JPH083191A - Antithrombotic peptide and medical device having the peptide immobilized - Google Patents

Abstract

(57) [Summary] [Structure] General formula: HX Tyr Gln Cys Gln Pro Leu Asn Gl
n Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala
YZ (Wherein, X and Y are single bonds, or Arg, Lys, As
n, Asp, Gln, Glu, His, Gly, Ile, Pro, Ala
, Ser is a single amino acid selected from the group consisting of Ser, or a peptide fragment having the above amino acids as a constituent and having 2 to 10 residues, and Z is a hydroxyl group or an amino group. ) A peptide represented by (4) and an immobilized antithrombotic medical device, wherein the peptide is immobilized. [Effect] The peptide of the present invention has a high inhibitory activity against blood coagulation, and is suitable for pharmaceutical use or antithrombogenic use for medical devices. In addition, the antithrombogenic medical device on which the peptide of the present invention is immobilized is also suitable as any medical device that is in contact with blood for a long period of time.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a peptide having an antithrombotic activity. More specifically, the present invention relates to a peptide that acts on a coagulation factor in blood and has an inhibitory effect on blood coagulation, and an antithrombotic medical device formed by immobilizing the peptide, and more specifically to a pharmaceutical use or a medical device. And a medical device that comes into contact with blood for use in antithrombogenic treatment.

[0002]

BACKGROUND OF THE INVENTION Many studies have been conducted on thrombomodulin or its similar peptides, including the examples shown below. Esmon et al. (J. Biol. Chem.
256 , 859 (1982)), thrombomodulin purified from rabbit lung was converted to protein C by thrombin.
Is described as being activated. Also, Salem et al. (J. Biol. Chem. 259 , 12246 (19
84)) describes that thrombomodulin isolated from human placenta requires calcium ions to bind thrombin and activate protein C. Zu
shi et al. (J. Biol. Chem. 264 , 1035.
1 (1989)) is derived from human thrombomodulin.
It is described that a peptide consisting of 15 amino acid residues activates protein C by thrombin.

[0003]

[Problems to be Solved by the Invention] Thrombomodulin or the above-mentioned peptide derived from thrombomodulin does not have sufficient stability of blood coagulation inhibitory activity, and therefore, it is necessary to use the blood coagulation inhibitory activity for the purpose of antithrombotic use of drugs or medical devices. It is desired to provide a peptide having higher activity and stability.

An object of the present invention is to provide a stable peptide having an inhibitory effect on the blood coagulation action, which is used for pharmaceutical use or antithrombogenic use of medical devices. Another object of the present invention is to provide an antithrombogenic medical device having the immobilization thereof.

[0005]

That is, the gist of the present invention is as follows.
(1) General formula: H-X-Tyr-Gln-Cys-Gln-Pro-Leu-Asn-
Gln-Thr-Ser-Tyr-Leu-Cys-Val-Cys-Ala
-Glu-Gly-Phe-Ala-Y-Z (In the formula, X and Y are single bonds, or Arg, Lys, Asn.
, Asp, Gln, Glu, His, Gly, Ile, Pro, Ala
, Ser is a single amino acid selected from the group consisting of, or the number of amino acid residues consisting of these amino acids is 2
Each of the 10 to 10 peptide fragments is represented, and Z represents a hydroxyl group or an amino group. ) A peptide represented by
(2) X and Y are both acidic peptide fragments, both basic peptide fragments, or one is a neutral peptide fragment and the other is an acidic or basic peptide fragment, and Lys-Lys-Lys, respectively. , Lys −Lys −Lys −Ly
s -Lys, Asp -Asp -Asp, Asp -Asp -Asp -Asp
-Asp, Asn-Asn-Asn, Asn-Asn-Asn-Asn-
Asn, Glu-Glu-Glu, Glu-Glu-Glu-Glu-Gl
u, Gln-Gln-Gln, Gln-Gln-Gln-Gln-Gln
The peptide according to (1) above, which is a peptide fragment selected from the group consisting of:
(3) A medical device using a polymer material as a base material, wherein the peptide according to (1) or (2) above is immobilized on the surface of the medical device that comes into contact with blood during use. Antithrombotic medical device, (4) Antithrombotic medical device is a polyurethane or polyvinyl chloride blood bag, blood circuit, or catheter, polycarbonate, methyl methacrylate, or polypropylene syringe or medical container, polyamide The antithrombotic medical device according to (3) above, which is a blood treatment membrane for artificial kidney made of artificial sulfone, cellulose, or polyvinyl alcohol.

