As shown in Example 1, screening for peptides that specifically bind to von Willebrand factor results in the isolation of peptides as shown in FIG. Here, in the vicinity of Epitope 1, since the peptide lacking the N-terminal side from the 669th and the C-terminal side from the 685th has been isolated, the region necessary for specific binding is from the 670th to the 684th. It is thought that. Therefore, hereinafter, the region from the 670th to the 684th is called Epitope 1. Similarly, even in the vicinity of Epitope 2 and Epitope 3, the regions necessary for specific binding can be identified as 484th to 507th and 618th to 641st, respectively. Call. Since these peptides corresponding to Epitopes bind specifically to von Willebrand factor, they can function as von Willebrand factor binding peptides.

  Furthermore, from the Examples, the peptides described in SEQ ID NOs: 2, 3, and 4 bind to von Willebrand factor and inhibit ADAMTS13 activity, that is, inhibit von Willebrand factor cleavage enzyme activity (FIG. 2). checking). Therefore, the peptide of the present invention can be used as an inhibitor of von Willebrand factor cleavage enzyme activity.

  Since these peptides having inhibitory activity correspond to Epitopes 1-3, any peptide that binds to any of Epitopes 1-3 is considered to inhibit ADAMTS13 activity. Therefore, is it part of the amino acid sequence of the peptide consisting of SEQ ID NO: 1 and human ADAMTS13 protein and consisting of the amino acid sequence of SEQ ID NO: 1 with 1 to 22 amino acids added to the N-terminal side? Or a peptide consisting of an amino acid sequence having 1 to 3 amino acids added to the C-terminal side, and an amino acid sequence of SEQ ID NO: 2 consisting of amino acids having 1 to 2 amino acids added to the N-terminal side, or A peptide consisting of an amino acid sequence to which 1 to 12 amino acids on the C-terminal side are added also specifically binds to vWF and inhibits the binding between vWF and ADAMTS13 as shown in the examples, thereby allowing ADAMTS13 (von Bill brand It is thought that the factor-cleaving enzyme) activity is inhibited.

  The von Willebrand factor cleaving enzyme activity inhibitor of the present invention can be used, for example, to study the function of von Willebrand factor or von Willebrand factor cleaving enzyme.

(2) Thrombosis model animal von Willebrand factor (vWF) has a multimer structure in which a plurality of one kind of subunits consisting of 2,050 amino acid residues are disulfide bonded. In a normal living body, vWF having such a multiple structure is appropriately decomposed by ADAMTS13.

  As shown in the Examples below, von Willebrand factor binding peptides inhibit vWF cleavage by ADAMTS13 (see FIG. 3). This means that administration of the von Willebrand factor binding peptide makes it impossible to degrade the multimeric structure of vWF, thereby promoting vWF-dependent platelet thrombus formation. Therefore, a thrombosis model animal can be produced by allowing the von Willebrand factor binding peptide to exist in the animal body. Here, the vertebrates to be model animals are not particularly limited as long as they are vertebrates other than humans, and examples thereof include mice, rats, guinea pigs, rabbits, dogs, monkeys, and the like.

The method for producing a thrombosis model animal is not particularly limited. For example, a von Willebrand factor-binding peptide in an amount that causes thrombosis and that does not lead to lethality is applied to vertebrates other than humans. Although it may be administered, the most preferred method is to create a transgenic mouse that expresses the von Willebrand factor binding peptide.
For thrombosis model animals, blood vessel strengthening agents (eg, adrenochrome, flavonoids, vitamin C, ethanesylate, estrogen), coagulation promoters (eg, vitamin K, purified snake venom preparation), anti-fibrinolytic agents (eg, antiplasmin) Thrombosis can be generated more efficiently by combining administration of drugs) and blood products.
The thrombosis model animal produced by the above method is useful for screening novel drugs such as anticoagulants, thrombolytic agents, and platelet aggregation inhibitors.

