JPH06116286A - Oligopeptide having anti-blood coagulation activity - Google Patents

Abstract

PURPOSE:To obtain the new oligopeptide useful as a constituent material of a therapeutic agent and a preventing agent for blood diseases such as disseminat ed intravascular coagulation and as a diagnostic reagent for blood diseases, showing strong anti-blood coagulation activity, having a specific amino acid sequence. CONSTITUTION:The oligopeptide of formula I and formula II which are parts of an amino acid sequence of annexin-V, an anti-blood coagulation protein isolated from human placenta are synthesized by using a peptide synthesizer and are separated from the peptide synthesizer by hydrogen fluoride method. Each of synthesized peptides is subjected to reverse phase high-performance liquid chromatography and purified to give the objective oligopeptide shown by formula I or formula II, which is an anti-blood coagulation action site of a placenta derived anti-blood coagulation protein, shown very strong anti-blood coagulation activity and is a therapeutic agent and a preventing agent for blood diseases such as DIC (disseminated intravascular coagulation) and is a diagnostic reagent for blood diseases.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel oligopeptide having anticoagulant activity.

[0002]

2. Description of the Related Art It is known that the blood coagulation reaction is controlled by a number of anticoagulants represented by serine protease inhibitor system and protein C / protein S / thrombomodulin system. It is thought that a part of the phospholipid surface is deeply involved in a key reaction in the blood coagulation chain reaction (cascade), and the peptide of such a part is physiologically or pharmacologically involved in blood coagulation. It is considered to be important as an area that enables control, and its research is expected. Similarly, tissue factor has been confirmed to initiate blood coagulation at the trauma site by binding to membrane phospholipids.
The factor IIa / tissue factor complex (lower case a means activated factor) is known to be complex-blocked by high concentrations of serum lipoproteins.

The extrinsic coagulation pathway (ex
There are distinct systems called the trinsic pathway and the intrinsic system coagulation pathway. The intrinsic coagulation pathway refers to a reaction that leads to thrombin formation by a factor existing in plasma. As an intermediate reaction, Factor IX is activated by Factor XIa and Calcium ion to activate Factor IXa. Factor IXa
Is a reaction that activates Factor X in the presence of Factor VIIIa, phospholipids and calcium ions. On the other hand, the extrinsic coagulation pathway refers to a reaction that induces a blood coagulation reaction from plasma factors and components present in a tissue extract, and is one of the blood coagulation factors, Factor VII.
a refers to a reaction that activates Factor X to Xa in the presence of tissue factor and calcium ions.

Recently, several anticoagulant factors have been isolated and identified from human placenta, and these have been named placenta-derived anticoagulant proteins (annexin-V to II / PAP-I to IV) [Funakoshi et al. , Biochemistry 26, p
5572-5578 (1987)]. Annexin-V
Is the most abundant of these and has been reported as a blood coagulation inhibitor, calphobindin I (ca).
lphobindin I / CPB-I) [Iwasaki et al.
Biochem. 102, p1261-1273 (19
87)] and an intravascular anticoagulant (vasculer
anticoagulant) [Reutlings
pager et al., Eur. J. Biochem. 15
1, p625-629 (1985)], and is considered to belong to the same group.

The above-mentioned annexin-V is capable of inhibiting both extrinsic and intrinsic blood coagulation reactions, and has the property of specifically binding to the surface of anionic phospholipids in the presence of calcium ions. .

[0006] Regarding the nucleotide sequence encoding the human placenta-derived anticoagulant protein, PP4, VAC and calphobinden II have already been reported [Grundmann et al., Proc. Natl. Ac
ad. Sci. USA 85, p3708-3712.
(1988); Maurer-Fogy et al., Eur.
J. Biochem. 174, p585-592 (19
88) and Iwasaki et al., J. Biochem. 106, p
43-49 (1989)]. These proteins and cDNA
According to A analysis, annexin-V is a calcium-dependent phospholipid-binding protein lipocortin / calpactin /
It was shown to be a protein belonging to the annexin family.