The abbreviations for amino acid residues used herein are as follows. Ala: L-alanine residue Arg: L-arginine residue Asn: L-asparagine residue Asp: L-aspartic acid residue Cys: L-cysteine residue Gln: L-glutamine residue Glu: L-glutamic acid residue Gly: L-glycine residue His: L-histidine residue Ile: L-isoleucine residue Leu: L-leucine residue Lys: L-lysine residue Met: L-methionine residue Phe: L-phenylalanine residue Pro : L-proline residue Ser: L-serine residue Thr: L-threonine residue Trp: L-tryptophan residue Tyr: L-tyrosine residue Val: L-valine residue

In the present specification, the amino acid sequence of a peptide is described according to a conventional method so that the N-terminal amino acid residue is located on the left side and the C-terminal amino acid residue is located on the right side.

The peptide of the present invention has the general formula: H-X-Ty.
r −Gln −Cys −Gln −Pro −Leu−Asn −Gln −Thr
−Ser −Tyr −Leu −Cys −Val −Cys −Ala −Glu −
It is represented by Gly-Phe-Ala-YZ. Here, X and Y are single bonds, or Arg, Lys, Asn, Asp, Gln,
A single amino acid selected from the group consisting of Glu, His, Gly, Ile, Pro, Ala, and Ser, or a peptide fragment having 2 to 10 amino acid residues containing these amino acids as constituents, and Z is a hydroxyl group. Or represents an amino group.

The peptide of SEQ ID NO: 1 in which X and Y are single bonds and Z is a hydroxyl group shows a sufficient inhibitory effect on blood coagulation, and the peptide of this amino acid sequence has the above-mentioned N-terminal and / or C-terminal side. Represented by X or Y in the general formula of Arg, Lys, Asn, Asp, Gln, Glu, Hi
A single amino acid selected from the group consisting of s, Gly, Ile, Pro, Ala, and Ser, or a peptide fragment containing these amino acids as a constituent is added to obtain a compound exhibiting a superior blood coagulation inhibitory effect. . The addition of these single amino acids or peptide fragments may be on the N-terminal side, the C-terminal side, or both. Alternatively, one may be the addition of a single amino acid and the other may be the addition of a peptide fragment. In this case, when amino acid residues such as Arg, Lys, Asp, Glu, Asn, and Gln are used as the added amino acid, the peptide can be made more hydrophilic and the solubility in blood becomes higher. As a result, the blood coagulation-inhibiting activity becomes higher, which is preferable. Furthermore, by utilizing the amino group of Lys residue or the carboxyl group of Asp residue and immobilizing it on the surface of medical containers that come into contact with blood, an excellent antithrombotic medical device can be obtained. It is particularly preferable because it can be produced.

Such peptide fragment X or Y is composed of 2 to 10 amino acid residues, preferably 2 to 5 amino acids, and its preferred examples are not particularly limited,
For example Lys-Lys-Lys, Lys-Lys-Lys-Lys-Ly
basic peptides such as s, Asp-Asp-Asp, Asp
-Asp -Asp -Asp -Asp, Glu -Glu -Glu, Glu-G
acidic peptides such as lu-Glu-Glu-Glu, and Asn-Asn-Asn, Asn-Asn-Asn-Asn-Asn
, Gln-Gln-Gln, Gln-Gln-Gln-Gln-Gln, and other neutral peptides.

The peptide fragment X or Y in the present invention
Is not limited to the peptide fragment consisting of the same plurality of amino acids as described above, and may be a peptide fragment consisting of two or more kinds of amino acids. For example, a peptide fragment containing two or more kinds of amino acids including these exemplified peptides is also included in the peptide fragment in the present application. For example, Lys-Lys-Lys-Ser-Asn-Ala-
Ile-Pro, Asp-Gly-Arg-Ser-Ser-Lys-Lys
-Lys-Lys-Lys, Lys-Lys-Lys-Lys-Lys-
Ser-Asn-Ala-Ile-Pro and the like. Also,
When the number of amino acid residues exceeds 10 as a peptide fragment,
It is not preferable because the synthesis becomes complicated and there is a risk that the antigenicity to the human body is developed. Also, the peptide fragment X,
Y may be the same or different peptide fragments. If the hydrophilicity of the entire peptide is increased by adding these peptide fragments X and / or Y, an increase in blood coagulation inhibitory activity can be expected. Therefore, when both peptide fragments X and Y are acidic or basic peptide fragments, it is particularly preferable that one of X and Y is neutral and the other is acidic or basic peptide fragment.