(3) Primary hemostatic thrombus formation promoter (3-1) Pharmacological action As described above, when the vascular endothelium is damaged by trauma or the like, platelets adhere and aggregate at the damaged site, and primary hemostatic thrombus formation is performed. Here, one of the proteins produced in vascular endothelial cells and secreted into plasma is von Willebrand factor (vWF). vWF binds the receptors on the platelet membrane to collagen in the blood vessels exposed by the damage and induces platelet adhesion (primary hemostatic thrombus formation).
The von Willebrand factor binding peptide of the present invention inhibits the cleavage of vWF by ADAMTS13 (see FIG. 3). This means that administration of the von Willebrand factor binding peptide makes it impossible to degrade the multimeric structure of vWF, thereby promoting vWF-dependent platelet thrombus formation. Therefore, the drug containing the von Willebrand factor binding peptide of the present invention can be used as a primary hemostatic thrombus formation promoter.

Here, the symptoms / diseases that need to promote the formation of primary hemostatic thrombus include, for example, bleeding from small blood vessels, capillaries and parenchymal organs that are difficult to stop by normal ligation (for example, bleeding due to trauma, surgery) Bleeding, bone bleeding, bladder bleeding, bleeding after tooth extraction, nasal bleeding, bleeding from the upper gastrointestinal tract, etc.).
In the case of hemostasis, in addition to the administration of the agent of the present invention, a blood vessel enhancer (for example, adrenochrome, flavonoid, vitamin C, ethanesylate, estrogen), a coagulation promoting agent (for example, vitamin K, purified snake venom preparation), anti-ray Combining administration of a lysing drug (for example, an antiplasmin drug), a blood product, etc. can stop hemostasis more efficiently.

(3-2) Method for Producing the Drug The drug only needs to contain the peptide of the present invention. The method for producing the peptide of the present invention is as described above.
As a pharmaceutically acceptable carrier that can be used in the drug of the present invention, various organic or inorganic carrier substances that are conventionally used as a pharmaceutical material may be used. For example, excipients, lubricants, binders and A disintegrant or a solvent in a liquid preparation, a solubilizing agent, a suspending agent, a tonicity agent, a buffering agent, a soothing agent and the like may be contained. Further, if necessary, additives such as usual preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like may be appropriately contained. As the dosage form, oral preparations include, for example, tablets, capsules, granules, powders, fine granules, syrups, sustained-release tablets / capsules / granules, cashews, chewable tablets or drops. Examples of the injection include solution injections, emulsion injections, solid injections, and the like, and examples of external preparations include removal powders, lotions, ointments / creams, sprays, tapes, and the like.

(3-3) Dosage and administration method of the drug The dose of the drug varies depending on age, weight, indication, or route of administration, but within the range where the above action can be exerted and the resulting side effects are acceptable. If there is no particular limitation. It should be noted that when the drug of the present invention is injected into the blood, the coagulation system of the whole body is promoted and a fatal result may be caused.
The route of administration of the drug is not particularly limited as long as the drug is delivered to the site of bleeding. For example, local administration is preferable in the case of bleeding due to trauma.
The administration method is not particularly limited as long as the drug is delivered to the bleeding site. For example, when bleeding due to trauma is observed in humans or non-human vertebrates, the drug is sprayed as it is. Or irrigate or distribute.

  Hereinafter, although the embodiment described above is specifically described using examples, this is an exemplification, and the present invention is not limited to these examples.

<Example 1: Epitope analysis of ADAMTS13 protein>
In order to identify the epitope of ADAMTS13 protein involved in binding to von Willebrand factor (vWF), the following experiment was performed.

(1) Preparation of ADAMTS13 epitope library
ADAMTS13 cDNA was obtained from Daiichi Pharmaceutical Co., Ltd. ADAMTS13 epitope library is available from known literature (Kuwabara I, Maruyama H, Mikawa YG, Zuberi RI, Liu FT, Maruyama IN. Efficient epitope mapping by bacteriophage lambda surface display. Nat Biotechnol 1997; 15: 74-8., Kuwabara I , Maruyama H, Kamisue S, Shima M, Yoshioka A, Maruyama IN.Mapping of the minimal domain encoding a conformational epitope by lambda phage surface display: factor VIII inhibitor antibodies from haemophilia A patients.J Immunol Methods 1999; 224: 89-99 Moriki T, Kuwabara I, Liu FT, Maruyama IN.Protein domain mapping by lambda phage display: the minimal lactose-binding domain of galectin-3. Biochem Biophys Res Commun 1999; 265: 291-6., Hagiwara H, Kunihiro S, Nakajima K, Sano M, Masaki H, Yamamoto M, Pak JW, Zhang Y, Takase K, Kuwabara I, Maruyama IN, Machida M. Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector. J Biochem (Tokyo) 2002; 132: 975-82. Specifically, it is as follows.