This annexin-V is composed of 319 amino acids and further has four repeating sequences. This amino acid sequence has been reported by Funakoshi et al. [Bi
chemistry 27, p6268-6276
(1988)]. In addition, each repeat of Annexin-V contains two regions normally present in phospholipid binding proteins, one region of which is KGXGTDEXXh.
It is a region consisting of 17 amino acids of hXhhXSR (X is an arbitrary amino acid, h is a hydrophobic amino acid), and such an amino acid sequence is an endonexin (endone).
xin), calelectrin
And a sequence often found in calcium ion-dependent membrane-bound proteins such as lipocortins. The other is a highly hydrophobic region consisting of 6 amino acids corresponding to the carboxy-terminal side of each repeat sequence. These two regions are thought to be directly involved in binding phospholipids.

In Annexin-V, in the absence of calcium ions, this protein has no anticoagulant activity or phospholipid-binding property, so that the calcium ion- or phospholipid-binding site is the site of action of this protein. It seems that there is an important relationship.

In such a situation, Annexin-V
Has been studied for the active site of the amino acid sequence of SEQ.
electrical and Biophysical
Research Communications, 1
68, p125-134 (1990)]. Furthermore, the present inventors have found that, among the amino acid sequences of Annexin-V, the oligopeptide having the amino acid sequence of SEQ ID NO: 3 has excellent anticoagulant activity, and previously filed a patent application (Patent application 3 -154811).

[0010]

The synthesis of oligopeptides derived from the amino acid sequence of annexin-V having such properties as anticoagulant activity and further excellent in anticoagulant activity is performed by the DIC (general pan) method. Intravascular coagulation syndrome)
It is important to develop a therapeutic / preventive drug for blood diseases such as Therefore, an object of the present invention is to provide an oligopeptide derived from the annexin-V anticoagulant active site, and further improving the activity to provide an oligopeptide having a stronger activity.

[0011]

In view of the above situation, the present inventors have improved a part of this amino acid sequence based on the oligopeptide derived from the annexin-V amino acid sequence previously found by the present inventors. When oligopeptides were synthesized and their anticoagulant activities were measured, it was found that the peptides having the amino acid sequences shown below have a strong anticoagulant activity, and the present invention was completed.

That is, the present invention provides an oligopeptide having the following amino acid sequence. His Asp Thr Leu Ile Arg (SEQ ID NO: 1), His Asp Thr Gln Pro Arg Val Leu Asp (SEQ ID NO: 2)

The oligopeptide of the present invention is an annexin-
It was discovered by paying particular attention to the amino acid sequence region of SEQ ID NO: 3 having a histidine residue in the course of research on the amino acid sequence of V and its anticoagulant activity, and partially improving this sequence. is there. That is, according to Miere et al., One of the phospholipase A ₂ inhibitory active sites of lipocortin I, which is one of the annexin family, has been reported to be a region of the amino acid sequence of HDMNKVLDL [Miere et al., Lucio Mieleet a.
l. , Nature 335, p726-730 (19).
88)]. As a result of comparing this amino acid sequence with the sequence of SEQ ID NO: 3 and further analyzing them by protein engineering and repeating the inference from various viewpoints, the present inventors have found that the amino acid of SEQ ID NO: 3 derived from Annexin-V. Of these, by replacing the Asp-His- sequence on the amino-terminal side with His-Asp-,
It was speculated that the anticoagulant activity may be further increased. In addition, regarding the amino acid sequence on the C-terminal side, Val and L
By extending the peptide with a hydrophobic amino acid such as eu, binding to phospholipids is made easier,
As a result, it was speculated that the anticoagulant activity could be increased. After such research and analysis, the following oligopeptides A and B were identified as oligopeptides of the present invention.
Was synthesized. Oligopeptide A (product of the present invention): His Asp Thr Leu Ile
Arg (SEQ ID NO: 1) Oligopeptide B (product of the present invention): His Asp Thr Gln Pro
Arg Val Leu Asp (SEQ ID NO: 2)

Further, oligopeptide D was synthesized as an oligopeptide having a sequence derived from the original annexin-V, which was the basis of the amino acid sequences of these oligopeptides. Furthermore, a known annexin-V-derived oligopeptide found by the present inventors previously was synthesized as a peptide for comparison and control. Oligopeptide C: Asp His Thr Leu Ile Arg (SEQ ID NO: 3) Oligopeptide D: Ser His Leu Arg Lys Val (SEQ ID NO: 4)

The region of Annexin-V of oligopeptides C and D consisting of these 6 amino acids is counted from the amino-terminal methionine of Annexin-V as 1.
They correspond to the 265th to 270th regions and the 203rd to 208th regions, respectively.