In the above general formula, Z may be either a hydroxyl group or an amino group. That is, whether the C-terminal amino acid is a normal amino acid or an amino acid amide, it has blood coagulation inhibiting activity.

Generally, when a biological synthesis method such as a chemical synthesis method or a genetic engineering method is used, disulfide bonds between cysteine residues often occur at random, and such a synthetic method does not necessarily have high activity. , A stable peptide cannot be obtained. In the case of natural thrombomodulin and the peptides described in the section of the prior art, there were problems in the stability of blood coagulation inhibitory activity due to the large number of cysteine residues. Therefore, the number of cysteine residues in the peptide of the present invention is preferably 3 residues.

Typical examples of the peptides provided by the present invention are shown below, but the peptides of the present invention are not limited thereto. Formula (1) Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Ty
r Leu Cys Val Cys AlaGlu Gly Phe Ala (SEQ ID NO:
1) Formula (2) Lys Lys Lys Tyr Gln Cys Gln Pro Leu Asn Gl
n Thr Ser Tyr Leu CysVal Cys Ala Glu Gly Phe Ala L
ys Lys Lys (SEQ ID NO: 2) Formula (3) Asn Asn Asn Tyr Gln Cys Gln Pro Leu Asn Gl
n Thr Ser Tyr Leu CysVal Cys Ala Glu Gly Phe Ala A
sp Asp Asp (SEQ ID NO: 3) Formula (4) Lys Lys Lys Tyr Gln Cys Gln Pro Leu Asn Gl
n Thr Ser Tyr Leu CysVal Cys Ala Glu Gly Phe Ala A
sp Gly Arg Ser Ser Lys Lys Lys (SEQ ID NO: 4) Formula (5) Lys Lys Lys Lys Asn Ala Ile Pro Tyr Gln Cy
s Gln Pro Leu Asn GlnThr Ser Tyr Leu Cys Val Cys A
la Glu Gly Phe Ala Lys Lys Lys (SEQ ID NO: 5) Formula (6) Lys Lys Lys Lys Lys Tyr Gln Cys Gln Pro Le
u Asn Gln Thr Ser TyrLeu Cys Val Cys Ala Glu Gly P
he Ala Asp Gly Arg Ser Ser Lys Lys Lys LysLys (SEQ ID NO: 6) Formula (7) Lys Lys Lys Lys Lys Ser Asn Ala Ile Pro Ty
r Gln Cys Gln Pro LeuAsn Gln Thr Ser Tyr Leu Cys V
al Cys Ala Glu Gly Phe Ala Lys Lys Lys LysLys (SEQ ID NO: 7) Formula (8) Glu Glu Glu Tyr Gln Cys Gln Pro Leu Asn Gl
n Thr Ser Tyr Leu CysVal Cys Ala Glu Gly Phe Ala G
lu Glu Glu (SEQ ID NO: 8) Formula (9) Asp Asp Asp Tyr Gln Cys Gln Pro Leu Asn Gl
n Thr Ser Tyr Leu CysVal Cys Ala Glu Gly Phe Ala G
lu Glu Glu (SEQ ID NO: 9) Formula (10) Glu Glu Glu Tyr Gln Cys Gln Pro Leu Asn
Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Al
a Asp Asp Asp (SEQ ID NO: 10) Formula (11) Asp Glu Asp Tyr Gln Cys Gln Pro Leu Asn
Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Al
a Glu Asp Glu (SEQ ID NO: 11) Formula (12) Lys Lys Lys Lys Lys Tyr Gln Cys Gln Pro
Leu Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gl
y Phe Ala His Lys Lys Lys Lys Lys (SEQ ID NO: 1
2) Formula (13) Lys Lys Lys Tyr Gln Cys Gln Pro Leu Asn
Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Al
a Lys Lys Lys-NH ₂ (SEQ ID NO: 13) Formula (14) Asn Asn Asn Tyr Gln Cys Gln Pro Leu Asn
Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Al
a Asp Asp Asp-NH ₂ (SEQ ID NO: 14) Formula (15) Lys Lys Lys Asn Ala Ile Pro Tyr Gln Cys
Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys Val Cys Al
a Glu Gly Phe Ala Lys Lys Lys-NH ₂ (SEQ ID NO: 1
5)

The peptide is synthesized by a known method. For example, a solid phase synthesis method, a stepwise extension method, a liquid phase synthesis method such as a fragment condensation method, or the like is used as the chemical synthesis method, but the solid phase synthesis method is convenient in operation. [Journal of the American Chemical Society
85 2149 (1963); Biochemistry Society of Japan, edited by "Biochemistry Experimental Course 1 Protein Chemistry IV Chemical Modification and Peptide Synthesis" (published by Tokyo Kagaku Dojin, November 15, 1977); Chemistry Experiment Lecture 2 Protein Chemistry (Bottom) "(Published by Tokyo Kagaku Dojin on May 20, 1987). Further, a biological synthetic method by genetic manipulation is also possible.