First, the full length ADAMTS13 cDNA was inserted into pcDNA3.1 myc-His (Invitrogen, Carlsbad, Ca). Thereafter, this vector was randomly decomposed with DNase I (Takara Bio Inc.), and the ends thereof were made blunt with T4 DNA polymerase (New England Biolabs, Beverly, MA). After ligation of the Sfi I adapter to the blunt end, 2.0% agarose gel electrophoresis (NuSieve; FMC, Rockland, ME) was performed to fractionate the DNA fragment. Agarose containing each DNA fragment ranging from 100 bp to 200 bp was cleaved, these DNA fragments were purified using a gel extraction kit (Qiagen Tokyo), and the purified DNA was inserted into the Sfi I site of the λfooDc vector. Subsequently, these vectors were packaged with MaxPlax (Epicentre Technologies, Madison, Wis.) And then infected with Escherichia coli strain Q447 to prepare a phage library.
As a result, a phage library of 1.1 × 10 ⁷ pfu (plaque forming unit) was obtained.

(2) Affinity selection and DNA sequencing
i) Affinity selection Phage that expresses various peptides derived from the ADAMTS13 plasmid exists in the phage library prepared by the method of (1) above. Therefore, affinity selection was performed for the binding of these phages to vWF. Affinity selection is performed using known literature (Kuwabara I, Maruyama H, Mikawa YG, Zuberi RI, Liu FT, Maruyama IN. Efficient epitope mapping by bacteriophage lambda surface display. Nat Biotechnol 1997; 15: 74-8., Kuwabara I , Maruyama H, Kamisue S, Shima M, Yoshioka A, Maruyama IN.Mapping of the minimal domain encoding a conformational epitope by lambda phage surface display: factor VIII inhibitor antibodies from haemophilia A patients.J Immunol Methods 1999; 224: 89-99 , Moriki T, Kuwabara I, Liu FT, Maruyama IN. Protein domain mapping by lambda phage display: the minimal lactose-binding domain of galectin-3. Biochem Biophys Res Commun 1999; 265: 291-6. . Specifically, it is as follows.

  First, PBS (containing 0.1% sodium azide) containing 7.5 μg / ml human vWF (Haematologic Technologies, Inc., River Road, VT) was placed in a microtiter plate (50 μl / well) and incubated at 4 ° C. overnight.

On the other hand, the phage prepared by the above method (1) and E. coli TG1 are placed in CY medium (Casamino Acid 10 g, Yeast Extract 5 g, NaCl 3 g, KCl 2 g, Distilled water to 1000 ml (pH 7.0)). (10 μl / ml) at 37 ° C. for 3-6 hours, and added with protease inhibitors (Pierce, Rockford, IL). Thereafter, polyethylene glycol (molecular weight 8000) is added to precipitate the phage, and the precipitated phage is bound to a binding buffer (5% skim milk, 0.25% bovine serum albumin (BSA), 0.1% Tween-20, and 0.1% sodium azide PBS. ). Here, since ADAMTS13 is a zinc-type metalloprotease, a divalent cation such as Ba ²⁺ or Ca ²⁺ is required to exert its action. Therefore, in order to investigate whether a divalent metal cation is involved in the binding of ADAMTS13 and vWF, a similar experiment was conducted using 5 mM EDTA-containing BSA or 5 mM EDTA-containing BSA instead of 5% skim milk. went. The phage suspension was then placed in the microtiter plate coated with human vWF and incubated overnight at 4 ° C.