The anticoagulant activity of the above four oligopeptides was measured as a delayed reaction in blood coagulation time using a kaolin-activated blood coagulation reaction. As a result, it was confirmed that the known oligopeptide D and the oligopeptide C previously invented by the present inventors have anticoagulant activity, but the oligopeptides A and B of the present invention have higher anticoagulant activity. It was confirmed to show activity. Specifically, from 203 to 208 of Annexin-V
It was confirmed that the oligopeptide C corresponding to the th amino acid has an extremely strong anticoagulant activity as compared with the known oligopeptide D. The oligopeptide of the present invention prepared by partially improving the sequence of oligopeptide C. Peptides A and B were found to exhibit much stronger anticoagulant activity than oligopeptide C, which has the original amino acid sequence derived from the annexin-V sequence. On the other hand, as a control, when histidine (His) in oligopeptide C was replaced with alanine (Ala), no anticoagulant activity could be found in this modified peptide [Funakoshi et al., Biochemical and Biophys.
social Research Communicat
Ions, 168, p125-134 (1990)].

Furthermore, it was confirmed that the oligopeptide of the present invention also exhibits anticoagulant activity in an in vivo activity confirmation test using a mouse pulmonary embolism model.

In the present invention, a peptide having only the above oligopeptide A or B as a substantial anticoagulant action site, and a peptide having no anticoagulant activity action site is also included.

Further, in the present invention, as a peptide having two or more anticoagulant sites, a peptide having a plurality of amino acid sequences of oligopeptide A or B provided by the present invention, and further oligopeptide A It also includes peptides having both amino acid sequences of B and B.

The method for producing the oligopeptide of the present invention is not particularly limited, but it can be easily synthesized using, for example, a peptide synthesizer.

[0021]

INDUSTRIAL APPLICABILITY The present invention provides improved oligopeptides derived from the most important region showing an anticoagulant activity of annexin-V, which is superior to the peptide having the original sequence, and these are extremely strong. It was confirmed to show anticoagulant activity. Therefore, such an oligopeptide enables the development of effective therapeutic and prophylactic agents for blood diseases such as DIC (generalized intravascular coagulation). Furthermore, since the oligopeptide of the present invention is used as a constituent material of a diagnostic agent even when diagnosing such a blood disease, it enables a more effective diagnostic method for the disease.

The present invention will be described in more detail below with reference to examples.

[0023]

【Example】

Example 1 Preparation of oligopeptide: The following oligopeptides C and D, which are part of the amino acid sequence of Annexin-V, were prepared using a peptide synthesizer and then separated from the synthesizer by the hydrogen fluoride method. Purification of each peptide was carried out by reverse phase high pressure liquid chromatography (HP
LC) and the degree of purification was confirmed by HPLC and amino acid analysis. Oligopeptide C: Asp His Thr Leu Ile Arg (SEQ ID NO: 3) Oligopeptide D: Ser His Leu Arg Lys Val (SEQ ID NO: 4)

Furthermore, peptides consisting of the following amino acid sequences A and B were similarly synthesized. Oligopeptide A (product of the present invention): His Asp Thr Leu Ile
Arg (SEQ ID NO: 1) Oligopeptide B (product of the present invention): His Asp Thr Gln Pro
Arg Val Leu Asp (SEQ ID NO: 2)

Test Example 1 In Vitro Anticoagulant Activity: The anticoagulant activity of oligopeptides A to D obtained in Example 1 was measured as follows. First, one vial of rabbit brain cephalin (Sigma) was suspended in 100 ml of physiological saline, and this was dispensed in 1 ml aliquots and stored at -20 ° C. This cephalin suspension was mixed with 33 mM calcium chloride solution before each test, and the kaolin solution (5
(Dissolved in physiological saline so as to have a concentration of 0 mM / ml) was added, and inhibition of endogenous blood coagulation by each peptide was measured. That is, 20 μl of plasma and 20 μl of a test sample (dissolved in 50 mM Tris-HCl solution (pH 7.9) containing 50 mM NaCl) were kept at 37 ° C. for 10 minutes, 20 μl of kaolin was added, and calcium chloride sephae was added. Each coagulation time was measured by adding a phosphorus mixture.