The peptide of the present invention thus obtained can markedly inhibit the coagulation of human plasma. Therefore,
In medical treatment, it is useful for preventing blood coagulation by adding a trace amount to blood or by treating the contact surface of a medical device that comes into contact with blood with the peptide of the present invention. That is, by immobilizing the peptide of the present invention on the surface that comes into contact with blood during use, an antithrombotic medical device useful for preventing blood coagulation can be obtained.

In the present invention, the coagulability of blood can be evaluated by a known method, that is, by measuring the activated partial thromboplastin time, the plasma recalcification time or the whole blood coagulation time. Specifically, the activated partial thromboplastin time was 100 μl of human plasma (15 nmole of peptide was added when measuring the effect of peptide).
Was placed in a glass test tube and incubated at 37 ° C. for 10 minutes, then 100 μl of activated partial thromboplastin (manufactured by Ortho) was added, further incubated for 3 minutes, and 100 μl of 25 mM calcium chloride solution was added, until coagulation It can be determined by measuring the time. The recalculation time for plasma calcium is 1
Add 00 μl (15 nmole peptide is added when measuring the effect of the peptide) to a glass test tube, 37
It can be determined by incubating at 0 ° C. for 10 minutes, adding 100 μl of 25 mM calcium chloride solution, and then measuring the time until coagulation. Whole blood clotting time is
It can be determined by placing 1 ml of human whole blood immediately after blood collection (150 nmol of peptide is added when measuring the effect of peptide) in a glass test tube and measuring the time until coagulation.

As the base material of the antithrombogenic medical device for immobilizing the peptide of the present invention, there is no problem as long as it is a polymeric material such as a synthetic resin which is a known medical material, and in particular, a hydroxyl group on the surface,
Those having an amino group and a carboxyl group are preferable. Further, it is also possible to use a material having no such functional group on the surface and having a hydroxyl group, an amino group or a carboxyl group introduced by surface treatment. Such medical devices include, for example, blood bags, blood circuits, catheters, etc. made of polyurethanes or polyvinyl chlorides;
Syringes, medical containers, etc. made of polycarbonates, acrylics such as methyl methacrylate, or polyolefins such as polypropylene; artificial blood vessels, etc. made of polyamides such as nylon; celluloses such as polysulfones, cellulose, cellulose acetate, etc. Examples thereof include blood treatment membranes such as artificial kidneys manufactured from polyvinyl alcohols such as ethylene-vinyl alcohol.

The surface treatment of the base material of the above antithrombogenic medical device can be carried out by a known method. For example, there is a method of performing plasma treatment in a gas containing oxygen gas or nitrogen gas and ammonia gas, or a method of chemically treating with a sulfuric acid solution containing permanganate. The method of performing plasma treatment is preferable because a large amount of medical equipment can be treated in a short time and the medical equipment can be kept clean because it is treated in gas. The method of chemical treatment has a wide range of application because it can treat even those having a complicated shape and those that cannot be plasma-treated such as the inner surface of a container. In general, many medical materials that come into contact with blood during use have complicated shapes such as tubes, hollow fibers and bottles, and the chemical treatment method is advantageous.

When plasma treatment is performed in oxygen gas,
Mainly hydroxyl groups are introduced on the surface, when plasma treatment in nitrogen gas and ammonia gas, mainly amino groups are introduced on the surface, when chemically treated with a sulfuric acid solution containing permanganate, etc., A hydroxyl group and a carboxyl group are mainly introduced.

By such treatment, the peptide can be immobilized on the surface of the substrate through a reagent having an epoxy group at both ends, a silane coupling agent having an epoxy group, and the like. When a hydroxyl group is present on the surface of the base material, it can be covalently immobilized by using a reagent having an epoxy group at both ends or a silane coupling agent having an epoxy group, and an amino group or a carboxyl group can be attached to the surface of the base material. When present, it is possible to similarly immobilize it by covalent bond by using a reagent having epoxy groups at both ends. That is, specifically, when a silane coupling agent is used, a 0.5 to 5% solution (water) of a silane coupling agent having an epoxy group (for example, (3-glycidoxypropyl) trimethoxysilane) is usually used. Or organic solvent solution)
Is contacted with a polymer material for 1 to 30 minutes, heated and dried for 1 minute to 2 days, and the silane coupling agent not cross-linked is washed and removed to prepare a polymer material into which an epoxy group is introduced. When using a reagent having an epoxy group at both ends, a reagent having an epoxy group at both ends (for example,
1,4-butanediol diglycidyl ether)
The polymer material is brought into contact with a 20% aqueous solution (pH 10 to 13) for 1 hour to 2 days, and then washed to prepare a polymer material into which an epoxy group has been introduced.