Then, it was washed 3 times with binding buffer, washed twice with buffer-1 (PBS containing 5% skim milk and 0.5% Tween 20), and buffer-2 (10 mM Tris-HCl (pH 7.4), 5 mM MgSO ₄ , 0.2 M NaCl and 10 mM CaCl ₂ ) were washed once, and finally, the phage bound to the microtiter plate was detached from the plate using collagenase (Sigma, Tokyo) (hereinafter, these experimental methods are referred to as “panning”). The titer of the obtained phage was measured by infecting E. coli JM105 with these phages in a medium containing X-gal. Panning was repeated until the number of white plaques reached 90% or more of the total number of plaques (4 times in total).
As a result, 12 phage clones specifically binding to vWF were obtained.

ii) Epitope mapping The nucleotide sequence of the inserted ADAMTS13 DNA fragment was examined for 12 phage clones obtained by the method described in i) above.
As a result, as shown in FIG. 1, 4 clones were mapped to the C-terminal side of the ADAMTS13 spacer domain, and 8 clones were mapped to the N-terminal and C-terminal centers of the ADAMTS13 cysteine-rich domain.
From this, it is found that the ADAMTS13 amino acid sequence specifically binds to human vWF is R ⁶⁷⁰ PDITFTYFQPKPRQ ⁶⁸⁴ (Epitope 1, SEQ ID NO: 1) and R ⁴⁸⁴ HMCRAIGESFIMKRGDSFLDGTR ⁵⁰⁷ (Epitope 2, SEQ ID NO: 2) It was.

iii) Panning with addition of peptide Furthermore, it was examined whether there was a peptide that specifically binds to vWF other than Epitope 1 and Epitope 2. The experimental method was the same as the above “i) except that“ PP1 (SEQ ID NO: 4) synthesized to a final concentration of 0.5 mM was added ”in the“ binding buffer ”described in“ i) Affinity selection ”. This is the same as the method described in “Affinity Selection”. A peptide corresponding to PP1 (SEQ ID NO: 4) was prepared by synthesis (Toray Research Center, Kamakura, Japan).
As a result, 12 phage clones specifically binding to vWF were obtained.

Furthermore, when the nucleotide sequence of the inserted ADAMTS13 DNA fragment was examined for these 12 phage clones, as shown in FIG. 1, all 12 clones were mapped to the center of the N-terminus and C-terminus of the ADAMTS13 spacer domain. It was done.
From this, it was revealed that a new site specifically binding to human vWF in the ADAMTS13 amino acid sequence is P ⁶¹⁸ SLLEDGRVEYRVALTEDRLPRLE ⁶⁴¹ (Epitope 3, SEQ ID NO: 3).

(3) Epitope analysis
In order to analyze the binding sites of Epitope 1, Epitope 2, and Epitope 3 on the ADAMTS13 amino acid sequence, a peptide corresponding to Epitope 1 (SEQ ID NO: 1) was designated as PP1 (SEQ ID NO: 4) and a peptide corresponding to Epitope 2 The peptide corresponding to PP2 (SEQ ID NO: 2) and Epitope 3 was PP3 (SEQ ID NO: 3), and various combinations of epitope peptides were added for panning. The experimental method is the same as the method described in “iii) Panning with addition of peptide”. In addition, peptides corresponding to PP2 (SEQ ID NO: 2) and PP3 (SEQ ID NO: 3) were prepared by synthesis (Toray Research Center, Kamakura, Japan). For the control, a peptide without addition of a peptide (PP (−)) and a peptide with a reverse of the amino acid sequence of PP1 (PP1R) were used. The results are as shown in Table 1.

  As shown in Table 1, when PP1 was added to the binding buffer, the number of phage clones expressing PP3 increased. In addition, when PP3 was added to the binding buffer, the number of phage clones expressing PP1 increased. On the other hand, addition of PP2 and PP1R to the binding buffer had no effect on the expression of phage clones. In addition, when the synthetic peptide was not put into the binding buffer, the number of phage clones expressing PP1 and PP2 was restored. Furthermore, addition of PP1, PP2 and PP3 to the binding buffer had no effect on the expression of phage clones.

  Thus, the ADAMTS13 amino acid sequence differs in the site that specifically binds to human vWF, but Epitope 1, Epitope 2 and Epitope 3 epitopes overlap in complex manner, and the binding site of PP1 is Epitope 3 It was shown that the site was exposed, making it more susceptible to PP3 attacks (see Figure 1).