Each peptide solution was prepared so as to have a final concentration of 1 mM to 5 mM, and this was added to blood to examine the delay of coagulation time. The results are shown in FIG. 1 as the ratio to the control clotting time without sample peptide (sample peptide / control). That is, with oligopeptides C and D, it was confirmed that the blood coagulation time becomes longer as the concentration of the added peptide is increased. On the other hand, it was confirmed that the oligopeptides A and B of the present invention had an extremely high blood coagulation delaying effect, which was more excellent than the oligopeptides C and D as the concentration of the added peptide was increased. By the way, the coagulation time of each blood when each peptide was added at the final concentration of 5 mM was about twice as long as that of peptide D, and that of oligopeptide C was twice that of the control without any addition. About 6
It was confirmed that the delay was doubled. Further, it was shown that with oligopeptides A and B of the present invention, the lag time of blood coagulation was delayed about 9 times or more as compared with the control at the final concentration of 5 mM. The coagulation time of the oligopeptides A and B of the present invention at a final concentration of 5 mM showed such high activity that coagulation did not occur within the preset measurement time and it was difficult to measure an accurate coagulation time. The results of measuring the anticoagulant activity of each oligopeptide at the final concentration of 4 mM are shown in Table 1 in terms of time.

[0027]

[Table 1]

Test Example 2 In Vivo Anticoagulant Activity: Using the oligopeptides A and B of the present invention obtained in Example 1 and oligopeptide C for pulmonary embolism model mice, the anticoagulant activity in vivo was determined as follows. Was measured. First,
Purified oligopeptides A, B and C were prepared in physiological saline at 2-5 mg / ml.

Male ddY mice (weight 25-40 g)
Each of the above oligopeptide solutions was administered by intravenous injection into the tail of a mouse at a volume of 10 ml / kg to a dose of 20 to 50 mg / kg. Then, within 30 seconds, 30 mg / kg of tissue factor (Tissue Factor: TF, lyoplastin, manufactured by Mochida Pharmaceutical, adjusted to 1 to 5 mg / ml with physiological saline), which is a blood coagulation factor, was administered within 30 seconds. After the administration of TF, the time course of the mice was observed and the time until death (survival time) was measured. The administration of this TF was also carried out by intravenous injection into the tail in a volume of 10 ml / kg.

In this model, when TF is intravenously administered to a mouse, the TF is carried in the bloodstream and is first sent from the heart to the lungs. Therefore, it was considered that blood clots were formed in the lungs, breathing was suppressed, and the mice died.

As a result, in the mice pre-administered with oligopeptide A or B of the present invention, no death was observed in the TF-administered mice in the 50 mg / kg administration group. Also, the dose is less than this, 20-40mg / kg
It was also confirmed that the mortality rate decreased as the dose increased in the oligopeptide-administered group.
With the oligopeptide B of the present invention, no death was observed in TF-administered mice even in the 40 mg / kg administration group.
On the other hand, in the non-administered mice (control) not pre-administered with the oligopeptide of the present invention, T
All deaths were confirmed after F administration. The results in each oligopeptide administration group are shown in Table 2 below. The numerical values in the table are shown by the number of dead mice / the number of TF-administered mice (mortality rate).

[0032]

[Table 2]

[0033]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 6 Sequence shape: Amino acid Sequence type: Peptide Sequence His Asp Thr Leu Ile Arg 1 5

SEQ ID NO: 2 Sequence length: 9 Sequence form: Amino acid Sequence type: Peptide sequence His Asp Thr Gln Pro Arg Val Leu Asp 15

SEQ ID NO: 3 Sequence length: 6 Sequence form: Amino acid Sequence type: Peptide sequence Asp His Thr Leu Ile Arg 1 5

SEQ ID NO: 4 Sequence length: 6 Sequence shape: Amino acid Sequence type: Peptide sequence Ser His Leu Arg Lys Val 1 5

[Brief description of drawings]

FIG. 1 is a view showing the anticoagulant activity of each oligopeptide in Test Example 1.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shoji Kojima 1301-6 Yuge, Tatsuta-cho, Kumamoto-shi, Kumamoto (72) Inventor Hiroshi Mizogami 1647-151 Ikutomi, Kushi-machi, Kikuchi-gun, Kumamoto

Claims (3)

[Claims] 1. An oligopeptide comprising any of the following amino acid sequences. His Asp Thr Leu Ile Arg, His Asp Thr Gln Pro Arg Val Leu Asp 2. The method according to claim 1, which is chemically synthesized.
The described oligopeptide. 3. A peptide having any of the following amino acid sequences as a substantial anticoagulant site. His Asp Thr Leu Ile Arg, His Asp Thr Gln Pro Arg Val Leu Asp