The polymer material prepared in this manner has a density of 0.
A 001 to 2% peptide aqueous solution (pH 10 to 12) is contacted for 1 hour to 2 days to covalently bond the amino group or carboxyl group of the peptide and the epoxy group introduced into the polymer material to obtain a peptide immobilization material. The immobilized amount of the peptide of the present invention is not particularly limited, but is usually 1
It is 0.01 nmol to 20 nmol per cm ² , and preferably 0.1 nmol to 5 nmol.

Although the antithrombogenic medical device has been described as the one to which the peptide of the present invention is immobilized, the present invention is not limited to this, and the medical device may come into contact with blood when used. The anti-thrombotic material obtained by immobilizing the peptide of the present invention on various materials described in (1) above is also included in the embodiments of the present invention. In this case, the antithrombotic material can be used as it is or after being processed into a desired shape.

[0024]

EXAMPLES The present invention will be described below based on Examples, Comparative Examples and Reference Examples, but the present invention is not limited to these.

Example 1 Tyr-Gln-Cys-Gln-Pro-Leu-Asn-Gln-Th
r −Ser −Tyr −Leu −Cys −Val −Cys −Ala −Glu
-Gly-Phe-Ala (SEQ ID NO: 1) was synthesized by the method shown below.

The peptide was synthesized by a solid phase synthesis method using an automatic peptide synthesizer (manufactured by Applied Biosystems, USA). Fm in which Fmoc-Ala is bound to benzyloxybenzyl alcohol type resin
Using 0.29 g of oc-Ala-resin (manufactured by Shimadzu Corporation),
According to a series of operations shown in Table 1, the target peptide was bound in the corresponding order from the C-terminal side toward the N-terminal direction. The amino acids used in this example and the following examples and comparative examples are all L-amino acids, and the amino group at the α-position is protected with an Fmoc group, and further protection of other substituents is required. About amino acids
g is a 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) group, Asp, Cys, Glu, Se.
r was protected with a t-butyl (tBu) group, and Lys was protected with a t-butoxycarbonyl (Boc) group (manufactured by Peptide Institute).

[0027]

[Table 1]

After the reaction procedure for all amino acids was completed, the obtained resin was washed successively with dichloromethane and methanol on a glass filter and then vacuum dried to obtain about 600 mg of a dried resin. 600 mg dry resin and 1 trifluoroacetic acid in a vial
0 ml, water 0.5 ml, thioanisole 0.5 ml, ethanediol 0.25 ml and phenol 0.75 g
Was mixed. After standing at room temperature for 20 hours, the mixture was filtered through a glass filter, diethyl ether was added to the filtrate, and the mixture was centrifuged to obtain a white precipitate. The obtained precipitate was vacuum dried, extracted with 2N aqueous acetic acid solution, and the extract was freeze-dried to obtain a peptide.

The obtained peptide was purified by a preparative high performance liquid chromatograph [apparatus: Millipore Waters]. The molecular weight of the obtained peptide was 2238 as determined by FAB mass spectrometry (theoretical value: 22).
38.5).

As a result of measuring the activated partial thromboplastin time (hereinafter abbreviated as APTT) of this peptide, 5
1.2 seconds (and the plasma recalcification time was 23
It was 2 seconds. ). This result indicates that the peptide of the present invention (SEQ ID NO: 1) significantly inhibits blood coagulation, when compared with the results of Reference Example 1 below. In addition, APTT
Was placed in a glass test tube containing 15 nmole of the peptide and 100 μl of human plasma, incubated at 37 ° C. for 10 minutes, added with 100 μl of activated partial thromboplastin (manufactured by Ortho Co.), and further incubated for 3 minutes to prepare 25 mM calcium chloride. After adding 100 μl of the solution, the time until coagulation was measured and determined. The plasma calcium re-addition time was 10 nm at 37 ° C. when 15 nmole of the peptide and 100 μl of human plasma were put into a glass test tube.
After incubation for a minute and adding 100 μl of 25 mM calcium chloride solution, the time until coagulation was measured and determined. The same method was used in the following Examples, Reference Examples, and Comparative Examples.