<Example 2: Evaluation of ADAMTS13 protease activity inhibition by addition of synthetic peptide>
As described above, ADAMTS13 protein is an enzyme that specifically cleaves von Willebrand factor (vWF). In Example 1, the vWF binding site in the ADAMTS13 protein could be identified, so in this example, the effect of the synthetic peptide on ADAMTS13 protease activity inhibition was examined.

  Recombinant ADAMTS13 protein is available from the literature (Uchida T, Wada H, Mizutani M, Iwashita M, Ishihara H, Shibano T, Suzuki M, Matsubara Y, Soejima K, Matsumoto M, Fujimura Y, Ikeda Y, Murata M; Research Project on Genetics of Thrombosis. Identification of novel mutations in ADAMTS13 in an adult patient with congenital thrombotic thrombocytopenic purpura. Blood 2004; 104: 2081-3.).

  The expression vector was prepared by inserting ADAMTS13 cDNA into pcDNA3.1 myc-His using FuGENE6 (Roche Molecular Biochemicals. Indianapolis, IN). This expression vector was introduced into HEK293 cells and cultured in DMEM medium containing 10% FBS and penicillin / streptomycin. After culturing for 24 hours, the medium was replaced with serum-free OPTI-MEM1 medium (Invitrogen), and further cultured for 48 hours. Thereafter, the cells were collected by centrifugation. The amount of the obtained recombinant ADAMTS13 protein was measured by ELISA (American Diagnostica, Stamford, CT).

  Next, using FRETS-VWF73 (a chemically synthesized substrate with a fluorescent group and a quenching group introduced on both sides of the cleavage site of the VWF73 peptide) assay kit (Peptide Institute, Osaka, Japan), the effect of the synthetic peptide on ADAMTS13 protease activity inhibition I investigated.

  First, in order to perform the FRETS-VWF73 assay, synthetic peptides (PP1, PP2, PP3) were dissolved in a reaction buffer to prepare a diluted series of synthetic peptides. Next, the recombinant ADAMTS13 protein prepared by the above method (hereinafter referred to as “r-ADAMTS13”) and the synthetic peptide prepared from the dilution series (0, 25, 50, 75, 100, 125 μM) were mixed together. FRETS-VWF73 (final concentration 4μM) was added to the mixture, and fluorescence was measured (Kokame K, Nobe Y, Kokubo Y, Okayama A, Miyata T. FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay. Br J Haematol 2005 129: 93-100.). The result is as shown in FIG.

All synthetic peptides inhibited FRETS-VWF73 cleavage by ADAMTS13 at a final concentration range of 25-125 μM. The inhibitory activity was weakened in the order of PP2>PP3> PP1.
Here, the decomposition of the vWF multimer structure can be visually observed using electrophoresis. Therefore, for PP1 and PP3, SDS-agarose gel electrophoresis was used to examine whether vWF has a multimeric structure (multimer analysis).

First, a medium containing 0.4 μg / ml recombinant ADAMTS13 in a low ionic strength buffer solution (5 mM Tris-EDTA pH 8.0) containing BaCl ₂ and 1.5 M urea, and a synthetic peptide that produced a dilution series ( PP1 and PP3) and 15 μg / ml purified vWF were reacted at 37 ° C. for 16 hours. Thereafter, EDTA (final concentration 10 mM) was added to stop the reaction, and then SDS agarose gel electrophoresis was performed. After electrophoresis, the electrophoretic product is transferred to a PVDF membrane, anti-vWF polyclonal antibody (Dako Japan, Kyoto) as the primary antibody, alkaline phosphatase-labeled anti-rabbit IgG antibody (Sigma) as the secondary antibody, and BCIP / NBT for color development. Western blotting was performed using The result is as shown in FIG.

  In general, those with low ADAMTS13 protease activity are known to retain the vWF multimeric structure, while those with high ADAMTS13 protease activity are known to lose this structure. As shown in FIG. 3, as the concentration of PP3 was increased, the cleavage of vWF by ADAMTS13 was inhibited. PP1 is not as strong as PP3, but it was found that the cleavage of vWF by ADAMTS13 is inhibited as the concentration of PP1 is increased.