Reference Example 1 APTT of human plasma to which the peptide of the present invention has not been added
Was 26.3 seconds. The recalcification time for plasma was 124 seconds. The whole blood clotting time was 7 minutes.
The whole blood coagulation time was determined by placing 1 ml of human whole blood immediately after blood collection and the peptide in a glass test tube and measuring the time until coagulation. The same method was used in the following examples.

Example 2 Instead of Fmoc-Ala-resin (manufactured by Shimadzu), Fmoc was added to benzyloxybenzyl alcohol type resin.
-Lys (Boc) -bound Fmoc-Lys (B
oc) -Example 1 except that a resin (manufactured by Shimadzu Corporation) was used.
The peptide of the following SEQ ID NO: 2 was synthesized in the same manner as in. The molecular weight determined by FAB mass spectrometry is 3
It was 008 (theoretical value: 3007.5). Lys −Lys −Lys −Tyr −Gln −Cys −Gln −Pro −Le
u −Asn −Gln −Thr −Ser −Tyr −Leu −Cys −Val
−Cys −Ala −Glu −Gly −Phe −Ala −Lys −Lys −
Lys (SEQ ID NO: 2)

15 nmole of this peptide was added to human plasma 100
When APTT was measured in addition to μl, it was 64.2 seconds, plasma recalcification time was 249.5 seconds, and whole blood coagulation time was 10.5 minutes. This result is obtained by adding Lys-Lys-Lys to both ends of the peptide of SEQ ID NO: 1.
It shows that the blood coagulation inhibitory activity was further enhanced.

Example 3 Instead of Fmoc-Ala-resin (manufactured by Shimadzu Corporation), benzyloxybenzyl alcohol type resin was used for Fmoc.
-Fmoc-Asp bound with -Asp (OtBu)
The following peptides were synthesized by the same method as in Example 1 except that (OtBu) -resin (manufactured by Shimadzu Corporation) was used. F
The molecular weight determined by the AB method mass spectrum was 2926 (theoretical value: 2926.0). Asn −Asn −Asn −Tyr −Gln −Cys −Gln −Pro −Le
u −Asn −Gln −Thr −Ser −Tyr −Leu −Cys −Val
−Cys −Ala −Glu −Gly −Phe −Ala −Asp −Asp −
Asp (SEQ ID NO: 3)

15 nmole of this peptide was added to human plasma 100
When APTT was measured in addition to μl, it was 63.1 seconds. This result shows that, even if a neutral peptide and an acidic peptide are added to both ends of the peptide of SEQ ID NO: 1, the hydrophilicity increases as a whole and the blood coagulation inhibitory activity is further enhanced. Show.

Example 4 The peptide synthesized in Example 2 was sterilized by autoclaving (1
(21 ° C., 20 minutes), 15 nmole of peptide was added to 100 μl of human plasma, and APTT was measured.
3.0 seconds and plasma calcium re-addition time is 245.0
Seconds, whole blood coagulation time was 10.5 minutes. This result is
It is shown that the peptide of the present invention (SEQ ID NO: 2) almost completely retains the blood coagulation inhibiting activity even under the condition of 121 ° C. for 20 minutes. This property enables the peptide of the present invention to be sterilized before being brought into contact with blood, and is also extremely advantageous when used after being immobilized on a medical device that requires sterilization before use. Can be said to be a special feature.

Comparative Example 1 Instead of Fmoc-Ala-resin (manufactured by Shimadzu Corporation), benzyloxybenzyl alcohol type resin was used as Fmoc.
-Lys (Boc) -bound Fmoc-Lys (B
oc) -Example 1 except that a resin (manufactured by Shimadzu Corporation) was used.
The following peptides were synthesized in the same manner as in. The molecular weight determined by FAB method mass spectrum was 3378 (theoretical value: 3377.9). Lys −Lys −Cys −Asn −Ala −Ile −Pro −Tyr −Gl
n −Cys −Gln −Pro −Leu −Asn −Gln −Thr −Ser
−Tyr −Leu −Cys −Val −Cys −Ala −Glu −Gly −
Phe-Ala-Lys-Lys-Lys (SEQ ID NO: 16)

15 nmole of this peptide was added to human plasma 100
When APTT was measured in addition to μl, it was 28.1 seconds, plasma calcium re-addition time was 128 seconds, and whole blood coagulation time was 7.5 minutes. This peptide has four
Since it has a Cys residue, it is considered that disulfide bonds are randomly generated and that many conformations that are disadvantageous to the blood coagulation inhibitory activity are formed.

Comparative Example 2 Instead of Fmoc-Ala-resin (manufactured by Shimadzu Corporation), benzyloxybenzyl alcohol type resin was used as Fmoc.
-Lys (Boc) -bound Fmoc-Lys (B
oc) -Example 1 except that a resin (manufactured by Shimadzu Corporation) was used.
The following peptides were synthesized in the same manner as in. The molecular weight determined by FAB method mass spectrum was 3711 (theoretical value: 3711.2). Lys −Lys −Cys −Ile −Pro −Tyr −Gln −Cys −Gl
n −Pro −Leu −Asn −Gln −Thr −Ser −Tyr −Leu
−Cys −Val −Cys −Ala −Glu −Gly −Phe −Ala −
Asp-Gly-Arg-Ser-Cys-Lys-Lys-Lys (SEQ ID NO: 17) 15 nmole of this peptide was added to 100 μl of human plasma,
The APTT was measured and found to be 26.6 seconds. Since this peptide also has 5 Cys residues in the molecule like the peptide of SEQ ID NO: 16, it is considered that a disulfide bond is randomly generated and a lot of conformation which is disadvantageous to the blood coagulation inhibitory activity is formed. .

Example 5 The inner surface of a vinyl chloride tube having an inner diameter of 10 mm was set to 0.4.
% Concentrated sulfuric acid solution containing potassium permanganate (25
After dipping at 5 ° C for 5 minutes), it was washed with distilled water for 30 minutes.
It was confirmed by X-ray photoelectron spectroscopy (ESCA method) that hydroxyl groups were introduced into the inner surface of the tube by this treatment.
Next, a 2% (3-glycidoxypropyl) trimethoxysilane aqueous solution was brought into contact with the inner surface of the tube at 25 ° C for 5 minutes, dried at 50 ° C for 2 hours, and washed with distilled water for 2 hours. 10 m of the peptide of SEQ ID NO: 2 obtained in Example 2
A solution obtained by dissolving g in 10 ml of distilled water and adjusting the pH to 11 was placed in the tube and left at 37 ° C. for 12 hours to obtain a peptide-immobilized tube. This tube is 10 cm
A tube for testing was prepared by cutting the sample into lengths and closing one end thereof. 2 ml of fresh blood from a healthy person was poured into it and kept at 25 ° C.
At 30 seconds, the condition of blood was observed and the time when blood fluidity was lost (coagulation time) was measured.
It was a minute. Comparing this result with the result of Comparative Example 3, it can be said that the peptide-immobilized tube of the present invention has a remarkable blood coagulation inhibiting ability.

Comparative Example 3 Internal diameter 10 not surface-treated with the peptide of the present invention
A mm vinyl chloride tube was cut into a length of 10 cm, and one end thereof was closed to prepare a test tube. 2 ml of fresh blood of a healthy person was poured therein, and the coagulation time was measured by the same method as in Example 5, and it was about 14 minutes.

[0042]

Industrial Applicability The peptide of the present invention has a high inhibitory activity against blood coagulation, and this activity is extremely stable even under sterilization conditions, and is suitable for pharmaceutical use or antithrombogenic use for medical devices. Further, the antithrombotic medical device obtained by immobilizing the peptide of the present invention also has high anticoagulant activity and stability, such as catheters, blood circuits, blood bags, hemodialysis membranes, artificial blood vessels and the like. It is suitable as any medical device that is used in contact with blood for a long period of time.

[0043]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala 1 5 10 15 Glu Gly Phe Ala 20

SEQ ID NO: 2 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Lys Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 1 5 10 15 Val Cys Ala Glu Gly Phe Ala Lys Lys Lys 20 25

SEQ ID NO: 3 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asn Asn Asn Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 1 5 10 15 Val Cys Ala Glu Gly Phe Ala Asp Asp Asp 20 25

SEQ ID NO: 4 Sequence length: 31 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Lys Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 1 5 10 15 Val Cys Ala Glu Gly Phe Ala Asp Gly Arg Ser Ser Lys Lys Lys 20 25 30

SEQ ID NO: 5 Sequence length: 31 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Lys Lys Asn Ala Ile Pro Tyr Gln Cys Gln Pro Leu Asn Gln 1 5 10 15 Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Lys Lys Lys 20 25 30

SEQ ID NO: 6 Sequence length: 35 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Lys Lys Lys Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 1 5 10 15 Leu Cys Val Cys Ala Glu Gly Phe Ala Asp Gly Arg Ser Ser Lys Lys 20 25 30 Lys Lys Lys 35