<Example 3: Affinity measurement of synthetic peptide for vWF>
BIAcore 2000 system (BIACORE, Tokyo) using surface plasmon resonance phenomenon to immobilize vWF covalently on CM-5 sensor chip coated with carboxyl dextran on gold film surface by covalent bond. (Set at 25 ° C.). Specifically, it is as follows.

First, the CM-5 sensor chip was activated with a 1: 1 mixture of 75 mg / ml 1-ethyl-3 (3-dimethylaminopropyl) carbodi-imide (EDC) and 11.5 mg / ml N-hydroxysuccinimide (NHS) for 7 minutes. . Next, vWF (240 μg / ml) was injected onto the CM-5 sensor chip at 25 ° C. at a flow rate of 10 μl / min for 2 minutes. The active groups remaining in the matrix on the CM-5 sensor chip were blocked with 1.0 M ethanolamine / HCl (pH 8.5) for 7 minutes. The vWF immobilized on the CM-5 sensor chip had a surface concentration of 1.76 ng / mm ² .

On the other hand, a dilution series (50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 μM) of synthetic peptide solution was prepared with HBS-N buffer (10 mM Hepes, pH 7.4, 150 mM NaCl), and 10 μl / min. Inject at a flow rate. After injecting each solution, the bound peptide was stripped using 100 mM NaOH for 1 minute before the next injection. The dissociation constant was measured using BIAevaluation software (version 2.1). The results are as shown in FIG. 4 and Table 2.

As a result of examining the affinity between the synthetic peptide and vWF, it was found that the synthetic peptides PP1, PP2, and PP3 had a stronger affinity than the control peptide (PP1R). The affinity for vWF was weak in the order of PP3>PP2> PP1 (FIG. 4, Table 2).
This revealed that the peptide of the present invention strongly binds to vWF. These peptides were also found to inhibit vWF cleavage by ADAMTS13.
From the above, it was shown that the peptide of the present invention plays an important role in binding to vWF.

In one Example of this invention, it is the figure showing the structure couple | bonded with the structure of ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs 13), and vWF (von Willebrand factor; von Willebrand factor). In one Example of this invention, it is the figure which investigated the effect of ADAMTS13 protease activity inhibition of the synthetic peptide. In one Example of this invention, it is the figure which showed the inhibitory effect of the cutting | disconnection of vWF polymer by a synthetic peptide. The result of Western blotting is shown. In one Example of this invention, it is the figure which investigated the affinity of the synthetic peptide with respect to vWF. The dissociation constant between the synthetic peptide and vWF was measured using surface plasmon resonance.

Claims (8)

The peptide of the following (a), (b) or (c).
(A) A peptide having the amino acid sequence of SEQ ID NO: 1.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and having a binding activity to von Willebrand factor (vWF).
(C) a part of the amino acid sequence of human ADAMTS13 protein, which consists of an amino acid sequence in which 1 to 22 amino acids are added to the N-terminal side in the amino acid sequence of SEQ ID NO: 1; A peptide consisting of an amino acid sequence to which three amino acids are added and having a binding activity to von Willebrand factor. The peptide of the following (a), (b) or (c).
(A) A peptide having the amino acid sequence of SEQ ID NO: 2.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2, and having a binding activity to von Willebrand factor.
(C) A part of the amino acid sequence of human ADAMTS13 protein, consisting of an amino acid having 1 to 2 amino acids added to the N-terminal side in the amino acid sequence of SEQ ID NO: 2, or 1 to 12 C-terminal side A peptide having an amino acid sequence to which is added an amino acid and having binding activity to von Willebrand factor. The peptide of the following (a) or (b).
(A) A peptide having the amino acid sequence of SEQ ID NO: 3.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3, and having a binding activity to von Willebrand factor.   A DNA encoding the peptide according to claim 1, 2 or 3.   An expression vector for expressing the peptide according to claim 1, 2 or 3.   6. A cell carrying the expression vector according to claim 5.   A von Willebrand factor cleaving enzyme activity inhibitor comprising the peptide according to claim 1, 2 or 3.   A primary hemostatic thrombus formation promoter comprising the peptide according to claim 1, 2 or 3.