SEQ ID NO: 7 Sequence length: 35 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Lys Lys Lys Ser Asn Ala Ile Pro Tyr Gln Cys Gln Pro Leu 1 5 10 15 Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Lys Lys 20 25 30 Lys Lys Lys 35

SEQ ID NO: 8 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Glu Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 1 5 10 15 Val Cys Ala Glu Gly Phe Ala Glu Glu Glu 20 25

SEQ ID NO: 9 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Asp Asp Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 1 5 10 15 Val Cys Ala Glu Gly Phe Ala Glu Glu Glu 20 25

SEQ ID NO: 10 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Glu Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 1 5 10 15 Val Cys Ala Glu Gly Phe Ala Asp Asp Asp 20 25

SEQ ID NO: 11 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Glu Asp Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 1 5 10 15 Val Cys Ala Glu Gly Phe Ala Glu Asp Glu 20 25

SEQ ID NO: 12 Sequence length: 31 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Lys Lys Lys Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 1 5 10 15 Leu Cys Val Cys Ala Glu Gly Phe Ala His Lys Lys Lys Lys Lys 20 25 30

SEQ ID NO: 13 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence features Characteristic symbols: modified site Location: 26 Other information: Lys-NH ₂ Sequence Lys Lys Lys Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 1 5 10 15 Val Cys Ala Glu Gly Phe Ala Lys Lys Xaa 20 25

SEQ ID NO: 14 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics: Symbol representing characteristics: modified site Location: 26 Other information: Asp-NH ₂ Sequence Asn Asn Asn Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys 1 5 10 15 Val Cys Ala Glu Gly Phe Ala Asp Asp Xaa 20 25

SEQ ID NO: 15 Sequence length: 30 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence features Characteristic symbols: modified site Location: 30 Other information: Lys-NH ₂ Sequence Lys Lys Lys Asn Ala Ile Pro Tyr Gln Cys Gln Pro Leu Asn Gln Thr 1 5 10 15 Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Lys Lys Xaa 20 25 30

SEQ ID NO: 16 Sequence length: 30 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Cys Asn Ala Ile Pro Tyr Gln Cys Gln Pro Leu Asn Gln Thr 1 5 10 15 Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Lys Lys Lys 20 25 30

SEQ ID NO: 17 Sequence length: 33 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Lys Cys Ile Pro Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 1 5 10 15 Leu Cys Val Cys Ala Glu Gly Phe Ala Asp Gly Arg Ser Cys Lys Lys 20 25 30 Lys

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location B01D 71/40 9538-4D 71/50 9538-4D 71/54 9538-4D 71/64 9538-4D 71/68 9538-4D C07K 14/745 17/08 // A61K 38/00 ACB

Claims (4)

[Claims] 1. A general formula: H-X-Tyr-Gln-Cys-Gln-Pro-Leu-Asn-
Gln-Thr-Ser-Tyr-Leu-Cys-Val-Cys-Ala
-Glu-Gly-Phe-Ala-Y-Z (In the formula, X and Y are single bonds, or Arg, Lys, Asn.
, Asp, Gln, Glu, His, Gly, Ile, Pro, Ala
, Ser is a single amino acid selected from the group consisting of, or the number of amino acid residues consisting of these amino acids is 2
Each of the 10 to 10 peptide fragments is represented, and Z represents a hydroxyl group or an amino group. ) The peptide represented by. 2. X and Y are both acidic peptide fragments, both basic peptide fragments, or one is a neutral peptide fragment and the other is an acidic or basic peptide fragment, and Lys-Lys, respectively. −Lys, Lys −Lys −
Lys-Lys-Lys, Asp-Asp-Asp, Asp-Asp-As
p −Asp −Asp, Asn −Asn −Asn, Asn −Asn −Asn
−Asn −Asn, Glu −Glu −Glu, Glu −Glu −Glu −
Glu-Glu, Gln-Gln-Gln, Gln-Gln-Gln-Gl
The peptide according to claim 1, which is a peptide fragment selected from the group consisting of n-Gln or a peptide fragment containing the same. 3. A medical device using a polymer material as a base material, wherein the peptide according to claim 1 or 2 is immobilized on the surface of the medical device that comes into contact with blood during use. Antithrombogenic medical device. 4. An antithrombogenic medical device is a blood bag, blood circuit, or catheter made of polyurethane or polyvinyl chloride, a syringe or medical container made of catheter, polycarbonate, methyl methacrylate, or polypropylene, artificial blood vessel made of polyamide, or Polysulfone,
The antithrombotic medical device according to claim 3, which is a blood treatment membrane for an artificial kidney made of cellulose or polyvinyl alcohol.